Animal liver tryptophan pyrrolases: Absence of apoenzyme and of hormonal induction mechanism from species sensitive to tryptophan toxicity. 1. Liver tryptophan pyrrolase exists as holoenzyme and apoenzyme in rat, mouse, pig, turkey, chicken and possibly man. 2. The apoenzyme is absent from cat, frog, gerbil, guinea pig, hamster, ox, sheep and rabbit. 3. The hormonal mechanism of induction of the pyrrolase is absent from species lacking the apoenzyme. 4. The concentrations of tryptophan in livers and sera of these species are lower than in species possessing the apoenzyme. 5. Species lacking the apoenzyme or the hormonal induction mechanism have a deficient kynurenine pathway and are sensitive to the toxicity of tryptophan. 6. It is suggested that these species are not suitable as models for studying human tryptophan metabolism. 7. The possible significance of these findings in relation to veterinary and human neonatal care is discussed.


The metabolism of steroids in the fatty liver induced by orotic acid feeding. 1. The metabolism of 4-[4-14C]androstene-3,17-dione, 4-[4-14C]pregnene-3,20-dione, 5alpha-[4-14C]androstane-3alpha,17beta-diol, [4-14C]cholesterol, 7alpha-hydroxy-4-[6beta-3H]cholesten-3-one, 5beta-[7beta-3H]cholestane-3alpha,7alpha-diol and [3H]lithocholic acid was studied in the microsomal fraction of livers from control and orotic acid-treated male rats. 2. As a result of the treatment the orotic acid-fed rats had fatty livers and subnormal concentrations of cholesterol and triglycerides in serum. 3. The 6beta- and 7alpha-hydroxylation of 4-androstene3,17-dione, and the 2alpha-, 2beta- and 18-hydroxylation of 5alpha-androstane-3alpha,17beta-diol, and the 5alpha-reduction of 4-androstene-3,17-dione and 4-pregnene-3,20-dione were decreased by 40--50% in orotic acid-fed rats. Other oxidative and reductive reactions of the steroid hormones were not significantly affected. 4. The 12alpha-hydroxylation of 7alpha-hydroxy-4-cholesten-3-one was decreased by about 50%, whereas the 7alpha-hydroxylation of cholesterol and the 26-hydroxylation of 5beta-cholestane-3alpha,7alpha-diol were not significantly decreased. The 6beta-hydroxylation of lithocholic acid was stimulated by 40%. 5. The results are discussed in relation to present knowledge of the heapatic drug-metabolizing enzymes and to the recent findings of an abnormal bile acid metabolism in liver disease.


The effect of insulin and glucocorticoids on the synthesis and degradation of phosphoenolpyruvate carboxykinase (GTP) in rat adipose tissue cultured in vitro. 1. Epididymal adipose tissue from the rat was maintained in culture for periods of up to 96h. 2. After an initial decrease in protein synthesis during the first 24h of culture, the adipose tissue recovered its capacity to synthesize and accumulate proteins of a relatively large size. 3. The activity of phosphoenolpyruvate carboxykinase decreased in a parallel manner, but increased again after 24h of incubation of the tissue in culture, to a value twice that noted in the tissue in vivo. This increase in enzyme activity was due to an increase in its rate of synthesis. 4. Both insulin and dexamethasone (9alpha-fluoro-16alpha-methyl-11beta,17,-21-trihydroxypregna-1,4-diene-3,20-dione) inhibited phosphoenolpyruvate carboxykinase synthesis, but dexamethasone also decreased total protein synthesis. 5. The half-life of phosphoenolpyruvate carboxykinase in adipose tissue cultured in vitro was 5--7h and was not altered by insulin or dexamethasone. 6. It is concluded that both insulin and glucocroticoids lower the activity of phosphoenolpyruvate carboxykinase in rat adipose tissue by decreasing its rate of synthesis.


Differences between newly formed and recycled free small ribosome subunits in liver cytoplasm. Heterogeneity among the free small ribosomal subunits of rat liver was studied. Newly made free small subunits differ from the bulk of the free subunits in the following ways. (1) The newly made free small subunits sediment slightly faster than most free small subunits and form a band at slightly lower densities. (2) Mature free small subunits, prepared under conditions which minimize the amount of loosely bound protein, readily dimerize in solutions of low ionic strength. Little dimerization is found with newly formed subunits. This permits the separation of fractions enriched in newly formed subunits. (3) The fraction enriched in newly formed subunits contains a distinctive additional protein as compared the most of the free small subunits.


Association of Sjögren's syndrome with HLA-B8. HLA antigens were examined by microdroplet lymphocyte cytotoxicity in 31 patients with Sjögrens syndrome. Fifty percent of 22 Causcasian patients (7 of 17 females and 4 of 5 males) had HLA-B8, as compared to 21% of 1205 controls (P=0.002). The presence of HLA-B8 was not correlated with parotid salivary flow, keratoconjunctivitis sicca, or focus score. Because of its association with a number of autoimmune diseases, including Sjögren's syndrome, it is suggested that HLA-B8 is genetically liked to an immune response gene(s) that predisposes the individual to autoimmune phenomena. The expression of autoimmunity may be determined by infectious or environmental factors.


Rheumatoid factor in acute bacterial endocarditis. Sera from 55 parenteral drug abusers with endocarditis due to Staphylococcus aureus were assayed for the presence and titer of rheumatoid factor. Thirteen (24%) of the 55 patients with endocarditis had sera positive for rheumatoid factor at one point or another in their courses; only 2 (7%) of 30 noninfected drug users were found to be positive. It appeared that more severe cases, as evidenced by duration of fever after initiation of antibiotic therapy, were more likely to develop rheumatoid factor.


Connective tissue synthesis by cultured scleroderma fibroblasts. I. In vitro collagen synthesis by normal and scleroderma dermal fibroblasts. The authors have been unable to demonstrate an increase in collagen synthesis by fibroblasts isolated from  sclerodermatous skin. In order to elucidate this problem further, scleroderma fibroblasts were biopsied from upper dermis, from lower (including subcutaneous) dermis, and from adjacent clinically noninvolved skin. All cell lines failed to show a significant increase in collagen synthesis when they were compared to control fibroblast lines. One difference among them was that fibroblasts from involved areas showed a rate of collagen synthesis equal to or less than cells isolated from adjacent clinically noninvolved sites.


Fibrous myopathy. A rheumatic complication of drug abuse. A progressive fibrous myopathy may result from chronic intramuscular drug abuse. This complication may mimic other rheumatic disorders and early recognition may prevent disability. The patient described here presented with fixed flexion and extension contracture of hips and knees, respectively, after abusing meperidine and other agents for 3 years. Soft tissues of thighs and buttocks were "wood hard," EMG showed absence of action potentials in affected muscles, and biopsy revealed extensive replacement of muscle with dense, acellular fibrous tissue. Possible mechanisms are discussed.


Intracellular distribution of radiogold. Localization to large granule membranes. The distribution of gold in Kupffer cells and in subcellular fractions of rat liver was studied at intervals following intraperitoneal injection of 195Au sodium thiomalate (Myochrysine). Kupffer cells, isolated by digestion of whole liver with Pronase, had radioactive gold counts per milligram of protein that were twice the counts in the digested liver supernatant. After fractionation of liver cells by differential centrifugation, radiogold was found predominantly in the nuclear, mitochondrial, and lysosomal fractions. When the distribution of isotope was related to the protein content, the highest gold concentration was found in the lysosomal fraction, where it was 28 times that in the soluble fraction. Most radiogold was nondialyzable, probably a result of binding to larger intracellular compounds. Approximately 95% of the nondialyzable gold was in the organelle membrane of attached to membrane-adsorbed material, as determined by treatment of dialyzed mitochondrial and lysosomal fractions with Triton X-100. These data suggest that the intracellular locus of gold action may reside in organelle membranes.


Carpo:metacarpal ratio. A new quantitative measure of radiologic progression of wrist involvement in rheumatioid arthritis. Analysis of 878 hand films shows the carpo : metacarpal ratio to be a sensitive quantitative index of progression of carpal involvement in rheumatoid arthritis. This index, easily calculated by dividing the lengths of the carpus by the third metacarpal, showed marked consistency in the ratio regardless of age, race, or sex in normal adults. A decreased ratio correlated highly with recognized radiographic stages of rheumatoid arthritis and thus suggested utility in longitudinal studies of patients with this disease.


Relationship between lipid composition and antibiotic-resistance to isoniazid, streptomycin, p-aminosalicylic acid, ethambutol, rifampicin in mycobacteria. The content of neutral lipids, peptido-glycolipids and phospholipids, separated by solvent extractions and TLC, was investigated and compared in various strains of Mycobacterium tuberculosis and in Mycobacterium avium. The examined strains could be divided into two groups: (i) those possessing a stable INH-resistance with a chromosomal location for the resistance genes, in which the content of total lipids and the distribution of lipid subfractions were modified with respect to the control strain H37Rv; (ii) those strains, such as the mutants resistant to streptomycin (Sm), ethambutol (EM), p-aminosalicylic acid (PAS) and rifampicin (R/AMP), uncorrelated drugs with different genetic location of the antibiotic-resistance, in which a disturbance of the lipid metabolism does not appeared to be significant. TLC and chemical analysis of INH-mutants suggested that the decrease of the cell lipids was associated with the peptidoglyco-phospholipid fraction, while the neutral lipids were characterized by a most elevated amount of both menaquinone-9 and free fatty acids, and by comparable levels of both glycerides and total fatty acids with respect to the sensitive strain. In the pattern of fatty acid shorter than C26 of all the fractions studied, no significant differences were observed between the H37Rv strain and the other resistant strains, including the M. avium. In the course of the lipid analysis no unusual phospholipids in addition to CL, PE and phosphatidylinositol-oligo-mannoside (PIM) were detected. Reasoning in a speculative way, the dramatic fall of the complex lipid elaboration in the INH-resistant mycobacteria, may underline a role for these substances in the envelope of the mycobacterial cell that defends itself from the selecting attack of the antibiotic molecule and that keeps this phenotypic expression of INH-resistance in the mutants.


Association of medullary carcinoma of the thyroid with carcinoembryonic antigen. To investigate the association between medullary carcinoma of the thyroid (MCT) and carcinoembryonic antigen (CEA), we assayed 78 sera from patients with thyroid diseases for CEA, employing the radioimmunoassay of double antibody technique. All 13 sera from patients with MCT had high levels of CEA, ranging from 14 to 170 ng/ml. Increased serum CEA was noted even in cases of small, localized carcinoma. By contrast, serum CEA levels were normal (below 10 ng/ml) in all other histological types of thyroid carcinoma (33 cases), except for one case of papillary adenocarcinoma. In 32 patients with non-malignant thyroid diseases, with few exceptions serum CEA levels remained within the normal range. The elevated serum levels of CEA in MCT returned to normal after successful operation. Furthermore, very high tissue concentrations of CEA were demonstrated in MCT. The results indicate that CEA is actively produced by MCT, and that its measurement is useful in the diagnosis and management of the disease. It is suggested that the highly specific association of CEA with MCT may well be related to a defect of neural crest origin.


Anti-tumour immunity in malignant melanoma assay by tube leucocyte adherence inhibition. Tumour antigen-induced inhibition of leucocyte adherence was modified for use in glass test tubes (Tube LAI assay) for the study of cell-mediated anti-tumour immunity to human malignant melanoma. Peripheral blood leucocytes (PBL) of 20 out of 25 patients (80%) with active malignant melanoma responded to an extract of malignant melanoma with LAI, whereas only 4-5% of 475 control subjects showed a response. The malignant melanoma patients reacted to both allogeneic and autologous extracts of malignant melanoma which indicates a common cross-reacting antigen. Malignant melanoma patients did not respond to unrelated tumour extracts. The LAI was mediated by PBL (monocytes) "armed" with cytophilic anti-tumour antibody specific for the sensitizing tumour antigen. The anti-tumour response of the malignant melanoma patients was dependent on the stage of the cancer, and 11 out of 13 Stage I patients had a positive NAI,  whereas patients with disseminated cancer had decreased response. The diminished LAI in patients with large tumour burdens appeared to be the result of release of tumour antigen systemically. Also, surgery and chemotherapy depressed LAI. Although LAI was depressed after surgical excision of the cutaneous melanoma, most patients showed LAI 1-3 months later. Tumour-free melanoma patients monitored for one year by the Tube LAI assay showed a decline in their anti-tumour immunity 5-6 months after surgery. The NAI was low or negative after the 8th post-surgical month in tumour-free patients. Patients with residual malignant melanoma showed persistent or  recurrent LAI after the 8th post-surgical month. LAI reactivity monitored after "curative" surgery for malignant melanoma may assist in determining whether the patient is tumour-free or has a recurrence.


Chemical transformation of Chinese hamster cells. I. A comparison of some properties of transformed cells. Fifty-one subclones from carcinogen-treated cells of 3 tissues (kidney, liver and prostate) of the male Chinese hamster have been studied to determine the relationships of 3 criteria of in vitro transformation: morphological change, increased plating efficiency and growth in soft agar. There was no correlation between increased plating efficiency and the other 2 parameters. Morphological change was not always easily recognisable, particularly in cells derived from liver, and was not always a stable feature of any given subclone. This may be due to the technique of isolation used (ring cloning) or may be due to chemically-treated cells requiring long periods of culturing before attaining a stable phenotype. When a stable morphological appearance was achieved, there was good correlation between transformed morphology and colony formation in soft agar. The problems of scoring morphological change as an assessment of malignant transformation, and the importance of spontaneous morphological changes are discussed.


Serum ferritin concentration in untreated Hodgkin's disease. Serum ferritin has been estimated in 125 untreated patients with Hodgkin's disease. Increasing concentrations are found at each advancing stage of the disease and high concentrations are found in patients with systemic symptoms. In all cases this is associated with a low serum Fe concentration and reduced transferrin saturation. There is no relationship between serum ferritin concentration and histological type of disease. The findings are compatible with a non-specific response of the reticuloendothelial system to malignancy, producing a secondary disorder of Fe metabolism.


Clinical trial of combination chemotherapy and specific active immunotherapy in disseminated melanoma. Fifty-six patients with disseminated malignant melanoma were randomly allocated to two treatment groups. The first group C received combination chemotherapy consisting of DTIC and ICRF 159. The second group (C+I) received the same chemotherapy but were also immunized with 2 X 10(7) irradiated allogeneic melanoma cells mixed with 50 mug of percutaneous BCG. The survival rates in both treatment groups C and (C+I) were not significantly different, and only minor enhancement of the chemotherapy was found in the (C+I) group. A similar pattern of tissue response was observed in both groups: lymph node, skin and, to some extent liver metastases, respond better than other sites.


Distribution of survival times of 12,000 head and neck cancer patients who died with their disease. The lognormal parametric statistical model can provide, for groups of carcinoma cervix patients, good estimates of long-term survival fractions several years earlier than would otherwise be possible. The present paper extends this model work to head and neck cancer by using a minimum chi-squared test for goodness of fit (P greater than 0-05), to study the distribution of survival times of patients who died with their cancer present. Some 12,000 case histories were available from 7 hospital registries, 4 regional cancer registries and one national registry (the OPCS). All histories were followed up for at least 10 years subsequent to treatment and could be grouped into one of 8 cancer sites: antrum, floor of mouth, larynx, nasopharynx, pyriform fossa, post cricoid, tonsil and tongue. The theoretical distributions investigated were the lognormal, negative exponential and skew exponential. The results showed that the lognormal provided the best overall fit to the data, although the range of optimum values for the lognormal parameter, S, differed with cancer site. The optimum range did, however, usually include the value S=0-45. These results will now permit the second stage of validation of the lognormal model to proceed for head and neck cancers.


Faecal steroids and bacteria and large bowel cancer in Hong Kong by socio-economic groups. In a study of three socio-economic groups in Hong Kong, the high income group had a high faecal concentration of bile acids, especially the dihydroxy bile acids, compared to the low income group. The faecal bile acids were also more highly degraded. The faecal flora contained more bacteroides and fewer eubacteria. Very few of the clostridia able to dehydrogenate the steroid nucleus were isolated. An epidemiological study based on street blocks indicated that the high income group also have a higher incidence of cancer of the large bowel and of the breast. The results are discussed in terms of theories on the aetiology of large bowel cancer.


Predictions of mortality from mesothelial tumours in asbestos factory workers. Using the accumulated data on deaths from mesothelial tumours among cohorts of male and female factory workers at a London asbestos textile factory, the mortality from this cause up to the year 2000 AD has been predicted. The limitations of the methods used are pointed out, but it is estimated that for men the mortality due to mesothelial tumours will be between 7% and 11% of the total mortality and somewhat higher for women. The highest number of deaths from mesothelial tumours will occur during the 1980s, thereafter the numbers will decline because of the decreasing size of the cohort resulting from general mortality.


Monitoring liver disorders in vinyl chloride monomer workers using greyscale ultrasonography. Recognition that vinyl chloride could be hepatotoxic led to a survey of workers to determine whether changes had been induced by past exposure, and to evaluate standard liver function tests as monitors of early liver abnormalities. Standard liver function tests were found to be unsuitable for the detection of such abnormalities in the population at risk. Of 487 workers examined, 102 (20-9%) had abnormalities on initial testing but only two were finally shown to have portal hypertension; in both cases, thrombocytopaenia provided the first diagnostic evidence since liver function tests were normal. Furthermore, 40 (35-7%) of 112 control subjects had initial test abnormalities. A sample of 19 workers with various exposures to vinyl chloride monomer were examined blind by greyscale ultrasonography. Five with minimal or no exposure were confirmed as normal but 12 of the remainder had abnormalities. These consisted of an enlarged portal vein (seven instances), splenomegaly (eight), and changes in hepatic texture (seven). Five of these 12 cases had previously been considered normal. It was concluded that greyscale ultrasonography had many advantages over standard methods for screening workers exposed to hepatotoxic chemicals, and should be the subject of a large scale evaluation.


Loss of pulmonary elastic recoil in workers formerly exposed to proteolytic enzyme (alcalase) in the detergent industry. Sixty-seven workers in the detergent industry whose exposure to proteolytic enzyme ceased in 1969 have been examined clinically and functionally. By comparison with 42 lightly and moderately exposed subjects, 13 heavily exposed subjects showed significant loss of pulmonary elastic recoil as evidenced by increased lung volumes and increased pulmonary compliance, but there were no differences in airways resistance or other parameters of lung function. No difference was found between the two groups in relation to symptoms on exposure, current exercise tolerance, skin reactivity to the proteolytic enzyme alcalase, trypsin inhibitor capacity, and other features. An increased clinical grade of breathlessness was associated with evidence of airways obstruction, but not of altered elastic recoil. Comparison of the data on lung mechanics with results obtained in 1970 suggests that partial recovery of pulmonary elastic recoil may have occurred in some cases. It is also suggested that diminished elastic recoil, in the absence of impairment of transfer factor at rest, may reflect altered physical properties of the lung fibre network without loss of effective surface area available for gas exchange.


Byssinosis: a follow-up study of cotton ginnery workers in the Sudan. A follow-up study of lung function tests and dust measurements was undertaken in ginnery workers employed in five ginning factories. Respiratory symptoms and respiratory function tests (FEV1 and FVC) were first recorded in 1967 on a total of 382 workers (323 permanently employed ginnery workers, 35 seasonal farfara workers, and 24 fire brigade men as controls). In 1969 after a six-month break from ginning before the start of the season, a follow-up study of lung function tests was undertaken on 96% of the same workers (306 ginnery workers, 35 farfara, and 24 fire brigade men). Because of the lapse of two years a new adjustment for age and height was made so as to compare the FEV1 measured in the follow-up study. The fire brigade men showed an expected fall in FEV1 during the two-year period, whereas the ginnery workers showed a rise presumably because they had had no dust exposure during the previous six months. The differences between the degree of change in these groups were statistically significant. In 1967 only the fine dust (less than 7 mum) was measured, while in 1969 the concentration of fine and medium dust, that is, less fly was measured. The factories were divided into three groups according to dust concentration. Comparison between the three factory groups and farfara shows a positive association between the level of dust concentration less fly and the prevalence of cough and phlegm. Since age did not appear to be a significant factor in the prevalence of byssinosis, comparisons between permanent workers in these three groups of factories and farfara workers taken separately were made without age standardization. The overall differences were statistically significant. There was a marked trend showing a positive association between prevalence of byssinosis and level of cotton dust concentration in the factories. The correlation between dust levels and the prevalence of byssinosis was nearly perfect when the time factor was included.


Acute inhalation toxicity of cotton plant dusts. The number of free lung-cells was studied in guinea-pigs after acute exposure to extracts of various cotton dusts. A good correlation was found between the increase in number of leucocytes in the airways and the number of Gram-negative bacteria in the different dusts. Experiments using the Shwartzmann reaction and the Limulus titration test demonstrated a relationship between the content of different endotoxins in the dusts and the pulmonary reaction. A model for the acute exposure effects after exposure to cotton dust is proposed.


Energy expenditure and physiological performance of Sudanese cane cutters. The thermal and exercise tolerances of 165 Sudanese cane cutters were measured in the laboratory and related to work performance and productivity in the cane fields. The results showed that the amount of cane cut per minute in the field was significantly correlated with changes in body weight (r = +0-53) during the third hour of work, aerobic energy expenditure (r = + 0-43), and cardiac frequency during work. These variables in turn were associated with predicted maximal power output (VO2 max) measured in the laboratory. The average energy expenditure during cane cutting was 1-66 +/- 0-33 1/min-1 (34-9 kJ/min-1) which represents approximately 60% of the workers predicted VO2 max. This rate of energy expenditure was sustained in the cane fields for at least three hours without significant pauses for rest. The sweat losses measured in 32 cane cutters during the two and three hours of work averaged 637 +/- 221 and 770 +/- 282 g/h-1 respectively, while the mean urine temperature immediately on cessation of effort was 37-74 +/- 0-46 degrees C. Despite the additional environmental heat load of the tropics, it would seem that cane cutters performing a self-paced task demanding heavy physical effort, are able to sustain work levels well in excess of those recommended for most European factory workers without obvious signs of fatigue or heat stress.


Urinary non-precipitable lead in lead workers. Sixty-six workers engaged in lead-glazing pottery with a presumed moderate exposure to lead were studied. The group comprised 20 men with long-term exposure to lead and positive laboratory signs of increased lead absorption (Group A); 22 with long-term exposure and negative laboratory signs (Group B); 11 with short-term exposure and positive laboratory signs (Group C); and 13 with short-term exposure and negative laboratory signs (Group D). In addition, 14 workers employed in casting the kelmet alloys with presumed heavy exposure to lead (Group E) and seven healthy individuals (Group F) were included. Urine samples from all the subjects were analysed to determine, first, the total lead using the ashing technique, and then the precipitable lead using the coprecipitation technique of Cholak, Hubbard, and Burkey (1948), but modified slightly by us. Thus, the non-precipitable lead fraction in urine was the difference between the two measurements and this was also expressed as a percentage of the total lead. The mean total lead and the mean proportion of non-precipitable lead were 0.62 mumol/l and 48.7%, 0.35 mumol/l and 44.9%, 0.40 mumol/l and 48.9%, 0.17 mumol/l and 24.6%, 1.43 mumol/l and 44.3%, 0.14 mumol/l and 18.8% for Groups A, B, C, D, E, and F respectively, showing that a large part of urinary lead was eliminated as precipitable lead in Groups D and F who had normal lead excretion, while about half was eliminated as non-precipitable lead in the other four groups who had excessive lead excretion. No essential difference in the proportion of non-precipitable lead among Groups A, B and C excluded the possibility that the proportion might be directly related to the period of exposure to lead and to the laboratory findings of excessive lead absorption. The mean proportion of non-precipitable lead for the physiological (up to 0.240 mumol/l), intermediate (0.241 to 0.721 mumol/l), and excessive (above 0.722 mumol/l) total lead levels was 26.7, 41.3, and 52.3% respectively, in the lead workers comprising Groups A, B, C, and E each showing increased lead excretion when grouped together. these data suggested that, when urinary lead is within the normal range, it is excreted largely as precipitable lead even in individuals exposed to lead, and that the principal conditions determining the excretion of non-precipitable lead would be the current or recent degree of lead absorption. The excretory mechanisms and the biological significance of the non-precipitable lead are also discussed.


Erythrocyte fluorescence and lead intoxication. Blood samples from people exposed to inorganic lead were examined by fluorescence microscopy for excess erythrocyte porphyrin. With continued lead absorption, fluorescent erythrocytes appeared in the circulation of workers handling this metal or its compounds, and they progressively increased in number and brilliance. These changes ensued if the blood lead concentration was maintained above 2-42 mumol/l (50 mug/100 ml), and preceded any material fall in the haemoglobin value. At one factory, 62-5% of 81 symptomless workers showed erythrocyte fluorescence attributable to the toxic effects of lead. Excess fluorocytes were found in blood samples from a child with pica and three of her eight siblings. These four were subsequently shown to have slightly increased blood lead concentrations (2-03 to 2-32 mumol/l). Fluorescence microscopy for excess erythrocyte porphyrin is a sensitive method for the detection of chronic lead intoxication. A relatively slight increase in the blood lead is associated with demonstrabel changes in erythrocyte porphyrin content. The procedure requires little blood, and may be performed upon stored samples collected for lead estimation. The results are not readily influenced by contamination, and provide good confirmatory evidence for the absorption of biochemically active lead.


Catalytic activation of transfer ribonucleic acid by a mammalian protein. A tRNA activator has been isolated from mammalian organs which increases the capability of tRNA to accept certain amino acids through the action of mammalian aminoacyl-tRNA synthetases. This activity may be separated from the aminoacyl-tRNA synthetases for isoleucine, lysine, serine, and methionine by fractionation of liver or pancreas cytosol with ammonium sulfate or by chromatography over Sephadex G-200. The tRNA activating material is nondialyzable and is destroyed by trypsin or short heating. It acts catalytically. A molecular weight of approximately 45,000 was obtained by chromatography of tRNA activator on a calibrated Sephadex G-150 column. Activator increases acceptance of yeast tRNA for the amino acids isoleucine, leucine, lysine, serine, and methionine. It shows higher activity on liver tRNAMet f, tRNAMet m, and tRNALys than on unfractionated liver tRNA. Removal of protein from mammalian tRNA by extra phenol extractions, chromatography, or proteinase treatment increases its response to activator.


Nearest neighbor analysis of heparin: identification and quantitation of the products formed by selective depolymerization procedures. Heparin was carboxyl-reduced with sodium boro[3H]hydride and converted to a mixture of oligosaccharides by treatment with nitrous acid at pH 2. The oligosaccharide mixture was aldehyde-reduced with sodium boro[3H]hydride and the mixture of products, labeled both in the hexoses formed in the carboxyl-reduction step and in the reducing sugars formed in the nitrous acid reaction, was separated and analyzed. The major product, L-idosyl 2-sulfate leads to anhydro-D-mannitol 6-sulfate (I), contained 60% of hexoses derived from the hexuronic acid residues in the original heparin. A second product, which contained 15% of the hexoses derived from the hexuronic acid residues in the original heparin, was identified as a tetrasaccharide composed of two L-idosyl 2-sulfate residues, one anhydro-D-mannitol 6-sulfate residue (the reducing end),and a hydroxymethylpentose sulfate residue formed by deamination of a disulfated D-glucosamine residue without bond cleavage. Several additional disaccharides derived from the regions of the polymer which contained D-glucuronic acid residues and lower degrees of O-sulfation were also identified among the deamination products. The oligosaccharides that were obtained accounted for 100% of the original carboxyl-reduced heparin, and paper chromatographic profiles of the oligosaccharide separations can be used as a fingerprint of the heparin preparation. The properties of I were examined in greater detail. The glycosidic bond of the L-idosyl 2-sulfate residue was found to be extremely labile to 0.1 N HCl at 100 degrees C, hydrolyzing with a t 1/2 of 18 min to give high yields of L-idose 2-sulfate and anhydro-D-mannitol 6-sulfate. L-Idofuranose was also identified as an intermediate in the conversion of L-idose 2-sulfate to L-idosan. The acid lability of the L-idosyl 2-sulfate bond in I offers a new route for the selective cleavage of carboxyl-reduced heparin.


2-Imino-2-methoxyethyl 1-thioglycosides: new reagents for attaching sugars to proteins. Cyanomethyl 1-thioglycosides ofD-galactose, D-glucose, 2-acetamido-2-deoxy-D-glucose, and D-mannose were prepared from the respective pseudothiourea derivatives and chloroacetonitrile. The nitrile group in these cyanomethyl thioglycosides can be converted to a methyl imidate group by treatment with sodium methoxide or HC1 in dry methanol to yield 2-imino-2-methoxyethyl 1-thioglycosides (IME-thioglycosides). The factors influencing the yield of IME-thioglycosides were investigated. The most convenient method of preparing IME-thioglycosides was treating 0.1 M cyanomethyl thioglycoside peracetate in dry methanol with 0.01 M sodium methoxide at room temperature for 24-48 h (50-60% yield). These IME-thioglycosides reacted readily with simple amines, amino acids, and proteins in mildly alkaline buffer solutions. Alpha-amylase and lysozyme modified with these reagents under appropriate conditions retained full activities. Thus the IME-thioglycosides constitute a new group of reagents for attaching sugars to proteins.


Attachment of thioglycosides to proteins: enhancement of liver membrane binding. Thioglycosides of D-galactose, D-glucose, N-acetyl-D-glucosamine, and D-mannose were covalently attached to Aspergillus oryzae alpha-amylase, hen's eggs lysozyme, and bovine serum albumin by amidination, diazo coupling, and amide formation. The binding of the newly formed glycoproteins (neoglycoproteins) to rabbit liver membranes was measured, using asialoorosomucoid as a reference. Attachment of D-galactosides by any of the three methods enhanced binding by several orders of magnitude. Coupling of a comparable number of D-mannosides or N-acetyl-D-glucosaminides had little or no effect. Attachment of D-glucosides also enhanced binding but to a variable extent depending on the method of attachment. Thus, the behavior of neoglycoproteins toward rabbit liver membranes closely paralleled that of serum glycoproteins (Ashwell and Morell, 1974) with respect to sugar specificity.


Biosynthesis of steroid sulfates by the boar testes. The steroidogenic enzymes present in boar testicular tissue have been shown to use steroid sulfates as substrates. Pregnenolone sulfate, doubly labeled with 3H in the nucleus and 35S, was incubated with the microsomal fraction of boar testicular tissue and a reduced triphosphopyridine nucleotide generating system and 17-hydroxypregnenolone sulfate as well as dehydroisoandrosterone sulfate were isolated. These products had the same 3H to 35S ratio as the substrate demonstrating that the conversions had taken place with the sulfate group intact. Thus testicular 17-hydroxylase and C-17,20-desmolase can convert delta5-3beta-yl sulfates into products which still contain the sulfate group at C-3. An unusual steroid sulfate, doubly labeled 5,16-androstadien-3beta-yl sulfate, was also isolated demonstrating that such an olefin can be biosynthetized via steroid sulfate pathways. In a second experiment small amounts of [3H]-21-hydroxypregnenolone sulfate were isolated from the incubation of [3H]pregnenolone sulfate with microsomes from boar testes. The isolation of the 21-hydroxylated steroid sulfate is taken as support for the hypothesis that delta16 steroids are biosynthesized from their C21 precursors by events that are initiated by oxygenation at C-21.


Role of cyclic electron transport in photosynthesis as measured by the photoinduced turnover of P700 in vivo. The light-induced turnover of P700 was measured spectrophotometrically in a wide variety of algae and some photosynthetic mutants. Analysis of the postillumination recovery of P700+ revealed that the apparent first-order rate constant for reduction via the cyclic pathway was much lower that that via the noncyclic pathway. After activation of photosystems 1 and 2 the half-time for reduction of P700+ was 5-20 ms, whereas after activation of primarily photosystem 1 a longer half-time of ca. 150 ms was observed. The extent of the photooxidation of P700 was the same in both regimes of illumination. The longer half-time was also noted after inhibition of photosystem 2 by 3-(3,4-dichlorophenyl)-1,1-dimethylurea or mild heat shock and in mutant algae known to lack a functional photosystem 2. No signal was observed in mutants lacking P700 itself but those strains lacking either plastocyanin or cytochrome f were capable of a very slow turnover (reduction t 1/2 greater than 500 ms at room temperature). This very slow turnover was not affected by carbonyl cyanide m-chlorophenylhydrazone or the plastoquinone antagonist, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, indicating that the pathway for reduction of P700+ in these mutants is not energy linked and does not utilize the intersystem electron transport chain. The slow, 150 ms, reduction of P700+ due to cyclic flow was not observed when cells were engaged in photosynthesis at high-light intensities. The data are interpreted as evidence for the involvement of the total functional pool of P700 in both electron transport pathways, and we suggest that cyclic electron transport does not contribute to photosynthesis in oxygen-evolving autotrophs.


Proton magnetic resonance study of the intramolecular association and conformation of the alpha and beta pyridine mononucleotides and nucleosides. The chemical shifts and coupling constants are reported for the proton nuclear magnetic resonance (NMR) spectra of the alpha and beta anomers of the oxidized and reduced pyridine mononucleotides and nucleosides. The pseudorotational conformational analyses of the ribose coupling constants indicate that the ribose conformation for beta-nicotinamide mononucleotide, beta NMN, can best be described by a 3:1 mixture of interconverting 3'-exo (S) and 2'-exo (N) conformers. Reduction of betaNMN to betaNMNH results in phase angles consistent with interconverting 2'-endo (S) and 3'-endo (N) conformers without changes in the conformer populations. Cleavage of the 5'-phosphate from betaNMN has a significant effect on the phase angles (becoming more like those for betaNMNH), conformer population (the N and S conformers become nearly equal), and the distribution of the rotational isomers around the ribose 4'-5' bond to the exocyclic methylene (the gauche-gauche population decreases by about 25%). In contrast, for betaNMNH these parameters are all insensitive to dephosphorylation. The pseudorotational analysis has been extended to define the conformational parameters of alpha nucleotides. Analysis of the coupling constants for the alpha anomers indicates that the phase angles, conformer populations, and rotational isomers are generally insensitive to dephosphorylation, whereas both the phase angle and conformer populations are strongly dependent on the redox state of the base, alphaNMN being predominantly 2'-endo and alphaNMNH exclusively 2'-exo. The rotational isomers around the 4'-5' and 5'- O bonds are found to be insensitive to.the large changes in ribose conformation in the absence of any interaction with the base. The results are discussed in terms of relative contributions from base-ribose, ribose-side chain, and base-side chain interactions to the general conformational restraints imposed by the cis-2'-3'-hydroxyl interaction in beta nucleotides and the additional cis-2'-hydroxyl-base interaction in alpha nucleotides. The significance of these interactions with respect to the enzymatic and nonenzymatic properties of the pyridine nucleotides is also considered.


The construction and testing of a simple, slow delivery-rapid quench apparatus. A simple, inexpensive, slow delivery-rapid quench apparatus is described. The apparatus can be used to mix small volumes (about 50 mul) of equilibrium mixtures of enzyme-substrate and enzyme-product complexes with a quenching solution, ideally to inactivate the enzyme more rapidly than such complexes can be inconverted. The efficiency of the apparatus is tested by (a) injecting basic solutions of an indicator dye into acid and observing the length of the unbleached plume of dye produced at the delivery tip and (b) by forming an enzyme-substrate complex with 32P-labeled phosphoglucomutase and measuring the extent of label transfer prior to inactivation by the quenching solution. Problems that may be encountered in attempts to inactivate equilibrium mixtures of enzyme-substrate and enzyme-product complexes without producing quenching artifacts are considered.


Thermodynamics and mechanism of the PO3 transfer process in the phosphoglucomutase reaction. The equilibria among the central complexes in the phosphoglucomutase system were evaluated by (a) using an excess of enzyme plus Mg2+ to prepare mixtures with glucose phosphates in which essentially no free glucose phosphates were present; (b) inactivating the enzyme in such mixtures by means of a procedure that prevents substantial interconversion of the central complexes; and (c) assaying the quenched mixture for glucose 1-P, glucose 1-6-P2, and glucose-6-P. The fractional amounts of Ep-Mg-Glc-1-P, ED-Mg-Glc-P2, and Ep-Mg-Glc-6-P present at pH 7.5 and 24 degrees C were 0.13, 0.54, and 0.33. (Ep and ED are the phospho and dephospho forms of the enzyme, respectively). From these fractions and the equilibrium isotope exchange constants for the three sugar phosphates, true dissociation constants can be calculated for each of the above complexes: 8.5 muM, 19 nM, and 57 muM, respectively. Relative to the rate of PO3 transfer to water, a 3 x 10(10)-fold rate increase is produced by binding glucose-1-P to the Mg2+-enzyme (Ray, jr., W.J., Long, J.W., and Owens, J.D. (1976), Biochemistry, the following paper in this issue). This "substrate-induced rate effect" is equivalent to a difference of some 14 kcal in Gibbs activation energies for transfer to chemically similar hydroxyl groups, and most of this energy difference ultimately must be rationalized in terms of binding interactions involving the phosphoglucosyl moiety. Three different mechanisms for using substrate binding energy to reduce the activation energy of the subsequent catalytic step are examined as possible explanations for the substrate-induced rate effect. These mechanisms emphasize (a) enthalpic destabilization and (b) (entropic) immobilization of reactant groups during formation of the enzyme-substrate complex, and (c) increased binding interactions of nonreactant groups during the subsequent approach to the transition state. As a test for enthalpic destabilization of the enzymic phosphate group, values of deltaG degrees' for the hydrolytic cleavage of this group in Ep and Ep-Glc-1-P are calculated from equilibria measured at pH 7.5 and 30 degrees C: about -1 and +1.4 kcal/mol, respectively. To test for destabilization of the acceptor hydroxyl group in the enzyme-substrate complex, deltaG degrees' for equilibrium, Ep-Glc-P in equilibrium ED-Glc-P2, is compared with that for the corresponding process involving the nonrigid acceptor, 1,4-butanediol monophosphate: about -0.9 and -1.9 kcal, respectively. These results are not consistent with a large enthalpic destabilization of the reactant groups in the Ep-Glc-1-P complex. To test for entropic immobilization of reactant groups, glucose-6-phosphate is considered as a bidentate ligand, and the chelate effect on the binding and subsequent enzymic transfer reaction that arises from covalently linked the sugar ring and the PO3 group is evaluated. Reference reactions involving xylose as a PO3 acceptor both in the presence and absence of bond (inorganic) phosphite are used...


An analysis of the substrate-induced rate effect in the phosphoglucomutase system. The rate constant for the catalytic transfer of the active-site PO3 group from rabbit muscle phosphoglucomutase to the hydroxyl group of a water molecule is about 3 x 10(-8) s-1 under optimal reaction conditions, but in the absence of the normal substrate, viz., at pH 7.5 and 30 degrees C, in the presence of saturating Mg2+; the corresponding constant for transfer to the 6-hydroxyl group of glucose 1-phosphate under analogous conditions, about 1000 s-1, is larger than this by some 3 x 10(10)-fold. Since no single factor appears to be capable of providing a rationale for a majority of this "substrate-induced rate effect" (Ray, jr., W.J., and Long, J.W. (1976), Biochemistry, the preceding paper in this issue), the change in the PO3-transfer rate produced by binding various parts of the phosphoglucosyl moiety to the enzyme, both separately and concurrently, was investigated. The rate of PO3 transfer to water is increased by up to 1000-fold by binding entities that provide the active site with a second PO3 group, e.g., ethyl phosphate or inorganic phosphite. Using an alcoholic acceptor further increases transfer efficiency (in the presence of bound phosphite): increase with methanol, about 2000-fold on a molar basis. The reactivities of ten other primary aliphatic alcohols vary by nearly 600-fold as the acidity of the PO3 acceptor is varied over a 4000-fold range. Although no straightforward relationship is observed between the efficiency of an alcohol as an acceptor and its acidity - presumably because of complications due to steric effects, for example - an increased transfer rate of 100-fold, relative to the water reaction, is estimated for a simple primary alcohol with a pKa similar to that expected for the 6-hydroxyl group of glucose 1-phosphate, when the alcohol is present at a concentration of 1 M. Joining an alcoholic acceptor and a PO3 group via five apparently inert bridging units changes PO3 transfer to an intramolecular process; in the case of 1,4-butanediol monophosphate the rate of transfer also increases by 240-fold, relative to the analogous reaction in the presence of 1 M propanol and bound inorganic phosphite. Comparable values also are obtained in comparisons of PO3 transfer rates for trans- 1,4-butenediol and 1,4-butynediol monophosphates relative to 1 M allyl and propargyl alcohols, respectively, in the presence of bound phosphite. An increased rate of transfer also is produced by binding the xylosyl part of the glucose ring, either when the acceptor is an hydroxyl group attached to the ring or when it is the hydroxyl group of a water molecule, e.g., as in the water reaction facilitated by bound xylose 1-phosphate. These and other results suggest that most of the differences between the rates of the water reaction and the glucose 1-phosphate reaction can be rationalized in terms of four fairly discrete factors whose approximate values are as follows: the PO4 factor, 1000-fold; the C-OH/H-OH factor, 100-fold; the nucleophile-binding factor, 250-fold; and the (CHOH)3-bridging factor, 200-fold...


The thermodynamic and structural differences among the catalytically active complexes of phosphoglucomutase: metal ion effects. When the identity of the metal ion activator, M, is changed within the series, Zn2+, Co2+, Mg2+, Ni2+, Mn2+, and Cd2+, the equilibrium distribution among the central complexes in the phosphoglucomutase system is markedly altered. (The central complexes are Ep-M-Glc-6-P, ED-M-Glc-1,6-P2, and Ep-M-Glc-1-P, where Ep and ED are the phospho and dephospho forms of the enzyme). This altered distribution is caused by a metal-specific change in the equilibrium constant for transfer of the enzymic PO3 group to bound glucose monophosphates: 65-fold as M is varied from Zn2+ to Cd2+. This change in equilibrium is related to metal-specific differences in chemical potential of the phosphate group in the Ep-M complex; these differences in chemical potential remain in the Ep-M-Glc-1-P and Ep-M-Glc-6-P complexes, but essentially disappear in the ED-M-Glc-1,6-P2 complex. If glucose monophosphates are considered as substrates, and glucose bisphosphate as the product, there is a direct relationship between the equilibrium concentration of enzyme-substrate and enzyme-product complexes (when these are varied by changing the identity of the bound metal ion) and the ultraviolet spectrum of the equilibrium mixture of complexes, as assessed by difference spectroscopy (Peck, E.J., Jr., and Ray, W.J., Jr. (1969), J. Biol, Chem. 244, 3754). These spectral changes apparently are caused by an alteration in the conformation of the enzyme during transfer of a PO3 group between the enzyme and the glucose phosphate moiety, or as the result of it. The extent to which conformational changes accompany group-transfer processes in other enzymic systems is not clear, but it is possible that analogous changes may help to account for the "half-of-the-sites reactivity" observed with a number of multimeric enzymes.


Synthesis of human [9-leucine-B] Insulin. The synthesis and isolation in purified form of [Leu9-B]insulin, a biologically active analogue of human insulin, is described. This analogue differs from the parent molecule in that the polar residue, serine, occupying position 9 in the B chain and located on the outside of the insulin molecule, has been replaced with the hydrophobic leucine residue. For the synthesis of this analogue the [Leu9]B chain of human insulin was chemically synthesized by the fragment condensation approach and isolated in the S-sulfonated form. Combination of this compound with the sulfhydryl form of human A chain afforded [Leu9-B]insulin. Separation of this analogue from the combination mixture and isolation as the hydrochloride in purified form were accomplished by chromatography on a carboxymethylcellulose column with an acetate buffer (pH 3.3) and an exponential sodium chloride gradient. [Leu9-B]Insulin possesses a potency of 13-14 IU/mg when assayed by the mouse convulsion method and 11-12 IU/mg by the radioimmunoassay method as compared to 23-25 IU/mg possessed by the natural hormone.


Synthesis of two biologically active insulin analogues with modifications at the N-terminal and N- and C-terminal amino acid residues. The synthesis and isolation in purified form of two analogues of insulin is described. [21-Isoasparagine-A] ([Iasn21-A]) insulin differs from the parent molecule in that the amino acid residue, asparagine, found at the C terminus of the A chain (A21) has been replaced by isoasparagine. [Sar1, Iasn21-A] insulin differs from insulin in that both the A1 and A21 amino acid residues, glycine and asparagine, have been substituted by sarcosine and isoasparagine, respectively. The synthesis of these analogues followed the pattern employed in this laboratory for the synthesis of insulin and its analogues. The S-sulfonated derivatives of the A chain analogues were chemically synthesized, converted to their sulfhydryl forms, and then combined with the S-sulfonated B chain to produce the respective insulin analogues. Isolation of the insulin analogues from the combination mixtures was effected by chromatography on a carboxymethylcellulose column with an exponential sodium chloride gradient. By the mouse convulsion assay method [Iasn21-A]insulin possessed a potency of 21 IU/mg and [Sar1, Iasn21-A] insulin 15 IU/mg. The radioimmunoassay method gave values of 16 IU/mg for the former and 7IU/mg for the altter analogue. The natural hormone has a potency of 23-25 IU/mg by both assay methods. These data indicate that the alpha- and beta-carboxyl groups of the A21 amino acid residue are nearly equivalent in terms of their contribution to the expression of the biological activity of insulin. Furthermore, these data strengthen the speculation (Cosmatos, A., Johnson, S., Breier, B., and Katsoyannis, P. G. (1975), J. Chem. Soc. Perkin Trans. 1, 2157) that the change in the relative positive charge at the N-terminal amino acid residue of the A chain is responsible for the considerable decrease in the immunoreactivity observed in such modified insulins.


Synthetic, cross-linked polypentapeptide fo tropoelastin: an anisotropic, fibrillar elastomer. Physical properties of the cross-linked polypentapeptide of tropoelastin are reported along with chemical characterization of key intermediates in its synthesis. 220 MHz proton magnetic resonance spectra are reported on the constituent pentamers and their respective high polymers which verify structural and conformational integrity. Scanning electron microscopy of the cross-linked material formed without orientation and with flow orientation is reported. The former demonstrates the inherent fibrillar and anisotropic nature of the synthetic product. Stress-strain studies show the cross-linked polypentapeptide to exhibit elastomeric properties that are dependent on the water content of the matrix. At high water contents the elastic modulus is less than that of wet native aortic elastin and becomes greater on drying.


Cross-linked polypentapeptide of tropoelastin: an insoluble, serum calcifiable matrix. The synthetic, cross-linked polypentapeptide of tropoelastin has been shown to calcify from serum alone even when separated from the serum medium by a dialysis membrane with a low-molecular-weight cut off. By microprobe analysis, it appeared that the only serum elements required for the calcification were calcium and phosphorus. Furthermore, thin sections of the calcified matrix showed the calcification to occur throughout the matrix, and thereby verified that it is a bulk property of the matrix and not an interfacial property. To our knowledge this is the first demonstration of an insoluble, synthetic polypeptide to function as a serum calcifiable matrix and by doing so it opens the door to potential medical applications.


Hybridization of maize chloroplast DNA with transfer ribonucleic acids. Hybridization of [125I] tRNA to chloroplast DNA indicates that 0.60-0.75% of maize chloroplast DNA contains sequences complementary to maize tRNA, corresponding to 20-26 tRNA cistrons. Green maize seedlings contain about twice the amount of chloroplast DNA-hybridizable tRNA as etiolated maize seedings. tRNA from green or etiolated maize seedlings was also aminoacylated in vitro with 21 labeled amino acids and then incubated with filters containing chloroplast DNA, tRNAs charging a total of at least 16 different amino acids hybridized with chloroplast DNA. Most of these plastid aminoacyl-tRNAs were present in higher concentrations in tRNA isolated from green maize seedlings, although there were several exceptions. The results are consistent with the hypothesis that a complete or nearly complete set or tRNAs can be transcribed from chloroplast DNA.


Poly(adenylic acid)-containing RNA from plastids of maize. Hybridization experiments with chloroplast DNA and 125I-labeled RNA from maize seedlings suggest that chloroplasts and etioplasts contain detectable amounts of RNA that contains poly(adenylic aicd) (poly (A)) and was transcribed from chloroplast DNA. About 6% of the total poly(A)-containing RNA isolated from maize seedlings hybridized to chloroplast DNA. Poly(A)-containing RNA could also be isolated directly from purified chloroplasts that were treated with ribonucleases to reduce cytoplasmic contamination. At least 65% of this poly(A)-containing RNA hybridized to chloroplast DNA. Chloroplast poly(A) tracts average about 45 nucleotides in length, one-half the average size of poly(A) tracts from whole cells. The poly (A) tracts themselves are probably added to plastid RNAs following their transcription, because maize chloroplast DNA was found not to contain poly(dT).


Inverted repetitive sequences in the human genome. A specific class of DNA sequences, the inverted repetitive sequences, forms hairpin-like structures in denatured DNA by the folding back of a single linear chain. The reassociation process of these sequences is unimolecular and the rate is extremely fast. Inverted repetitive sequences comprise 6% of the total human genome. They appear to be heterogeneous in length with an overall average length of 190 nucleotides. The inverted sequences are represented in almost all families of repetition frequencies, highly repetitive as well as very few copies per genome. They are not localized at unique sites on metaphase chromosomes. It is estimated that there are approximately 2 X 10(6) inverted repeats per haploid human genome. The biological function of this class of sequences is unknown.


Histone-histone interactions within chromatin. Preliminary characterization of presumptive H2B-H2A and H2B-H4 binding. Cyanogen bromide peptide analyses of cross-linked histones show that histone 2B possesses separate binding sites for histones 2A and 4. Various polymorphic forms of H2A and H4 in chromatin are bound to H2B through these binding sites. These interactions are thus a fundamental structural feature of chromatin. Additional data strongly suggest that H2B is bound to H2A and H4 simultaneously in vivo. Mouse L cells were treated with either tetranitromethane (TNM) or ultraviolet (uv) light at 280 nm in order to induce the cross-linking of histone 2B in their chromatin to either histone 4 or 2A. The characteristics of these cross-linking agents and the reactions they induce suggest that the cross-links define loci within the actual binding sites between these histones. Characterization of the cyanogen bromide peptide fragments of the H2B-H4 and H2B-H2A dimers showed that the C terminal half of H2B contains the link to H4 (also near its C terminus), whereas the N-terminal half contains the link to H2A. Treatment of the cells with both uv light and TNM results in the appearance of a single new trimer which we presume to be H2A-H2B-H4. We conclude form this that H2B interacts simultaneously with H2A and H4 in chromatin through separate binding sites. Further analysis shows that several acetylated subspecies of H4 are represented in the H2B-H4 dimer and that at least two of the polymorphic forms of H2A are present in the H2B-H2A dimer. These data reveal that the H2A-H2B-H4 association is an important underlying structural feature of chromatin.


An experimental method correcting for absorption flattening and scattering in suspensions of absorbing particles: circular dichroism and absorption spectra of hemoglobin in situ in red blood cells. An experimental approach to interpretation of the anomalous absorption and circular dichroism (CD) spectra of hemoglobin in situ in red blood cells is reported. Absorption flattening effects have been overcome by use of high cell concentratons in very short light path cuvettes. Differential scattering contributions to circular dichroism have been resolved using a CD instrument capable of variable detection geometry. Scattering effects have also been resolved using media of high refractive index to match that of the red blood cell. The results are in agreement with a parellel calculational analysis of red blood cell CD spectra, which predicted the relative magnitudes of the flattening and differential scattering CD contributions. An experimental absorption spectrum has been obtained for hemoglobin in the red blood cell with scattering and flattening eliminated. This quantitatively simulates the spectrum of a hemoglobin solution. The methods described should be widely applicable to conformational studies of macromolecules in their native environment.


The role of methionine-192 of the chymotrypsin active site in the binding and catalysis of mono(amino acid) and peptide substrates. We find that specific oxidation for the Met-192 residue in delta-chymotrypsin to methionine sulfoxide results in a twofold increase in Km(app) and unchanged kcat in the hydrolysis of N-acetyl mono(amino acid) amide substrates. However, the catalyzed hydrolyses of N-acetyl dipeptide amide substrates by (methionine sulfoxide)-192-delta-chymotrypsin (MS-delta-Cht) shows a four- to fivefold decrease in kcat and unchanged Km(app) with respect to delta-chymotrypsin. Hydrolysis of alpha-casein by MS-delta-Cht shows a similar 4.2-fold decrease in kcat. These results imply that the Met-192 acts differently with substrates that bind only in the primary, S1, binding site (i.e., AcPheNH2) from those that bind to more extended regions of the enzyme active site. In the binding of c+AcPheNH2  and AcTrpNH2, the results support a mechanism in which the Met-192 acts to slow the rate of sustrate dissociation from the Michaelis complex to free substrate and enzyme. This is in agreement with the x-ray crystallographic structure of dioxane inhibited alpha-chymotrypsin (Steitz, T., et al. (1969), J. Mol. Biol. 46, 337). However, this mechanism is not apparent when peptide and protein substrates bind. The decrease in kcat on Met-192 modification of approximately fivefold in the hydrolysis of polypeptide substrates show a small, but significant, catalytic contribution of the Met-192 toward the lowering of the energy of activation polypeptide substrate hydrolysis by chymotrypsin. This may support the crystallographic model of Fersht et al. (Fersht, A., et al. (1973), Biochemistry 12, 2035) in which it is proposed that the Met-192 participates in the distortion of bound polypeptide substrates toward the reaction transition-state configuration and, thus, plays a role in catalysis. However, if this mechanism occurs, the effect is small, only contributing about 1 kcal/mol to the lowering of the reaction activation energy.


Histidine decarboxylase of Lactobacillus 30a: inactivation and active-site labeling by L-histidine methyl ester. Histidine decarboxylase from Lactobacillus 30a is rapidly and irreversibly inactivated upon incubation with L-histidine methyl ester. The rate of inactivation is first-order with respect to remaining active enzyme and exhibits saturation kinetics with a kinact of 1.2 mM and an apparent first-order rate constant of 0.346 min-1 at pH 4.8 and 25 degrees C. On complete inactivation, 3 mol of [14C]histidine (from L-[14C]histidine methyl ester) and 2 mol of 14C (from L-histidine [14C]methyl ester) are bound in nondialyzable form per mol (190 000 g) of protein inactivated with a corresponding loss of three of the five DTNB-titratable--SH groups that are essential for activity of the native enzyme. Imidazole propionate, a competitive inhibitor of the enzyme, protects against inactivation, loss of --SH groups, and incorporation of radioactivity from both the histidine and the methyl ester moieties of the labeled inhibitor, and kinetic evidence indicates that imidazole propionate and histidine methyl ester compete for binding at the active site of histidine decarboxylase in a mutually exclusive manner. Treatment of the labeled protein with either alkali or hydroxylamine results in the quantitative release of radioactivity. These data suggest that inactivation of histidine decarboxylase by L-histidine methyl ester results from two different modes of interaction between the inhibitor and the active site of histidine decarboxylase; the major interaction involves an essential -SH group.


Maleylacetone cis-trans-isomerase: affinity chromatography on glutathione-bound sepharose. Two-substrate-binding sequence from inhibition patterns. Maleylacetone cis-trans-isomerase isolated from Vibrio 01 binds glutathione strongly; Km = 1.4 X 10(-4) M. Oxidized glutathione and S-methylglutathione are competitive inhibitors, KI = 9.4 X 10(-4) and 1.2 X 10(-3) M, respectively. Based on these interactions, three different glutathione-bound agarose affinity adsorbents were synthesized and tested. Affinity chromatography of the isomerase with one of these affords 70- to 100-fold purifications. In separate syntheses, portions of the affinity arm were prepared and examined as to their inhibitory properties in the enzyme-catalyzed reaction. The fragment, containing glutathione bound through its sulfur to the carbon chain, is a powerful competitive inhibitor for glutathione (KI = 6 X 10(-5) M). The results described suggest that the isomerase binds glutathione through the backbone of the tripeptide and that the thiol group is required for activity. The initial velocity patterns of the enzyme-catalyzed reaction resulting from simultaneous variation of glutathione and maleylacetone concentrations were examined in the absence and presence of inhibitors resembling glutathione. The observed kinetic patterns suggest an ordered sequence of binding maleylacetone first followed by glutathione.


Interactions between the subfractons of calf thymus H1 and nonhistone chromosomal proteins HMG1 and HMG2. The nonhistone chromosomal proteins, HMG1 and HMG2, interact with the various subfractions of calf thymus H1 with a high degree of specificity. Subfractions 1b and 2 interact very strongly with HMG1 to form heterodimers. In contrast, subfractions 3a and 3b interact much more weakly. HMG2 interacts with 3a and 3b but shows no detectable complexing with 1a and 2.


Tubulin-nucleotide interactions during the polymerization and depolymerization of microtubules. The interactions of nucleotides and their role in the polymerization of tubulin have been studied in detail. GTP promotes polymerization by binding to the exchangeable site (E site) of tubulin. The microtubules formed contain only GDP at the E site, indicating that hydrolysis of E site GTP occurs during or shortly after polymerization. Tubulin prepared by several cycles of polymerization and depolymerization will polymerize in the presence of ATP as well as GTP. Polymerization in ATP is preceded by a distinct lag period which is shorter at higher concentrations of ATP. As reported by others ATP will transphosphorylate bound GDP to GTP. Under polymerizing conditions the maximum level of GTP formation occurs at about the same time as the onset of polymerization, and the lag probably reflects the time necessary to transphosphorylate a critical concentration of tubulin. The transphosphorylated protein can be isolated and will polymerize without further addition of nucleotide. The transphosphorylated GTP is hydrolyzed and the phosphate released during polymerization. About 25% of the phosphate transferred from ATP is noncovalently bound to the subunit as inorganic phosphate and this fraction is also released during polymerization. The nonhydrolyzable analogue of GTP, GMPPNP, will promote microtubule assembly at high concentration. GMPPNP assembled microtubules do not depolymerize in Ca concentrations several fold greater than that which will completely depolymerize GTP assembled tubules; however, addition of Ca prior to inducing polymerization in GMPPNP prevents the formation of microtubules. Thus GTP hydrolysis appears to promote depolymerization rather than polymerization. GDP does not promote microtubule assembly but can inhibit GTP binding and GTP induced polymerization. GDP does not, however, induce the depolymerization of formed microtubules. These experiments demonstrate that tubulin polymerization can not be treated as a thermodynamically reversible process, but must involve one or more irreversible steps. Exchange experiments with [3H]GTP indicate that the "E" site on both microtubules and ring aggregates of tubulin is blocked and does not exchange rapidly. However, during polymerization and depolymerization induced by raising or lowering the temperature, respectively, all the E sites become transiently available and will exchange their nucleotide. This observation does not suggest a direct morphological transition between rings and microtubules. The presence of a blocked E site on the rings explains the apparent transphosphorylation and hydrolysis of "N" site nucleotide reported by others.


Protein rotational relaxation as studied by solvent 1H and 2H magnetic relaxation. Earlier studies of the magnetic field dependence of the nuclear spin magnetic relaxation rate of solvent protons in solutions of diamagnetic proteins have indicated that this dependence (called relaxation dispersion) is related to the rotational Brownian motion of solute proteins. In essence, the dispersion is such that 1/T1 (the proton spin-lattice relaxation rate) decreases monotonically as the magnetic field is increased from a very low value (approximately 10 Oe); the dispersion has a point of inflection at a value of magnetic field which depends on protein size, shape, concentration, temperature, and solvent composition. The value of the proton Larmor precession frequency nu(c) at the inflection field appears to relate to tau (R), the rotational relaxation time of the protein molecules. We have measured proton relaxation dispersions for solutions of various proteins that span a three-decade range of molecular weights, and for one sample of transfer ribonucleic acid. We have also measured deuteron relaxation dispersions for solutions of three proteins: lysozyme, carbonmonoxyhemoglobin, and Helix pomatia hemocyanin with molecular weight 900 000. A quantitative relationship between both proton and deuteron dispersion data and protein rotational relaxation is confirmed, and the point is made that magnetic dispersion measurements are of very general applicability for measuring the rotational relaxation rate of macromolecules in solution. It has been previously shown that the influence of proton motion on the relaxation behavior of the solvent is not due to exchange of solvent molecules between the bulk solvent and a hydration region of the protein. In the present paper, we suggest that the interaction results from a long range hydrodynamic effect fundamental to the situation of large Brownian particles in an essentially continuum fluid. The general features of the proposed mechanism are indicated, but no theoretical computations are presented.


Conformational properties of the complexes formed by proteins and sodium dodecyl sulfate. Circular dichroism spectra have been obtained for albumin, alpha-chymotrypsinogen, collagen, concanavalin A, elastase, hemoglobin, histone f2b, alpha-lactalbumin, lactate dehydrogenase, beta-lactoglobulin, lysozyme, myoglobin, papain, ribonuclease A, and thermolysin in the presence of sodium dodecyl sulfate and dithiothreitol. While all spectra have the shape anticipated for a mixture of random coil and alpha helix, the intensities differ markedly ([theta]222 ranges from --1400 to --15 000 deg cm2/dmol). The variation in the circular dichroism can be quantitatively explained by a model which assumes that the arginyl, histidyl, and lysyl residues have an enhanced probability of propagating a helical segment in the presence of the detergent. The model also permits the computation of dimensional properties (unperturbed end-to-end distance and radius of gyration) for polypeptides of known amino acid sequence. Such computations have been performed for 67 proteins. The computed dimensions are compatible with experimental values and with the molecular weight dependence of the transport properties of the complexes. Furthermore, the model can account for the abnormal transport properties of the sodium dodecyl sulfate complexes formed by ribonuclease A, collagen fragments, and histones f2a1, f2a2, f2b, and f3. Even though some of the protein--sodium dodecyl sulfate complexes have helical contents as high as 50%, their overall conformation more closely approximates that of a random coil than a rod.


Nuclear estradiol receptor in the adult rat uterus: a new exchange assay. A protamine exchange assay has been developed to measure uterine nuclear estrogen receptor in mature rats exposed to estradiol (E). After ovariectomized-adrenalectomized mature rats are injected with E, estrogen receptor (RnE) is extracted from uterine nuclei with 0.6 M potassium chloride, diluted, and quantitatively precipitated with protamine sulfate. The precipitate is subjected to a ligand exchange with radiolabeled estradiol (E), with or without unlabeled diethylstilbesterol, to determine nonspecific binding. At 37 degrees C complete exchange of E for E in RnE is observed at 2.5 h; virtually no receptor degradation occurs up to at least 5 h. Exchange does not occur at 4 degrees C. Using the protamine assay, the depletion of cytoplasmic estrogen receptor (Rc) and the accumulation of RnE were studied at various doses of E at specific time points. Increasing doses of E result in a decrease of Rc with an equal increase of RnE. At the highest dose of E (10 mug) Rc is completely depleted within 10 min, by 6 h it is 25% replenished, and by 24 h returns to slightly above control levels. Within 10 min after the injection, RnE increases to 80-90% of the original cytoplasmic level of receptor (approximately 2-3 pmol/mg of DNA or approximately 1.5 pmol/100 mg of uterus). At 6 h RnE is 75% depleted and it is completely absent at 24 h. The protamine assay permits precise quantitative studies of nuclear estrogen receptor and avoids the problems of receptor degradation and excessive nonspecific binding often found in exchange reactions at elevated temperatures.


Synthesis and properties of poly(O6-methylguanylic acid) and poly(O6-ethylguanylic acid). The nucleotide analogues, O6-methyl- and O6-ethylguanosine diphosphate, have been synthesized and polymerized to high-molecular-weight homopolymers with polynucleotide phosphorylase. The ultraviolet spectra of these polymers show marked hypochromicity, which suggests that they possess considerable secondary structures. Graphs of optical denisty vs. temperature in 0.15 M NaC1 indicate that cooperative melting occurs for both polymers, and that the secondary structure of poly(O6-methylguanosine monophosphate) is somewhat more stable than that of poly (O6-ethyl-guanosine monophosphate). Mixing experiments show that these analogue polymers no longer form helical structures with poly(C), nor do thye form helices with poly(U). We would conclude from these data that environmental mutagens and carcinogens which react at the O6 position of guanine not only disrupt normal base-pairing relationships, but may also affect the secondary structure of nucleic acids.


Binding of platinum and palladium metallointercalation reagents and antitumor drugs to closed and open DNAs. The interaction of platinum and palladium complexes with closed and nicked circular and linear DNAs was investigated by a variety of methods. Cationic metal complexes containing flat, aromatic ligands, such as 2,2',2''-terpyridine, o-phenanthroline, and 2,2'-bipyridine, interfere with the usual fluorescence enhancement of ethidium bromide by competing for intercalation sites on calf-thymus DNA. Metal complexes having kinetically exchangable ligands, including the antitumor drugs cis-[(NH3)2PtCl2] and [(en)PtC12], inhibit noncompetitively the DNA-associated ethidium fluorescence enhancement by binding covalently to the bases and blocking potential intercalation sites. Only the metallointercalators were capable of altering the DNA duplex winding, as judged by the effects of these reagents upon the electrophoretic mobility and sedimentation behavior of PM-2 DNAs. Long-term (t greater than 120 h) interactions of metal complexes with PM-2 DNAs I, I0, and II, corresponding to superhelical, closed relaxed, and nicked circles, respectively, showed that covalent binding occurs the most readily to DNA I, possibly because of the presence of underwound duplex regions in this tightly wound superhelical DNA. The active antitumor drugs cis-[(NH3)2PtC12] and [(en)PtC12] bind covalently to DNA I under conditions where the inactive trans- [(NH3)2PtC12] does not. Most of the complexes studied were capable of producing chain scissions in PM-2DNA I. Exceptions are the kinetically inert complexes [(bipy)Pt(en)]2+ and (terpy)Pt(SCH2CH2OH)]+, suggesting that covalent binding might be a prerequisite for nicking.


The effect of hydrogen peroxide on CO2 fixation of isolated intact chloroplasts. Low concentration of hydrogen peroxide strongly inhibit CO2 fixation of isolated intact chloroplasts (50% inhibition at 10(-5) M hydrogen peroxide). Addition of catalase to a suspension of intact chloroplasts stimulates CO2 fixation 2--6 fold, indicating that this process is partially inhibited by endogenous hydrogen peroxide formed in a Mehler reaction. The rate of CO2 fixation is strongly increased by addition of Calvin cycle intermediates if the catalase activity of the preparation is low. However, at high catalase activity addition of Calvin cycle intermediates remains without effect. Obviously the hydrogen peroxide formed at low catalase activity leads to a loss of Calvin cycle substrates which reduces the rate of CO2 fixation. 3-Phosphoglycerate-dependent O2-evolution is not influenced by hydrogen peroxide at a concentration (5x10(-4) M) which inhibits CO2 fixation almost completely. Therefore the inhibition site of hydrogen peroxide cannot be at the step of 3-phosphoglycerate reduction. Dark CO2 fixation of lysed chloroplasts in a hypotonic medium is not or only slightly inhibited by hydrogen peroxide (2,5x10(-4) M), if ribulose-1,5-diphosphate, ribose 5-phosphate or xylulose 5-phosphate were added as substrates. However, there is a strong inhibition of CO2 fixation by hydrogen peroxide, if fructose 6-phosphate together with triose phosphate are used as substrates. This indicates that hydrogen peroxide interrupts the Calvin cycle at the transketolase step, leading to a reduced supply of the CO2-acceptor ribulose 1,5-diphosphate.


Concentration-dependent effects of salicylaldoxime on chloroplast reactions. Salicylaldoxime has been found to have a variety of concentration-dependent effects on chloroplast activities. At low concentrations (less than 10 mM), salicyladoxime reversibly inhibits all reactions which involve Photosystem II. Since the DCMU-insensitive silicomolybdate Hill reaction is also inhibited, one site of inhibition is definitely located before the DCMU-sensitive site, possibly before the photoact. The inhibition kinetics and the response of chloroplast fluorescence may indicate another site in the DCMU-sensitive region. At almost exactly the same concentrations (less than 10 mM), salicylaldoxime uncouples phosphorylation reversibly, whether it is supported by Photosystem II or by Photosystem I. At higher concentrations (approx. 20 mM) salicylaldoxime inhibits Photosystem II irreversibly, uncouples irreversibly, and begins to cause changes in chloroplast light scattering which could be manifestations of membrane damage. At very high concentrations (approx. 45 mM) salicylaldoxime irreversibly inhibits Photosystem I activity in the region of plastocyanin. This is indicated by the ability of salicylaldoxime to inhibit the photooxidation of cytochrome f but not the photooxidation of P-700.


Phosphorylation of nucleotides bound to chloroplast membranes and their role in photophosphorylation. 1. The initial rapid phosphorylation of membrane-bound ADP yields [gamma- 32P]-ATP. Long term illumination of chloroplasts resulted in the introduction of label in the beta position of ADP and ATP. 2. It is concluded that the initial acceptor in photophosphorylation is ADP and not AMP. The appearance of 32Pi in the beta position of the nucleotide fractions is probably the result of side reactions not directly involved in the photophosphorylation mechanism. 3. The inhibitor phlorizin affects similarly the phosphorylation of bound ADP and the net photophosphorylation reaction. 4. The uncoupler nigericin has different effects on the phosphorylation of membrane-bound ADP and the net photophosphorylation reaction. Dissipation of energy by low concentrations of the uncoupler affects primarily the step(s) of ATP release to the medium, while steps leading to the interconversion of Pi and ADP to form bound ATP are less sensitive and are inhibited by relatively higher uncoupler concentrations.


Anisotropy of photosynthetic membranes and the degree of fluorescence polarization. The degree of fluorescence polarization, P, of unoriented and magnetically oriented spinach chloroplasts as a function of excitation (400-680 nm) and emission wavelengths (675-750 nm) is reported. For unoriented chloroplasts P can be divided into two contributions, PIN and PAN. The latter arises from the optical anisotropy of the membranes which is due to the orientation with respect to the membrane plane of pigment molecules in vivo. The intrinsic polarization PIN, which reflects the energy transfer between different pigment molecules and their degree of mutual orientation, can be measured unambiguously only if (1) oriented membranes are used and the fluorescence is viewed along a direction normal to the membrane planes, and (2) the excitation is confined to the Qy (approximately 660-680 nm) absorption band of chlorophyll in vivo. With 670-680 nm excitation, values of P using unoriented chloroplasts can be as high as +14%, mostly reflecting the orientational anisotropy of the pigments. Using oriented chloroplasts PIN is shown to be +5+/-1%. The excitation wavelength dependence studies of PIN indicate that the carotenoid and chlorophyll Qy transition moments tends to be partially oriented with respect to each other on a local level (within a given photosynthetic unit or its immediate neighbors).


Kinetics and stoichiometry of light-induced proton release and uptake from purple membrane fragments, Halobacterium halobium cell envelopes, and phospholipid vesicles containing oriented purple membrane. We have used flash spectroscopy and pH indicator dyes to measure the kinetics and stoichiometry of light-induced proton release and uptake by purple membrane in aqueous suspension, in cell envelope vesicles and in lipid vesicles. The preferential orientation of bacteriorhodopsin in opposite directions in the envelope and lipid vesicles allows us to show that uptake of protons occurs on the cytoplasmic side of the purple membrane and release on the exterior side. In suspensions of isolated purple membrane, approximately one proton per cycling bacteriorhodopsin molecule appears transiently in the aqueous phase with a half-rise time of 0.8 ms and a half-decay time of 5.4 ms at 21degreesC. In cell envelope preparations which consist of vesicles with a preferential orientation of purple membrane, as in whole cells, and which pump protons out, the acidification of the medium has a half-rise time of less than 1.0 ms, which partially relaxes in approx. 10 ms and fully relaxes after many seconds. Phospholipid vesicles, which contain bacteriorhodopsin preferentially oriented in the opposite direction and pump protons in, show an alkalinization of the medium with a time constant of approximately 10 ms, preceded by a much smaller and faster acidification. The alkalinization relaxes over many seconds. The initial fast acidification in the lipid vesicles and the fast relaxation in the envelope vesicles are accounted for by the misoriented fractions of bacteriorhodopsin. The time constants of the main effects, acidification in the envelopes and alkalinization in the lipid vesicles correlate with the time constants for the release and uptake of protons in the isolated purple membrane, and therefore show that these must occur on the outer and inner surface respectively. The slow relaxation processes in the time range of several seconds must be attributed to the passive back diffusion of protons through the vesicle membrane.


Light-dependent inhibitory action of p-nitrothiophenol on photosystem II in relation to the redox state of electron carriers. The photosystem-II activity of chloroplasts was inhibited by the treatment with p-nitrothiophenol (NphSH) in the light, and the inhibition was accompanied by a change of the fluorescence spectrum. Aromatic mercaptans examined were active in causing this inhibition and fluorescence change. These effects of p-nitrothiophenol were highly accelerated by blocking the electron transport of the oxidation side of photosystem II by carbonyl-cyanide-m-chlorophenylhydrazone (CCCP) or Tris-HC1 or heat pre-treatment, whereas these were suppressed by blocking the transport on the reduction side by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). It was deduced that the site of NphSH action in the electron transport chain is closer to the reaction center of photosystem II that the blocking site of CCCP or Tris-HC1 or heat, and that such a site in photosystem II is exposed to be modified with NphSH when electron carriers on the oxidation side of photosystem II are oxidized by illumination.


A highactivity ATP translocator in mesophyll chloroplasts of Digitaria sanguinalis, a plant having the C-4 dicarboxylic acid pathway of photosynthesis. The effect of exogenous adenine nucleotides on CO2 fixation and oxygen evolution was studied with mesophyll protoplast extracts of the C4 plant Digitaria sanguinalis. Exogenous ATP was found to stimulate the rate of pyruvate and pyruvate+oxalacetate induced CO2 fixation, as well as reverse the inhibition of CO2 fixation of carbonyl cyanide m-chlorophenyl hydrazone and several electron transport inhibitors. The ATP-dependent stimulation of CO2 fixation varied from 40 to 70 mumol CO2 fixed/mg chlorophyll per h, suggesting that ATP was crossing the chloroplast membranes at rates of 80-140 mumol/mg chlorophyll per h, since 2 ATP are required for each CO2 fixed. Fixation of CO2 could also be induced in the dark by exogenous ATP, in which case ADP accumulated outside the chloroplasts...


Binding of radioactively labeled carboxyatractyloside, atractyloside and bongkrekic acid to the ADP translocator of potato mitochondria. 1. The inhibition of the ADP-stimulated respiration of potato mitochondria by carboxyatractyloside is relieved by high concentration of ADP or by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Atractyloside is a much less potent inhibitor than carboxyatractyloside. The inhibition of the ADP-stimulated respiration required about 60-times more atractyloside than carboxyatractyloside. 2. [35S]carboxyatractyloside and [3H]bongkrekic acid bind to potato mitochondria with high affinity (Kd = 10 to 20 nM, n=0.6-0.7 nmol per mg protein). Added ADP competes with carboxyatractyloside for binding; on the contrary ADP increases the amount of bound bongkrekic acid. [3H]atractyloside binds to potato mitochondria with a much lower affinity (Kd=0.45 muM) than carboxyatractyloside or bongkrekic acid. 3. Bound [3H]atractyloside is displaced by ADP, carboxyatractyloside and bongkrekic acid. The displacement of bound [35S]carboxyatractyloside by bongkrekic acid and of bound [3H]bongkrekic acid by carboxyatractyloside is markedly increased by ADP. 4. Bongkrekic acid competes with [35S]carboxyatractyloside for binding. Addition of a small concentration of ADP considerably enhances the inhibitory effect of bongkrekic acid on [35S]carboxyatractyloside binding. 5. The adenine nucleotide content of potato mitochondria is of the order of 1 nmol per mg protein. ADP transport in potato mitochondria is inhibited by atractyloside 30- to 40-times less efficiently than by carboxyatractyloside.


Deuterium oxide effects on excitation-contraction coupling of skeletal muscle. 2H2O (99.8%) Ringer's solution greatly reduces the twitch and tetanus of frog sartorius muscle and, as specially shown here, slows the onset features of the mechanical output of the twitch by: (a) increasing the time (LR) from stimulus to start of latency relaxation; (b) slowing the development of the latency relaxation, and (c) greatly decreasing the rate of onset of tension development. These changes reflect effects of 2H2O on excitation-contraction coupling and they represent the critical direct effects of 2H2O on muscle since it does not depress either the action potential or the intrinsic myofibrillar contractility. The increase in LR is attributed to slowed inward electrical propagation in the T-tubule. But the critical effect of 2H2O on frog muscle is to greatly depress mobilization of activator Ca2+. The depression of the Ca2+ mobilization and of its effects on the activation of contraction evidently result from (a) a lowered rate of release of Ca2+ from the sar coplasmic reticulum, as indicated by the slowed development of the latency relaxation, (b) a decreased amount of Ca2+ released in a twitch, and (c) a reduced speed of diffusion of the Ca2+ to the contractile filaments. The depressed mobilization of Ca2+ is apparently the essential  cause of 2H2O's general depression of twitch and tetanus output.


Multiple-flash development of thermoluminescence bands in dark-grown spruce leaves. Spruce leaves greened in darkness were devoid of three of the five thermoluminescence bands found for mature leaves. These bands were developed rapidly by exposure of the dark-grown leaves to continuous light. The development of these bands was studied by illumination with repetitive flashes at varied intervals. Flashes at intervals of 1 s were the most effective in inducing these bands. Those at shorter or longer intervals were less effective. It was deduced from these data that the development of these bands is a multiquantum process which involves at least two photo-events with a dark reaction between them.


Maturation of ribosomes in yeast. I Kinetic analysis by labelling of high molecular weight rRNA species. To study the maturation of ribosomes in Saccharomyces carlsbergensis, protoplasts were pulse labeled with [5-3H]uridine at 15 degrees C. Investigation of the cellular location of pulse-labelled ribosomal RNA precursor and mature ribosomal RNA shows that both the 37-S precursor RNA, common to both 17-S and 26-S rRNA, as well as the 29-S RNA, the direct precursor of 26-S rRNA, are located in the nucleus. Most of the 18-S RNA, the direct precursor of 17-S rRNA, is found in the cytoplasmic fraction. Apart from 37-S and 29-S RNA the nucleus also contains an appreciable amount of 26-S rRNA as well as a small quantity of 18-S RNA. These data indicate that processing of 29-S to 26-S RNA occurs in the nucleus, whereas the conversion of 18-S RNA to 17-S rRNA takes place in the cytoplasm. The kinetics of appearance of pulse-labelled 26-S and 17-S rRNA in the various cytoplasmic ribosomal particles indicate, that newly formed 40-S ribosomal particles are almost immediately incorporated into 80-S ribosomes and polysomes. On the other hand, there appears to exist a fairly large cytoplasmic pool of newly synthesized ribosomal particles containing 26-S rRNA and sedimenting at about 60 S. The kinetics of appearance of newly formed 26-S and 17-S rRNA in mature ribosomes show that the maturation of the large ribosomal subunit takes about twice as much time as that of the small subunit.


Maturation of ribosomes in yeast. II. Position of the low molecular weight rRNA species in the maturation process. Yeast protoplasts were pulse labelled with [5-3H] uridine and the labelling kinetics of the low molecular weight rRNA species were determined in order to gain more insight into the position of those small rRNAs in the process of ribosome maturation. 7-S RNA, the immediate precursor of 5.8-S rRNA, is found to be present only in the nucleus, indicating that the conversion of 7-S to 5.8-S RNA is a nuclear event. 5.8-S rRNA is observed in the cytoplasm almost immediately after its formation. This as well as the presence of only a small amount of 5.8-S RNA in the nucleus, shows that the ribosomal precursor particles of the large ribosomal subunit are very rapidly transported into the cytoplasm once 5.8-S rRNA is formed. Most of the newly synthesized 5-S RNA is found in the nucleus. This nuclear 5-S rRNA is mainly present in the ribosomal precursor particles. However, a small pool of free 5-S rRNA is probably also present.


Evidence for the synthesis of tubulin on membrane-bound and free ribosomes from rat forebrain. Evidence is presented that tubulin is synthesized in cell-free systems derived from rat forebrain containing either membrane-bound or free ribosomes. Both systems were supplemented with saturating amounts of messenger RNA (from either membrane-bound of free ribosomes) and soluble enzyme and initiation factors from whole forebrain. Synthesized products were chracterized by two biochemical purification methods for brain tubulin isolation. These methods of tubulin analysis indicated that both cell free systems synthesized the same relative amounts of tubulin.


Effects of ionizing radiation of RNA metabolism in cultured mammalian cells. III. Specific reduction in mRNA synthesis during resumed division and plateau phases following exposure to 800 rads of x irradiation. The cell growth response of cultured Chinese hamster ovary cells, line CHO, to 800 rads of X irradiation involves a period of division delay, followed by a period of resumed division which terminates in a plateau phase. Over 95% of the cells die eventually. No direct effects of RNA or protein metabolism are evident during the delay period. During the resumed division and beginning of plateau phases, however, a specific and relatively constant reduction in mRNA synthesis relative to messenger-like RNA and heterogenous nuclear RNA synthesis is evidenced. The ratio of mRNA to messenger-like RNA synthesis ranges from 0.8 to 0.65 during these phases. The effect is not due to altered cell-cycle distribution, and evidence is presented to indicate that it is probably not a compensatory response to the unbalanced growth that occurs during the division delay period.


Culture conditions affect photoreactivating enzyme levels in human fibroblasts. Photoreactivation of pyrimidine dimers in mammalian cells occurs under our experimental conditions but has not been observed under conditions used by others. We have tested three possible differences in experimental procedures including dimer separation and analysis methods, illumination conditions and cell culture techniques. We show that out methods of dimer separation and analysis indeed measure cis-syn pyrimidine dimers and give results in quantitative agreement with the methods of others. We find that while light pre-illumination of fibroblasts from the xeroderma pigmentosum line XP12BE or of normal cells does not affect the cellular capacity for dimer photoreactivation. However, we show that cell culture conditions can affect photoreactivating enzyme levels and thus, cellular dimer photoreactivation capacity. Cells grown in Eagle's minimal essential medium (supplemented with 15% fetal bovine serum) contain very low levels of photoreactivating enzyme and cannot photoreactivate dimers in their DNA; however, companion cultures maintained in Dulbecco's modified Eagle's minimal medium do contain photoreactivating enzyme and can photoreactivate cellular dimers.


Partial purification and properties of the reticulocyte guanylating enzyme. The guanylating enzyme which catalyzes the insertion of a guanine residue into one of the isoacceping tRNAHis of rabbit reticulocytes has been purified approximately one-hundred fold. It is free of nuclease activity. The enzyme does not catalyze the replacement of inserted radioactive guanine by unlabeled guanine, indicating that the reaction is irreversible. We have separated the histidyl-tRNA of reticulocytes into three isoacceptors. Previous work showed that the last histidyl-tRNA to elute from RPC-5 columns was the product of the guanylation reaction. This reports shows that the same late-eluting peak also contains the substrate for the guanylating enzyme, indicating that the guanine insertion reaction is chromatographically silent. The isoaccepting tRNAHis that is the substrate for the guanylating enzyme does not contain the hypermodified base known as Q. It is the other major reticulocyte tRNAHis that coantains Q, showing that at least in the reticulocyte the role of the guanylating enzyme is not the conversion of the Q form of tRNA to the homogeneic G form. The purified enzyme does not insert any base other than guanine into tRNA.


Incorporation of radioactive amino acids into protein in isolated rat hepatocytes. The incorporation of radioactivity from a 14C-labelled amino acid mixture (algal protein hydrolysate) into protein in isolated rat hepatocytes has been studied. The incorporation rate declined with increasing cell concentration, an effect which could be explained partly by isotope consumption, partly (and largely) by isotope dilution due to the formation of non-labelled amino acids by the cells. At a high extracellular amino acid concentration, the rate of incorporation into protein became independent of cell concentration, because the isotope dilution effect was now quantitatively insignificant. The time course of protein labelling at various cells concentrations correlated better with the intracellular than with the extracellular amino acid specific activity, suggesting that amino acids for protein synthesis were taken from an intracellular pool. With increasing extracellular amino acid concentrations both the intracellular amino acid concentration, the intracellular radioactivity and the rate of incorporation into protein increased. Protein labelling exhibited a distinct time lag at high amino acid concentrations, presumably reflecting the time-dependent expansion of the intracellular amino acid pool. The gradual increase in the rate of protein labelling could be due either to an increased intracellular specific activity, or to a real stimulation of protein synthesis by amino acids, depending on whether the total intracellular amino acid pool or just the expandable compartment is the precursor pool for protein synthesis.


Arterial elastin synthesis in the young chick. In the growing chick a marked stimulation in soluble and mature arterial elastin synthesis occurs 2 and 5 weeks after hatching. Measurement of [3H]valine and [3H]proline incorporation into arterial soluble protein during this period indicated that most of the label is found in a 70 000 dalton protein subunit. The labeled soluble subunit had the characteristics of native soluble elastin or tropoelastin. During the period in which the greatest percentage increase in mature elastin occurs, the highest specific activities of soluble [3H]valine-labeled protein were observed. These changes were striking and suggest a developmental period for the growing chick in which factors related to elastin metabolism may be more easily studied.