Isolation and characterization of X-linked mutants of Drosophila melanogaster which are sensitive to mutagens. Thirteen X-linked mutants have been isolated in Drosophila melanogaster which render male and homozygous female larvae sensitive to the mutagen methyl methanesulfonate. Their characterization and preliminary assignment to functional groups is described. Four of these mutants are alleles of mei-41 (Baker and Carpenter 1972). Like previously isolated alleles of this locus, these mutants reduce fertility and increase loss and nondisjunction of the X-chromosome in homozygous females. The remaining mutants have been tentatively assigned to six functional groups (two mutants to the mus(1)101 locus, two to mus(1)102 , two to mus(1)103, and one each to mus(1)104, mus(1)105 , and mus(1)106). Several of the complementation groups can be distinguished on the basis of nondisjunction and cross sensitivity to mutagens. Females homozygous for the mei-41, mus(1)101 and mus(1)102 mutants exhibit elevated levels of nondisjunction. Mutants belonging to complementation groups mei-41, mus(1)101, and mus(1)104 are sensitive to nitrogen mustard (HN2) in addition to their MMS sensitivity. Among these mutants there is currently a direct correlation between sensitivity to HN2, sensitivity to 2-acetylaminofluorene and a deficiency in post-replication repair ( Boyd and Setlow 1976). Only the mei-41 mutants are hypersensitive to UV radiation, although several of the mutants exhibit sensitivity to gamma-rays. Semidominance is observed in female larvae of the mei-41, mus(1)104, and mus(1)103 mutants after exposure to high concentrations of MMS. The properties of the mutants generally conform to a pattern which has been established for related mutants in yeast. Additional properties of these mutants are summarized in Table 9.


[Effect of 2,6-diaminopurine resistance mutations on adenine and adenosine assimilation by cells of an adenine-dependent strain of Escherichia coli K-12]. In purine-requiring strain of Escherichia coli K-12 defective in purine nucleoside phosphorylase (pur, pup) mutants (designated apt) have been obtained that are resistant to 2,6-diaminopurine on guanine-containing medium and incapable to utilize adenine for their growth at 42degreesC, but they are still sensitive to the analogue and can utilize adenine at 28degreesC. It has been shown that the introduction of the corresponding apt mutations in the genome of adenine-requiring strains impaired the ability of these strains to grow on both adenine and adenosine at 42degreesC.


[The radiology of Egyptian mummies (author's transl)]. The technique used for the radiological examination of a well preserved mummy is described. Problems of identification and determination of age, size and sex are discussed. Post mortem changes consist of subluxation of the cervical spine and fractures of the ribs and fibulae. Of medical interest, it was possible to diagnose definitely Scheuermann's disease with a scoliosis and marked reactive spondylotic and spondylo-arthritic changes. The radiological findings provided further information regarding the technique of mummification and the method of burial. Some findings of mythological significance could be demonstrated radiologically, such as the fact that the heart and kidneys had been left in the body, the presence of a scarab, various gold plates and amulets and a fayence-decorated shroud.


[Studies on the regulatory factors of 3-hydroxy-3-methylglutaryl CoA reductase (HMG CoA reductase) activity]. HMG CoA reductase, which catalyzes the reaction, HMG CoA + 2 NADAPH2 leads to mevalonate + CoA-SH + 2 NADP, is considered to be the rate-limiting enzyme on cholesterol biosynthetic pathway. Since a degree in activity of this enzyme is almost proportional to the rate of cholesterol synthesis from acetate, elucidation of factors that regulate reductase activity would provide insight into the control mechanisms on the cholesterol biosynthesis. In the present study, attempts were made to establish standard assay conditions of HMG CoA reductase activiy, and to qualify the factors affecting the activity of the enzyme. The results obtained were as follows: (1) As standard assay conditions of HMG CoA reductase activity, 85, muM were chosen for substrate concentration, 25-80 mug for microsomal enzyme protein, and 20 min for incubation time in a final volume of 0.1 ml. (2) HMG CoA reductase activity of rat liver microsomes was exhibited diurnal variation. The level of reductase activity at night was 4 fold higher than that of at daytime. (3) Either ATP or insulin administration stimulated hepatic HMG CoA reductase activity. But, cyclic AMP had no effect on reductase activity. The stimulatory effect of ATP or insulin on reductase activity was inhibited by a preadministration of glucagon. These results suggested that an interplay of hormone might regulate reductase activity and consequently cholesterol biosynthesis. (4) HMG CoA reductase activity was increased by preincubation of microsomes with cytosol. Presence of ATP or Mg++ intensified this effect. When digested by trypsin or degenerated by heat treatment, cytosol lost the stimulating activity. These results suggested as existence of protein factors in cytosol, which might modulate the enzyme interconversion from inactive to active forms.


Glucose metabolism in isolated fat cells: enhanced response of larger adipocytes from older rats to epinephrine and adrenocorticotropin. The effects of insulin and of two lipolytic hormones (epinephrine and ACTH1) on the rate and pattern of glucose metabolism were compared during incubation of isolated fat cells, obtained from epididymal fat pads of rats of varying age and degrees of adiposity. Glucose metabolism and the intracellular free fatty acid levels were expressed on a per cell basis and in relation to adipocyte size. The data for total glucose metabolism show that, in contrast to the declining insulin effect observed with adipocyte enlargement, the stimulation of glucose uptake and metabolism by these lipolytic hormones was significantly greater in the larger fat cells from the older fatter rats than in the smaller ones from the younger leaner rats. Lipolytic hormones suppressed, whereas insulin enhanced, fatty acid synthesis; moreover the lipolytic hormones stiumlated glucose ce effect of epinephrine on the intracellular free fatty acid levels was greater in the small fat cells than in the large ones; this effect of epinephrine was markedly curtained by the presence of glucose in the incubation medium, making it unlikely that acceleration of glucose metabolism by the lipolytic stimulus was mediated by an elevation of the intracellular free fatty acid level. The present results show a markedly enhanced capacity of the large adipocytes to accelerate glucose metabolism in response to these liplytic hormones. Thus, in contrast to prevailing notions of declining hormonal responsiveness with expanding fat cell size in older and more obese animals, this study documents an instance of increased hormonal response in enlarged adipocytes and points to the need for a more comprehensive reevaluation of the various hormonal effects in adipocytes of different size.


Adrenal insufficiency secondary to hypothalamic corticotropin releasing factor (CRF) insufficiency with hyperpigmentation: a case report. Partial adrenocortical insufficiency as a result of an insufficiency of the hypothalamic corticotropin releasing factor (CRF) was demonstrated in a 53-year-old female patient. Somatotropic, gonadotropic and thyreotropic functions of the pituitary gland were shown to be normal by a simultaneous pituitary stimulation test. This held true especially for the adrenocorticotrophic function: administration of lysine-vasopressin induced a normal rise in immunoreactive plasma-ACTH. Thus, a pituitary defect as a primary cause of the disease could be excluded and evidence was provided that there was a lack in hypothalamic stimulae absence of elevated ACTH levels hyperpigmentation of the skin existed. Possible explanations are discussed.


Cyclic AMP, glucose and cortisol in plasma during surgery. Cyclic AMP, glucose and cortisol in plasma were measured before, during and after major surgery (hysterectomy, six patients) and minor surgery (tympanoplasty, 10 patients). During major surgery cyclic AMP as well as glucose and cortisol showed a pronounced increase. During minor surgery cyclic AMP, glucose and cortisol levels were significantly lower than in the group undergoing major surgery. It is concluded that the increase of plasma cyclic AMP during operative procedures is related to the severity of the trauma.


Parathyroid hormone and calcitonin: no direct effect on vitamin D3-mediated, intestinal calcium absorptive mechanism. Embryonic chick duodenum maintained in organ culture responds to vitamin D3 in the culture medium by increased cyclic AMP concentration, de novo synthesis of a specific calcium-binding protein and by increased uptake and transmucosal transport of radiocalcium. The presence of bovine PTH, porcine or salmon calcitonin had no effect on these intestinal responses suggesting that these hormones may have no direct effect on the vitamin D3-mediated, intestinal calcium absorptive mechanism.


Reversal of the unresponsiveness of neonatal rat ovary to LH in cAMP synthesis by estrogen. The rat ovary during the 1st postnatal week is unresponsive to luteinizing hormone (LH), but responds to prostaglandin E1 with increase of cyclic adenosine 3',5'-monophosphate synthesis. In the present experiments unresponsiveness of ovaries of 6-day-old rats to LH in synthesis of cAMP was effectively reversed by injection of depot estradiol and diethylstilbestrol on the 2nd and 4th postnatal day. Administration of testosterone, progesterone, deoxycorticosterone, pregnant mare's serum gonadotropin and human chronic gonadotropin had no stimulatory effect. The lack of response to LH also failed to be reversed when estradiol was injected 21 h before killing of the animals or the ovaries were preincubated with estradiol. These results suggest that the development of an ovarian cell system responsive to LH in newborn rat may be accelerated by long-term action of estradiol.


Hereditary hemolytic anemia with erythrocyte pyrimidine 5'-nucleotidase deficiency in Spain. Clinical, biological and familial studies. We report a hereditary hemolytic anemia associated with a severe erythrocyte pyrimidine 5'-nucleotidase deficiency in a Spanish family of five members in which the parents are first cousins. Both parents exhibited decreased nucleotidase activity without clinical or hematologic abnormalities. Two children (a male and a female) showed severe pyrimidine 5'-nucleotidase deficiency with hemolytic anemia. The remaining child (a male) showed no signs of the disease. The findings strongly suggest an autosomal recessive mode of inheritance in this enzymopathy. This seems to be the first report of pyrimidine 5'-nucleotidase deficiency in Spain.


Experimental herpes simplex virus carditis in mice. Herpes simplex virus types 1 and 2 induced acute and chronic cardiac damage in suckling and weaning mice after intranasal inoculation. Signs of virus replication were detected by light, immunofluorescent, and electron microscopy techniques. Virtually all of the cardiac tissues appeared to be susceptible to herpes simplex virus. The myocardium, however, was most regularly affected. The viral lesions were discrete during the acute phase of infection. The cardiac damage, however, was more extensive in some of the chronically infected mice. Morphologically, these lesions either resembled the acute ones or were associated with inflammatory granulomatous and sclerotic changes.


Effects of beta-adrenergic blockade on plasma cyclic AMP and blood sugar responses to glucagon and isoproterenol in man. In healthy humans glucagon infusion resulted in a significant increase in blood sugar and in plasma cyclic AMP. No discernible hemodynamic effects were found. Isoproterenol infusion on a mole per mole basis in the same subjects induced a significant, although less pronounced rise in plasma cyclic AMP, heart rate, and a fall in diastolic blood pressure but had no effect on blood sugar. Propranolol administration abolished the hemodynamic effects of isoproterenol and significantly decreased the response of plasma cyclic AMP; the same blocking dosage had little effect on plasma cyclic AMP changes induced  by glucagon wheras the response in blood sugar was significantly reduced. These data in vivo are compatible with the in vitro demonstration of separate receptors for glucagon and isoproterenol.


In vitro evaluation of some latent radioprotective compounds. In tissue culture, protection against X-irradiation by a number of cysteamine derivatives was studied and the results were compared with data obtained in mice. Compounds with a covered SH group, like WR 638, cysteamine phosphate, WR 2721, and AE 48527, showed practically no protection when dissolved in tissue-culture medium, but developed a protective activity when dissolved in rat blood. Thiol measurements demonstrated that in rat blood the compounds were partly hydrolysed to thiols. C511 was also hydrolysed in culture medium and was slightly less effective than cysteamine in culture medium. Cysteamine phosphate was hydrolsed more easily than cysteamine sulphate and the protective activity in rat blood was better. WR 2721 was also partly hydrolysed in rat blood. The in vitro protection of this compound was disappointing when compared with results in vivo. Its SH form (WR 1065) also showed less protection than expected from in vivo experiments. Thus, the little protection by WR 2721 in vitro in rat blood is not only due to its incomplete conversion into its thiol. Longer incubation times and the use of rat blood as a solvent brought the protective activity of WR 1065 almost up to the level of cysteamine. This may indicate that WR 1065 penetrates the cells poorly. WR 1065 was the only compound we studied whose protective activity in vitro was improved appreciably by dissolving it in rat plasma.


Studies of short-lived radicals in the gamma-irradiated aqueous solution of uridine-5'-monophosphate by the spin-trapping method and the liquid chromatography. An aerated aqueous solution of uridine-5'-monophosphate was gamma-irradiated with 2-methyl-2-nitrosopropane as a spin-trapping reagent. Liquid chromatography was applied to separate the stable nitroxide radicals in the irradiated solution. The radicals were detected by U.V. and e.s.r. spectrometry. The e.s.r. detection showed four peaks in the chromatogram. The orcinol method for detection of the residual sugar moieties was applied before and after reduction of the base to determine the existence of the 5,6-double bond for the molecules in each fraction. From the combined results of the e.s.r. and orcinol methods, the short-lived radicals which were trapped by 2-methyl-2-nitrosopropane were identified as radicals of N-1 and C-6 positions of the base moiety and t-butyl radical which was the radiolytic product of the trapping reagent.


Polyadenylation of ribonucleic acids in mouse L cells infected by encephalomyocarditis virus. Polyadenylation of cytoplasmic RNAs was investigated in L cells infected with encephalomyocarditis virus. During the early stage of infection, the mean size of newly made poly(A) chains attached to cell mRNAs decreased gradually, in parallel with the shut-off of RNA synthesis. Later on, poly(A) segments, an average of 50 nucleotides long, were associated with viral RNA isolated either from polysomes or whole cytoplasm. These segments seemed to be processed differently in the course of time, and RNA molecules containing short poly(A) tracts (less than 20 bases) were incorporated into the virions.


Uncontrolled nuclear division in murine cells abortively transformed by simian virus 40. Cytochalasin B (CB) prevents cytoplasmic cleavage without directly affecting nuclear division. Secondary cultures of mouse embryo fibroblasts or 3T3 cells show controlled nuclear division when treated with CB: only binucleated cells are formed. Many CB-treated transformed cells show uncontrolled nuclear division and become highly multinucleated. When CB-treated normal cells are concurrently infected with high inputs of SV40, many of these cells become highly multinucleated. It is suggested that these highly multinucleated cells represent abortively transformed cells since the actual number of transforming units (focus-forming units) of simian virus 40 (SV40) is too low to account for the appearance of these cells. Also, if the CB treatment is begun 6 days after SV40 inoculation, the large increase in highly multinucleated cells is not observed. Most cells stably transformed by SV40 or adenovirus show uncontrolled nuclear division when treated with CB. However, 3T3 cells transformed by SV40 or adenovirus and analyzed shortly after transformation are an exception and show controlled nuclear division. This property of 3T3 cells is apparently overcome by high inputs of SV40 since abortively transformed cells become highly multinucleated.


Lacalization of the SV40 T antigen in hamster cells transformed by PARA(3ct)-adenovirus 7. The localization of the SV40 T antigen in hamster cells transformed by the PARA(3ct)-adenovirus 7 variant and, for comparison, by SV40 was examined. The H50 cell strain (transformed by SV40) and the P7-/BL/SV/OD strain (transformed by PARA(3ct)-adenovirus 7) showed a preferential localization of the SV40 T antigen in the nucleus both by immunofluorescence and by complement fixation. On the other hand, two different PARA(3ct)-adenovirus 7-transformed cell lines (P7-/Ar/d/15A and P7/Ar/m/14A) showed an almost equivalent concentration of the SV40 T antigen in the nucleus and in the cytoplasm both by immunofluorescence and complement fixation. The H50, P7-/BL/SV/3D, P7/Ar/m/14A cells were oncogenic for hamsters; the P7/Ar/d/15A line was not.


Replicating DNA of herpes simplex virus type 1. Newly synthesized herpes simplex virus type 1 DNA yielded a heterogeneous sedimentation profile in neutral sucrose gradients, with the main peak occurring at approximately 40S. Components sedimenting slower than virion DNA and a rapidly sedimenting intracellular HSV DNA were also observed. Both the low-molecular weight and the rapidly sedimenting components seemed to be precursors of virion DNA: they almost completely disappeared after a 60-min chase of a 3-min pulse of 3H-thymidine, and were converted into DNA which cosedimented with virion 32P-labeled DNA. However, sedimentation analysis in alkaline sucrose gradients showed that a 60-min period was insufficient for completing the maturation of HSV DNA. Cleavage of parental DNA molecules was observed in neutral sucrose gradients after infection with 3H-thymidine-labeled virions. No evidence for the formation of covalently closed circles during the replication process was obtained. The presence of single-stranded regions in the replicative form of HSV DNA was revealed. Some of the short-pulse (30 sec) labeled HSV DNA (26.1%) was eluted from hydroxylapatite columns with the properties of single-stranded DNA, and 22% of its trichloroacetic acid precipitability was susceptible to single-strand specific S1 nuclease treatment. Pulse-chase experiments indicated that the life-time of this single-stranded component in nascent DNA was probably not longer than 3 min. A small proportion of single-stranded regions, however, survived for longer periods. Almost all of the newly synthesized short-pulse-labeled HSV DNA exhibited an affinity for nitrocellulose filters. This affinity, which was S1 nuclease-sensitive, gradually decreased with prolongation of the time of the chase. After chasing the pulse for 1 h, the attachment of newly synthesized DNA was comparable with virion DNA.


Comparison of DNA polymerase activities induced by herpes simplex virus types 1 and 2. HSV-2-induced a salt-stimulated DNA polymerase activity in HEp-2 cells at similar levels to that induced by HSV-1 virus at multiplicites of infection from 2 to 20 PFU/cell. De novo synthesis of protein and mRNA was required for DNA polymerase induction by both types. DNA polymerase activity of three strains of each type was compared by response to various salt concentrations and by heat inactivation. Strain-dependent, but not type-dependent differences were found by both tests. Considerable cross-neutralization of polymerase activity was obtained with specific antisera. The DNA polymerase activity induced by HSV-1 and HSV-2 cannot be readily differentiated by these criteria.


Size of the poly(A) sequences in encephalomyocarditis virus RNA. Poly(A) sequences were demonstrated in the RNA of encephalomyocarditis (EMC) virus by affinity chromatography on poly(U)-Sepharose, treatment with RNases, and determination of the ability of the RNA to prime for reverse transcription. The size of the poly(A) sequences was estimated as 10-20 nucleotides in length by determination of the fraction resistant to pancreatic RNases plus T1 RNases, followed by analysis of electrophoretic mobility on polyacrylamide gels and of the AMP-to-adenosine ratio after alkaline hydrolysis. The poly(A) sequences are located at the 3' end of the RNA as shown by mild digestion by snake venom phosphodiesterase.


Effect of pasteurization and heat treatment on bovine leukemia virus. Four sheep inoculated with mixtures of bovine leukemia virus (BLV) and milk that was treated by a simulated high-temperature short-time pasteurization procedure did not become infected with BLV or develop tumor. Four sheep inoculated with unpasteurized BLV-mil mixtures became infected with BLV and 3 died with tumor at 15, 21, and 27 months, respectively, after inoculation. Fluid from a BLV-infected cell culture was heated to 56, 60, 65, and 73 C for 1/2 minute and 1 minute (in separate trials) and transferred to noninfected cells. Culture fluid heated to 60 C and higher did not infect the cells.


Natural infection of pigs with bovine viral diarrhea virus and its differential diagnosis from hog cholera. Natural infection of pigs with bovine viral diarrhea virus (BVDV) through contact with infected cattle has caused problems in diagnosing hog cholera (HC). Low cross-reacting serum antibody titers against HC caused by BVDV infection were found in clinically normal pigs as well as those suspected of having HC. Bovine viral diarrhea virus was isolated from specimen tissues and initially identified as HC virus (HCV), using the fluorescent antibody cell culture technique. Additional cell cultures, as well as pig and calf trials, were necessary to identify it as BVDV. The isolate caused clinical signs of illness in the calves, whereas the pigs remained healthy. Bovine viral diarrhea virus may be detected in tissue sections or isolated in cell cultures and confirmed as HCV, using the HC fluorescent antibody conjugate. Laboratories performing the neutralization test for HC should use discretion when interpreting HC titers unless BVD titers are determined on the same serums.


Pathologic features of polybrominated biphenyl toxicosis in the rat and guinea pig. Young male rats were fed a diet containing 0, 1, 10, 100, or 500 ppm of a commercial mixture of polybrominated biphenyls (PBB) that had been accidentally incorporated into a mineral mixture and fed to Michigan livestock and poultry. After 30 days, 9 of the 12 rats in each group were killed and tissues were examined. Liver weight to body weight ratios were significantly increased at all feeding levels; at 500 ppm, liver weight had more than doubled. Kidney weight was not affected. Microscopic lesions were mostly confined to the liver and consisted of extensive swelling and vacuolation of hepatocytes in rats fed diets containing 100 and 500 ppm of PBB. Slight swelling and vacuolation were seen in rats fed the diet containing 10 ppm, and lesions were not found at 0 or 1 ppm. There was a significant increase in hepatic mitochondrial size at 1 ppm, and smooth endoplasmic reticulum was markedly increased at 100 and 500 ppm. Myelin bodies were present at 100 and 500 ppm, and vacuoles were numerous. Rats killed at 60 days had similar lesions. The activity of hepatic microsomal enzymes increased at all levels of feeding of PBB. Rat pups nursing dams fed a diet containing 10 ppm of PBB had microscopic and ultrastructural hepatic lesions. When guinea pigs were fed PBB at the same amounts as were rats, the results were strikingly different. Guinea pigs fed a diet containing 500 ppm of PBB died within 15 days; at 100 ppm, only 2 of 6 survived for 30 days. Effects on liver weight were inconsistent, but 2 of 6 fed a diet containing 10 ppm had enlarged livers.


Carbohydrate metabolism of heat-acclimated hamsters. V. Control of gluconeogenesis. The rate of gluconeogenesis was similar in liver of both fed heat-acclimated and control hamsters. Twenty-four hours of fasting calsed 6 times elevation in hepatic gluconeogenesis of the control animals whereas only 4 times enhancement of this pathway was found in heat-acclimated animals. Thus, significant difference existed between the two experimental groups in fasting. Triiodothyronine stimulated the rate of gluconeogenesis only in fed heat-acclimated hamsters whereas dibutyryl cyclic AMP caused elevation of this pathway in liver slices of fed control hamsters only. The results suggest that a decrease in hepatic gluconeogenesis in heat acclimation occurs only in fasted animals and it is controlled by thyroid hormones.


Mitochondrial biogenesis during fungal spore germination: respiratory cytochromes of dormant and germinating spores of Botryodiplodia. The mitochondrial respiratory cytochrome contents of dormant and germinating conidia of Botryodiplodia theobromae were examined. Oxidized versus reduced difference spectra at 77 degrees K of whole mitochondria from physiologically mature germinated spores showed a typical a-band pattern for cytochromes c, b, and a, with absorption maxima at 549, 554 + 559, and 604 nm, respectively, whereas the difference spectrum of the counterpart mitochondrial fraction from dormant spores showed no cytochrome a bands. However, a fraction prepared from dormant spore mitochondria by detergent extraction and (NH4)2SO4 fractionation contained readily detectable quantities of cytochromes c and b (as shown by the a and Soret absorption bands), but it did not contain the a or Soret bands of cytochrome a observed in a counterpart preparation from germinated spores. The pyridine hemochromogen preparation from the dormant spore mitochondria contained no material that is spectroscopically characteristic of a-type heme and protoheme. These results suggest that cytochrome a is not present as a functional molecule in dormant spores. The first spectroscopically detectable cytochromes were observed in whole mitochondria at 210 min of spore germination, and the amount of each of the cytochromes increased with cell growth. A precursor of the heme porphyrin, delta-[4-14C]aminolevulinic acid, was first incorporated (at accelerating rates) into acid-insoluble spore material at 180 min of germination, which appears to be the approximate time of organization of new mitochondria in these spores.


Purification and characterization of cytochrome c3, ferredoxin, and rubredoxin isolated from Desulfovibrio desulfuricans Norway. Different electron carriers of the non-desulfoviridin-containing, sulfate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) have been studied. Two nonheme iron proteins, ferredoxin and rubredoxin, have been purified. This ferredoxin contains four atoms of non-heme iron and acid-labile sulfur and six residues of cysteine per molecule. Its amino acid composition suggests that it is homologous with the other Desulfovibrio ferredoxins. The rubredoxin is also an acidic protein of 6,000 molecular weight and contains one atom of iron and four cysteine residues per molecule. The amino acid composition and molecular weight of the cytochrome c3 from D. desulfuricans (strain Norway 4) are reported. Its spectral properties are very similar to those of the other cytochromes c3 (molecular weight, 13,000) of Desulfovibrio and show that it contains four hemes per molecule. This cytochrome has a very low redox potential and acts as a carrier in the coupling of hydrogenase and thiosulfate reductase in extracts of Desulfovibrio gigas and Desulfovibrio desulfuricans (Norway strain) in contrast to D. gigas cytochrome c3 (molecular weight, 13,000). A comparison of the activities of the cytochrome c3 (molecular weight, 13,000) of D. gigas and that of D. desulfuricans in this reaction suggests that these homologous proteins can have different specificity in the electron transfer chain of these bacteria.


Specific deficit in the synthesis of 6-sulfoquinovsyl diglyceride in Chlorella pyrenoidosa. It was found that when Chlorella pyrenoidosa was grown on cysteine as the sole sulfur source, it lost the ability to grow photoautotrophically. When grown in the presence of glucose, cysteine-grown cells displayed a doubling time in the light or dark of 45 h, which is identical to that of cells grown on glucose and SO4 in the dark. This suggests that cells grown on cysteine as sole sulfur source can only grow heterotrophically. In support of this hypothesis, it was found that cysteine-grown cells were defective both in vivo and in vitro in CO2 fixation, although O2 evolution in such cells was normal. Assays of the enzymes of the Calvin cycle indicated that the deficit in CO2 fixation could be ascribed to a lowered phosphoribulokinase activity. A total lipid analysis of Chlorella grown on cysteine revealed that such cells showed a 100-fold deficiency in the purportedly chloroplast-associated 6-sulfoquinovsyl diglyceride. This agrees with earlier reports that cysteine could not serve as a precursor of sulfolipid in Chlorella. No other polar lipid was affected. Large amounts of triglyceride, however, were found in cysteine-grown cells. The biosynthesis of triglyceride provides a means of utilizing reduced nicotinamide adenine dinucleotide reducing equivalents not being used for CO2 fixation.


Presence of cytochrome c in Desulfomonas pigra. Desulfomonas pigra, a gram-negative, nonmotile anaerobic, sulfate-reducing bacillus isolated from human feces, was found to have cytochrome c and a desulfoviridin-like pigment.


Internally generated reduced nicotinamide adenine dinucleotide as a substrate for glycine transport by membrane vesicles of Paracoccus denitrificans. Internally generated reduced nicotinamide adenine dinucleotide was the most efficient substrate for glycine transport by membrane vesicles of Paracoccus denitrificans.


Role of the rel gene product in the control of cyclic adenosine 3',5'-monophosphate accumulation. The presence of a relA mutant allele affects the kinetics of cyclic adenosine 3',5'-monophosphate accumulation during downshift from glucose to succinate. The nucleotide accumulates at the normal rate early in the downshift transition but continues to accumulate for a longer time in the relA mutant, leading to a two- to threefold excess by the end of the diauxic lag. Evidence is presented that this effect occurs independently of the accumulation of ppGpp.


Cyclic adenosine 3',5'-monophosphate levels and activities of adenylate cyclase and cyclic adenosine 3',5'-monophosphate phosphodiesterase in Pseudomonas and Bacteroides. A modified Gilman assay was used to determine the concentrations of cyclic adenosine 3',5'-monophosphate (cAMP) in rapidly filtered cells and in the culture filtrates of Pseudomonas aeruginosa, Escherichia coli K-12, and Bacteroides fragilis. In P. aeruginosa cultures, levels of cAMP in the filtrate increased with the culture absorbance (3.5 to 19.8 X 10(-9) M) but did not vary significantly with the carbon source used to support growth. Intracellular concentrations (0.8 to 3.2 X 10(-5) M) were substantially higher and did not vary appreciably during growth or with carbon source. Sodium cAMP (5 mM) failed to reverse the catabolite repression of inducible glucose-6-phosphate dehydrogenase (EC 1.1.1.49) synthesis caused by the addition of 10 mM succinate. Exogenous cAMP also had no discernible effect on the catabolite repression control of inducible mannitol dehydrogenase (EC 1.1.1.67). P. aeruginosa was found to contain both soluble cAMP phosphodiesterase (EC 3.1.4.17) and membrane-associated adenylate cyclase (EC 4.6.1.1) activity, and these were compared to the activities detected in crude extracts of E. coli. B. fragilis crude cell extracts contain neither of these enzyme activities, and little or no cAMP was detected in cells or culture filtrates of this anaerobic bacterium.


Characterization of one neutral and two acidic glycoasparagines isolated from the urine of patients with aspartylglycosylaminuria (AGU). One neutral and two acidic glycoasparagines were isolated from the urine of patients with aspartylglycosylaminuria (AGU). The neutral one was identified as beta-Gal-(1 leads to 4)-beta-GlcNAc-Asn. The acidic ones were composed of 1 mole of sialic acid and 2 moles each of galactose and N-acetylglucosamine, attached to asparagine, and were isomeric with respect to the position of sialic acid attachment since they produced the same glycoasparagine on incubation with the neuraminidase [EC 3.2.1.18] from Clostridium perfringens. The structure of the resulting sialic acid-free glycoasparagine was determined to be beta-Gal-beta-GlcNAc-beta-Gal-(1 leads to 4)-beta-GlcNAc-Asn based mainly on the results of sequential enzymatic degradations.


Generation of the superoxide radical during autoxidation of oxymyoglobin. Autoxidation of bovine oxymyoglobin to metmyoglobin induces co-oxidation of epinephrine to adrenochrome. This co-oxidation is markedly inhibited by superoxide dismutase [EC 1.15.1.1]. Electron transfer from oxymyoglobin to ferricytochrome c is partially inhibited by superoxide dismutase. These results indicate that autoxidation of oxymyoglobin results in generation of superoxide radicals. Autoxidation of oxymyoglobin is accelerated by superoxide dismutase and partially inhibited by catalase [EC 1.11.1.6].


Conformational transitioons of polypeptide chain elongation factor Tu. II. Further studies by electron spin resonance. The conformational transitions of polypeptide chain elongation factor Tu (EF-Tu) associated with the ligand change from GDP to GTP and also with the displacement of GDP by elongation factor Ts (EF-Ts) have been investigated using the spin-labeling technique. Of the two reactive sulfhydryl groups in EF-Tu, the one essential for interaction with aminoacyl-tRNA was selectively labeled with various kinds of iodoacetamide or maleimide spin-labeling reagents. The electron spin resonance (ESR) spectra of EF-Tu-GDP labeled with these reagents generally consisted of two components, one narrow and one broad, corresponding to labels relatively weakly and strongly immobilized, respectively. The degree of immobilization and the ratio of the narrow to the broad components were different for each kind of label used. The spectra of spin-labeled EF-Tu-GDP changed markedly when its GDP moiety was replaced by GTP through incubation with phosphoenolpyruvate and pyruvate kinase [EC 2.7.1.40], the broad component increasing at the expense of the narrow component. The reversible nature of the conformational change was confirmed with EF-Tu labeled with a maleimide reagent. The GTP-induced spectral change was reversed upon conversion of labeled EF-Tu-GTP to EF-Tu-GDP by addition of excess GDP. A similar type of spectral change was also observed when spin-labeled EF-Tu-GDP was incubated with EF-Ts to form labeled EF-Tu-EF-Ts complex. The extent of the spectral change induced by EF-Ts was even greater than that induced by GTP. These results, together with those obtained by studies with hydrophobic and fluorescent probes (Arai, Arai, Kawakita, & Kaziro (1975) J. Biochem. 77, 1095-1106) indicate that a reversible conformational change is induced in EF-Tu near the sulfhydryl group that is essential for interaction with aminoacyl-tRNA.


Conformational transition of polypeptide chain elongation factor G as determined by electron spin resonance. The conformational transition of the polypeptide chain elongation factor G (EF-G) induced by interaction with guanine nucleotide has been investigated by means of the spin-labeling technique. Various spin-label probes were attached specifically to the sulfhydryl group of the protein that is essential for binding to ribosomes, and the effects of these ligands on the electron spin resonance (ESR) spectra were examined. It was found that the ESR spectra of EF-G labeled with nitroxide maleimide reagents were modified by the addition of various guanine nucleotides such as GDP, GTP and, to a lesser extent, by Gpp(NH)p and Gpp(CH2)p, indicating that conformational changes accompany the binding of nucleotide ligand. However, the ESR spectra of labeled EF-G-GDP and EF-G-GTP were almost identical. On the other hand, when EF-G was labeled with nitroxide iodoacetamide reagents, a clear difference in the ESR spectra of EF-G-GDP and EF-G-GTP derivatives was observed. In this case, the spectral shape of the spin-labeled EF-G in the presence of GTP or its analogs, Gpp(NH)p or Gpp(CH2)p, was quite similar to that of free, unliganded EF-G derivative. These results, together with those previously obtained using hydrophobic probes (Arai, Arai, & Kaziro (1975) J. Biochem. 78, 243-246) demonstrate the existence of an EF-G-guanine nucleotide binary complex. They also indicate that there is a substantial difference in conformation between free EF-G, EF-G-GDP, and EF-G-GTP near the active site essential for interaction with ribosomes.


Studies on the turnover rates of ornithine aminotransferase in Morris hepatoma 44 and host liver. The ornithine aminotransferase [EC 2.6.1.13] content of Morris hepatoma 44 is about 15 times higher than that in normal liver. The turnover rates of ornithine amino-transferase in hepatoma 44 and host liver were determined using L-[14C]leucine. Studies on the incorporation of radioactive leucine into ornithine aminotransferase in rats bearing hepatoma 44 showed that the rate of synthesis of this enzyme in the hepatoma was about 5-fold higher than that in host liver. The half-life of ornithine aminotransferase in host liver was 0.98 day, which was the same as that in normal liver, whereas that in hepatoma 44 was 3.5 days. The rate constant of degradation of ornithine aminotransferase in hepatoma 44 was significantly less than that in host liver. These results show that the high ornithine aminotransferase content of hepatoma 44 is due to both increase in its rate of synthesis and decrease in its rate of degradation.


The role of tryptophanyl residues in heavy meromyosin as studied by chemical modification with 2-hydroxy-5-nitrobenzyl bromide. 1. Two moles of 2-hydroxy-5-nitrobenzyl group bound selectively to one mole of heavy meromyosin when it was treated with 2-hydroxy-5-nitrobenzyl bromide, a specific reagent for tryptophanyl residues. The binding with ADP, the size of the initial burst of Pi liberation and the difference absorption spectrum with and without ADP of the bound 2-hydroxy-5-nitrobenzyl groups were measured with heavy meromyosin modified with various amounts of reagent. The properties of the modified heavy meromyosin did not change until the molar binding ratio of the reagent, rH, was about 1, but the properties changed remarkably when rH increased from 1 to 2. 2. Subfragment-1 was prepared from the modified heavy meromyosin by trypsin [EC 3.4.21.4] digestion. The molar binding ratio of the reagent in subfragment-1, rS, was found to be less than 0.1 when rH of the starting heavy meromyosin was less than 0.8. However, rS was about 0.5 in subfragment-1 prepared from heavy meromyosin of rH about 2. The results indicate that only one mole of 2-hydroxy-5-nitrobenzyl group, which was bound with lower reactivity than the other, was bound to a head part of heavy meromyosin. 3. Subfragment-1 fraction prepared from the modified heavy meromyosin could be separated into two fractions by DE-32 cellulose column chromatography; the subfragment-1 portion which eluted later showed a higher rS than that eluted in front. The binding with ADP, the size of the initial burst of Pi liberation and the difference absorption spectrum induced by ATP were measured with the modified subfragment-1 separated by DE-32 cellulose column chromatography. The ADP-binding ability and the size of the initial burst were not dependent on rS, and coincided with those of subfragment-1 prepared from unmodified heavy meromyosin. 4. The results of ADP binding studies suggest that heavy meromyosin is constituted from nonidentical subunits, and that there is an interaction between them which controls the ADP binding. Two tryptophanyl residues having specific reactivity toward 2-hydroxy-5-nitrobenzyl bromide are assumed to be involved in the interaction.


The isolation and characterization of glycopeptides and mucopolysaccharides from plasma membranes of an ascites hepatoma, AH 130 FN. Plasma membranes were isolated from an ascites hepatoma, AH 130 FN, a free-cell type subline of AH 130, by the fluorescein mercuric acetate (FMA) method. Glycopeptides and mucopolysaccharides were prepared from the membranes by pronase digestion then fractionated chromatographically and electrophoretically. Isolated fractions were analyzed for amino acid and carbohydrate compositions. The results were compared with those for corresponding fractions from AH 66 and AH 130 ((1974) J. Biochem. 76, 319-333; (1975) ibid., 78, 863-872). The fraction excluded from Sephadex G-50 contained mucopolysaccharides and a series of glycopeptides. The mucopolysaccharides were identified as chondroitin sulfate A on the basis of their chemical composition, electrophoretic behavior on cellulose acetate and digestibility with chondroitinase AC [EC 4.2.2.5]. This contrasts with previous findings that mucopolysaccharides from the corresponding fractions from AH 130 and AH 66 were heparan sulfate. The chemical composition of the glycopeptides, which showed high contents of threonine, serine, galactose, galactosamine, glucosamine, and sialic acid, indicated the presence of glycopeptides with O-glycosidic linkages. The glycopeptides also contained a small but significant amount of aspartic acid, suggesting that N-glycosidic glycopeptides were also contained in this fraction. The fraction included in Sepnadex G-50 contaoned N-glycosidic glycopeptides as major components, since the carbohydrate moieties were composed of fucose, galactose, mannose, glucosamine, sialic acid, and a smaller amount of galactosamine. The presence of galactosamine suggested that O-glycosidic glycopeptides were present as minor components. Glycopeptides with both O- and N-glycosidic linkages were isolated from AH 130, but not from AH 66.


Evidence for the presence of di- and triphospho pyridine nucleotide dehydrogenase derivatives as consistent contaminants of purified beef heart cytochrome-c oxidase. Purified beef heart cytochrome-c oxidase preparations derived by three different laboratories contain NADH-K3 Fe (CN)6, NADH-nitrobluetetrazolium, and NADPH-nitrobluetetrazolium reductases. This is true of preparations exhibiting heme aa3 to protein ratios considered indicative of an excellent purity. An apparent association of cytochrome-c oxidase and one or more of the contaminants persists through immunodiffusion and nondenaturing electrophoresis and, in addition, in one instance copurification of NADH-K3Fe(CN)6 reductase and cytochrome-c oxidase to a constant ratio of specific activities was demonstrated. Cytochrome-c oxidase can be freed of the contaminants by equilibration with an NAD+-affinity matrix. As aconcomitant of equilibration with the matrix, the KM of cytochrome-c oxidase for ferrocytochrome-c is invariably decreased. Rat constants at low ferrocytochrome-c concentrations are consistently enhanced in all oxidase preparations upon equilibration with the NAD+ matrix. However, the effects of such equilibrations on the extrapolated Vmax varies from one preparation to another. Polyacrylamide gel electrophoresis in SDS-urea systems establishes that each of the preparations contains a minimum of three contaminants, each of an apparent formula weight of greater than 40,000 Daltons. NADH-NBT reductase was found to have a formula weight of approximately 46,000 Daltons. Their properties establish that NADH-K3Fe(CN)6 and NADH-NBT reductases are separate proteins; the separate identity of NADPH-NBT reductase has not yet been determined.


Chromatin receptors for thyroid hormones. Interactions of the solubilized proteins with DNA. Thyroid hormone-responsive tissues contain chromatin "receptor" proteins that are concentrated in chromatin subfractions enriched in DNA. These receptors appear to be DNA-binding proteins. In the present study, we utilized a DNA-cellulose binding assay to further examine the interactions of solubilized receptors with DNA. [125I]Triiodothyronine associates with receptors bound to DNA-cellulose, whereas free [I]triiodothyronine and [125I]triiodothyronine associated with other proteins does not. The DNA-receptor interactions appear to be strong enough to exist at physiological ionic strength since binding is 50% maximal ag 0.175 M NaCl and is only partly inhibited by Ca2+ and Mg2+ in the 1 to 5 mM range. Most, if not all, of the receptors are capable of DNA binding, and there are at least 80,000 receptor binding sites/diploid DNA (assuming one triiodothyronine binding site/receptor). Binding of the receptor-[125I]triiodothyronine complexes to other DNAs and analogs was examined using a competition assay. There is similar binding by native and denatured DNA, gy eukaryotic DNA from different species and by prokaryotic DNA (Bacillus subtilis). Binding by natural DNAs is more avid than by cytoplasmic RNA, nuclear RNA, poly(dA-dT)-poly(dA-dT), or poly(dG-dC)-poly(dG-dC). Under these conditions, binding by tRNA and poly(dA) is insignificant, and the nucleotide monomers ATP and GTP have no detectable binding. These studies support the idea that the thyroid hormone receptor is a DNA-binding protein and that the interaction is a major determinant for receptor localization in chromatin. The competition studies suggest that the polynucleotide composition and/or conformation can have marked influences on the binding, and that multiple orders of binding affinity can exist. The presence of specific sequences cannot be excluded. However, the finding that receptors bind extensively and tightly to DNA suggests that receptors in chromatin may randomly bind to any available DNA, resulting in some of the receptors being at physiologically unimportant sites. If so, the several thousand hormone receptors present in each target cell may be required to enhance the possibility that some of the receptors are present at the actual sites of action.


Gonadotropin receptors in plasma membranes of bovine corpus luteum. I. Effect of phospholipases on the binding of 125I-choriogonadotropin by membrane-associated and solubilized receptors. The ability of bovine corpus luteum plasma membranes to bind 125I-choriogonadotropin has been examined after prior treatment of the membranes with phospholipases A, C, and D. Treatment of the purified membranes with low concentrations of phospholipases A and C resulted in the inhibition of the binding of 125I-choriogonadotropin to its receptors, whereas phospholipase D had no effect. Receptor activity was decreased by low concentrations of phospholipase A from either bee venom, Vipera russelli or Crotalus terrificus terrificus. Similarly, low concentrations of phospholipase C from Clostridium perfringens and Clostridium welchii also inhibited the binding activity while comparatively higher concentrations of phospholipase C from Bacillus cereus were required to achieve comparable inhibition. The time required to produce 50% inhibition of in vitro binding by phospholipases A and C was found to be 6 and 23 min, respectively. Upon either removal or chelation of calcium ions by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) both enzymes were completely inhibited as evidenced by the complete retention of the membrane binding activity. The decrease in the specific binding of choriogonadotropin to membranes after phospholipase digestion resulted in a decrease in the number of binding sites and was not accompanied by a change in the affinity of the hormone-receptor complex. The rates of association and dissociation of the 125I-choriogonadotropin-receptor complex and the equilibrium dissociation constant (Kd) were nearly identical in untreated and phospholipase-treated membranes. Phospholipases did not have any effect on the preformed hormone-receptor complex or on solubilized receptor. Filtration through Sepharose 6B of solubilized 125I-choriogonadotropin-receptor complex from untreated membranes or membranes which had been pretreated with phospholipase C prior to carrying out hormone binding did not alter the profile (Kav 0.38). Gel filtration of membranes treated with phospholipase A showed two peaks of bound radioactivity with distribution coefficients (Kav) of 0.08 and 0.35, respectively.


Gonadotropin receptors in plasma membranes of bovine corpus luteum. II. Role of membrane phospholipids. The role of phospholipids in the binding of 125I-choriogonadotropin to bovine corpus luteum plasma membranes has been investigated with the use of purified phospholipase A and phospholipase C to alter membrane phospholipids. The phospholipase C-digested plasma membrane preparation showed 85 to 90% inhibition of 125I-choriogonadotropin binding activity when 70% of the membrane phospholipid was hydrolyzed. Similarly treatment of plasma membranes with phospholipase A resulted in 45 to 55% hydrolysis of membrane phospholipid and almost 75% inhibition of receptor activity. Both these enzymes hydrolyzed membrane-associated phosphatidylcholine to a greater extent than phosphatidylethanolamine and phosphatidylserine. Phosphorylaminoalcohols of phospholiphase C end products were completely released into the medium, while phospholipase A by-products remained associated with plasma membranes. Addition of a phospholipids suspension or liposomes to plasma membranes pretreated with phospholipase A and C did not restore gonadotropin binding activity. Soluble phosphorylcholine, phosphorylethanolamine, and phosphorylserine and insoluble diglyceride products of phospholipase C action had no effect on receptor activity. In contrast, end products of the phospholipase A action, such as lysophosphatides and fatty acids, inhibited both on the membrane-associated and solubilized receptor activity. Lysophosphatidylcholine was the most effective end product inhibiting the binding of gonadotropin to the receptor, followed by lysophosphatidylethanolamine and lysophosphatidylserine. The inhibitory effects of phospholipase A or lysophosphatides were completely reversed upon removal of membrane-bound phospholipid end products by washing the membranes with defatted bovine serum albumin. However, phospholipase C inhibition could not be overcome by defatted albumin washings. Solubilization of plasma membranes with detergents which had been pretreated with phospholipase C partially restored the inhibited activity. It is concluded that the phospholipase-mediated inhibition of gonadotropin binding activity was due to hydrolysis and alterations of the phospholipid environment in the case of phospholipase C and by direct inhibition by end products in the case of phospholipase A.


Angiotensin II binding to mammalian brain membranes. High affinity binding sites for angiotensin II in bovine and rat brain membranes have been identified and characterized using monoiodinated Ile5-angiotensin II of high specific radioactivity. Degradation of labeled and unlabeled peptide by washed brain particulate fractions was prevented by adding glucagon to the final incubation medium and including a proteolytic enzyme inhibitor (phenylmethylsulfonyl fluoride) in preincubation and incubation procedures. 125I-Angiotensin II binding can be studied using either centrifugation or filtration techniques to separate tissue-bound radioactivity. 125I-Angiotensin II binding to calf brain membranes is saturable and reversible, with a dissociation binding constant of 0.2 nM at 37 degrees. A similar binding constant is found in rat brain membranes. Analogues and fragments of angiotensin II compete for these brain binding sites with potencies which correlate with both their in vivo potencies and their binding inhibition protencies at adrenal cortex angiotensin II receptors. Angiotensin I is 1 to 2 orders of magnitude weaker than angiotensin II; the 3-8 hexapeptide and 4-8 pentapeptide are much weaker still. (desAsp1) angiotensin II (angiotensin III) is slightly more potent than angiotensin II, as are several antagonists of angiotensin II with aliphatic amino acids substituted at position 8. In calf brain 125I-angiotensin II binding is restricted almost exclusively to the cerebellum (cortex and deep nuclei). In rat brain, angiotensin II binding is highest in the thalamus-hypothalamus, midbrain, and brainstem, areas which are believed to be involved in mediating angiotensin II-induced central effects. These findings illustrate the presence of high affinity specific binding sites for angiotensin II in rat and bovine brain and suggest a physiological role for angiotensin peptides in the central nervous system.


Spin-labeled stearates as probes for microenvironment of murine thymocyte adenylate cyclase-cyclic adenosine 3':5'-monophosphate system. The interaction of various spin-labeled compounds with the murine thymocyte adenylate cyclase-cyclic AMP system was investigated. Electron paramagnetic resonance spectra from spin-labeled compounds were used to calculate the order parameter, S, and indicated that the thymocyte plasma membrane is a relatively rigid structure. Increasing concentrations of spin-labeled stearates, but not their corresponding methyl esters, resulted in increased membrane fluidity, partial lysis, and concomitant complete inhibition of cholera toxin-mediated increases in cyclic AMP content. Upon subsequent isolation of plasma membranes from these cells, cholera toxin-stimulated adenylate cyclase activity was also completely inhibited. Direct addition of spin-labeled stearates, but not spin-labeled methyl stearates, to thymocyte homogenates caused a dramatic reduction of basal, cholera toxin-, isoproterenol-, NaF-, and prostaglandin E1-stimulated adenylate cyclase activity. Inhibition was complete within the first minute of addition to homogenates and required approximately 0.2 mM spin-labeled stearate I(12,3) for half-maximal inhibition. This inhibition occurred in the presence or absence of an ATP-regenerating system and was not readily reversible. Furthermore, since the membrane cyclic phosphodiesterase activity was not altered by spin-labeled stearates, their inhibition was attributed to a direct action of stearate spin labels on adenylate cyclase. Neither stearate, methyl stearate, spin-labeled methyl stearates nor 2,2,6,6,-tetramethylpiperidine-1-oxyl (Tempo) altered cell viability or enzyme activities at the concentrations studied. Spin-labeled stearates seemed to intercalate into different areas of the plasma membrane than their corresponding methyl esters. Furthermore, the action of spin-labeled stearates appeared to be on the exterior of the plasma membrane rather than the interior. These results illustrate the presence of multilipid domains and the importance of selected lipids and lipid-protein interactions in the adenylate cyclase-cyclic AMP system. Thymocyte adenylate cyclase is described in terms of a current model for membrane proteins.


On the nature of the three intermediate species formed after reaction of reduced cytochrome oxidase with oxygen. Spectral examinations of the reaction of reduced cytochrome oxidase with molecular oxygen has revealed the formation of at least three intermediates, which are designated as Compounds I, II, and III according to the order of their appearance. From the difference spectrum against the oxidized oxidase, Compound I is characterized by a maximum at 605 nm, Compound II at 578 nm, and Compound III by double peaks at around 600 and 580 nm. In the Soret region, Compound I shows a peak at 435 nm and a trough at 412 nm, Compound III exhibits a peak at 442 to 443 nm and a trough at 418 nm. In the absence of cytochrome c, the spontaneous decay of Compound I precedes that of Compound II; the first order rate constants have been found to be 4 X 10(-3) s(-1) and 8 X 10(-4) s(-1) for Compounds I and II, respectively. Compound III, however, does not revert back to the oxidized form even after several hours. The decay of Compound I is accelerated in the presence of ferrocytochrome c by a factor of 10(3) to 10(4) depending on the concentration of the latter. The time for sequential differentiation between Compound I and Compound II becomes less clear in the presence than in the absence of ferrocytochrome c. On the contrary ferricytochrome c does not show such an accelerating effect. These and other observations lead us to postulate Compound I as an active intermediate, the true oxygenated compound in the cytocchrome oxidase reaction.


Platelet cyclic 3':5'-nucleotide phosphodiesterase released by thrombin and calcium ionophore. Contact of rat platelets with thrombin or the divalent cation ionophore A-23187, in the presence of extracellular calcium, resulted in the secretion of adenosine 3':5'-monophosphate (cyclic AMP) and guanosine 3':5'-monophosphate (cyclic GMP) phosphodiesterases. Significant association of calcium with platelets occurred during platelet surface contact with thrombin. Thrombin concentration to induce association of calcium virtually agreed with that to release the enzyme. The finding that A-23187 (5 to 20 muM) also provoked a rapid and marked association of extracellular calcium with platelets suggests that calcium mobilization into the intracellular environment may account, at least in part, for this association between platelet and calcium. Two different phosphodiesterases, a relatively specific cyclic AMP and a relatively specific cyclic GMP phosphodiesterase were secreted from platelets into the plasma in soluble form. The amounts of the phosphodiesterases secreted were dose- or time-dependent on thrombin (0.1 to 2 units) or A-23187 (5 to 20 muM) within 30 min. The enzyme release by thrombin was completely inhibited by heparin but the release by A-23187 was not. The two phosphodiesterases secreted seemed to correspond to the two enzymes isolated from platelet homogenates in many respects. Rat platelets contained, at least, three cyclic 3':5'-nucleotide phosphodiesterases, namely, two relatively specific cyclic AMP phoshodiesterases and a relatively specific cyclic GMP phosphodiesterase which were clearly separated from each other by Sepharose 6B or DEAE-cellulose column chromatography or sucrose gradient centrifugation. The two platelet cyclic AMP phosphodiesterase (Mr = 180,000 and 280,000) had similar apparent Km values of 0.69 and 0.75 muM with different sedimentation coefficient values of 4.9 S and 7.1 S, respectively. They did not hydrolyze cyclic GMP significantly. A cyclic GMP phosphodiesterase (Mr - 260,000) exhibited abnormal kinetics for cyclic GMP with an apparent Km value of 1.5 muM and normal kinetics for cyclic AMP with a Km of 300 muM. The properties of a platelet cyclic AMP phosphodiesterase (Mr = 180,000) and a platelet cyclic GMP phosphodiesterase were found to agree with those of the two phosphodiesterases released from platelets by thrombin or A-23187. Depletion of extracellular calcium by an addition of citrate, EDTA, or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) to the blood or platelet suspension resulted in a loss of the activity of the smaller form of platelet cyclic AMP phosphodiesterase (Mr = 180,000) and addition of calcium restored the activity of this cyclic AMP phosphodiesterase. Thus, calcium seemed to be involved in the mechanism of an occurrence of this smaller form of cyclic AMP phosphodiesterase as well as the secretion of this enzyme. Contact of human platelets with thrombin also resulted in the secretion of cyclic nucleotide phosphodiesterase which was dependent on the concentration of calcium. No species difference was observed in this respect.


Mechanism of self-phosphorylation of adenosine 3':5'-monophosphate-dependent protein kinase from bovine cardiac muscle. The adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase purified from bovine cardiac muscle catalyzes the transfer of up to 2 mol of 32P from [lambda-32P]ATP to seryl residues in its cyclic nucleotide-binding protein component (Erlichman, J., Rosenfeld, R., and Rosen, O. M. (1974) J. Biol. Chem. 249, 5000-5003). We now present three lines of evidence to support our conclusions that the undissociated holoenzyme does not catalyze the phosphorylation of exogenous substrates but can undergo self-phosphorylation by an intramolecular reaction: (a) addition of either cAMP-binding protein or the protein kinase inhibitor (Walsh, D. A., Ashby C. D., Gonzales, C., Calkins, D., Fischer, E. H., and Krebs, D. G. (1971) J. Biol. Chem. 241, 1977-1985) does not inhibit self-phosphorylation as it does phosphorylation of exogenous substrates in the presence or absence of cAMP; (b) addition of catalytic subunit to an excess of cyclic nucleotide-binding protein results in phosphorylation equivalent to the amount of holoenzyme so generated; (c) the rate of self-phosphorylation is not affected by dilution of the holoenzyme.


Effect of Ca2+ binding on troponin C. Changes in spin label mobility, extrinsic fluorescence, and sulfhydryl reactivity. The Ca2+ binding component (TnC) of troponin has been selectively labeled with either a spin label, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetamide, or with a fluorescent probe, S-mercuric-N-dansyl cysteine, presumably at its single cysteine residue (Cys-98) in order to probe the interactions of TnC with divalent metals and with other subunits of troponin. The modified protein has the same Ca2+ binding properties as native TnC (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628), viz. two Ca2+ binding sites at which Mg2+ appears to compete (Ca2+-Mg2+ sites, KCa = 2 X 10(7) M-1) and two sites at which Mg2+ does not compete (Ca2+-specific sites, KCa = 2 X 10(5) M-1). Either Ca2+ or Mg2+ alters the ESR spectrum of spin-labeled TnC in a manner that indicates a decrease in the mobility of the label, Ca2+ having a slightly greater effect. In systems containing both Ca2+ and Mg2+ the mobility of the spin label is identical with that in systems containing Ca2+ alone. The binding constants for Ca2+ and Mg2+ deduced from ESR spectral changes are 10(7) and 10(3) M-1, respectively, and the apparent affinity for Ca2+ decreases by about an order of magnitude on adding 2 mM Mg2+. Thus, the ESR spectral change is associated with binding of Ca2+ to one or both of the Ca2+-Mg2+ sites. Addition of Ca2+ to the binary complexes of spin-labeled TnC with either troponin T (TnT) or troponin I (TnI) produces greater reduction in the mobility of the spin label than in the case of spin-labeled TnC alone, and in the case of the complex with TnI the affinity for Ca2+ is increased by an order of magnitude. The fluorescence of dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-labeled TnC is enhanced by Ca2+ binding to both high and low affinity sites with apparent binding constants of 2.6 X 10(7) M-1 and 2.9 X 10(5) M-1, respectively, calculated from the transition midpoints. The presence of 2 mM Mg2+, which produces no effect on dansyl fluorescence itself, in contrast to its effect on the spin label, shifts the high affinity constant to 2 X 10(6) M-1. Spectral changes produced by Ca2+ binding to the TnC-TnI complex furnish evidence that the affinity of TnC for Ca2+ is increased in the complex. The reactivity of Cys-98 to the labels and to 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) is decreased by Ca2+ or Mg2+ both with native TnC and in 6 M urea. The reaction rate between Cys-98 and Nbs2 decreases to one-half the maximal value at a Ca2+ concentration that suggests binding to the Ca2+-Mg2+ sites. Formation of a binary complex between TnI and TnC reduces the rate of reaction, and there is a further reduction by Ca2+. The effect of Ca2+ takes place at concentrations that are 1 order of magnitude lower than in the case of TnC alone. These results suggest that the Ca2+ binding site adjacent to Cys-98 is one of the Ca2+-Mg2+ binding sites.


Agonist-specific refractoriness induced by isoproterenol. Studies with mutant cells. The beta-adrenergic catecholamine isoproterenol produces a large, rapid, but often a transient, elevation in cellular content of cyclic AMP. We have used the S49 mouse lymphoma cell line, in which genetic variants with specific defects in the pathway of cyclic AMP generation and function have been isolated, to study the increase and subsequent decrease in cyclic AMP levels (termed refractoriness) following incubation of cells with isoproterenol. In wild type S49 cells, isoproterenol produces a peak response in the cellular content of cyclic AMP within 30 min, but the cyclic AMP level falls rapidly thereafter, approaching basal levels by 6 h. Neither inactivation of the drug nor secretion of a nonspecific inhibitor of adenylate cyclase appears to account for the refractoriness. Because isoproterenol refractory cells can still be stimulated by cholera toxin, refractoriness to isoproterenol does not represent a generalized decrease in cellular cyclic AMP response. Particulate preparations from refractory cells have a selective loss of isoproterenol-responsive adenylate cyclase activity, but their activation constants and stereoselectivity for (-)- and (+)-isoproterenol are unaltered. In addition, refractory cells have decreased specific binding of the beta-adrenergic antagonist [125I]iodohydroxybenzylpindolol. This decrease appears to represent a reduction in the number, but not the affinity, of beta-adrenergic receptor sites. Similar studies in an S49 clone that lacks the enzyme cyclic AMP-dependent protein kinase yield essentially identical findings. Because kinase-deficient cells do not induce the cyclic AMP-degrading enzyme phosphodiesterase after the cellular content of cyclic AMP is increased, induced of phosphodiesterase cannot account for refractoriness to isoproterenol. Cyclic AMP-dependent protein kinase does not appear to be required for either the decrease in beta-adrenergic receptors and isoproterenol-responsive adenylate cyclase, nor does it appear to be required for the development of refractoriness to isoproterenol. In contrast, an S49 clone lacking hormone-responsive adenylate cyclase activity but retaining beta-adrenergic receptors does not appear to lose receptors after being incubated with isoproterenol, either alone or together with dibutyryl cyclic AMP. Therefore, in this clone, receptor occupancy alone or in combination with elevated cyclic AMP levels is insufficient to cause refractoriness. Refractoriness thus appears to require intact adenylate cyclase. This suggests that adenylate cyclase may exert regulatory controls on beta-adrenergic receptors in addition to generation of cyclic AMP.


Spectral evidence for a flavin adduct in a monoalkylated derivative of pig heart lipoamide dehydrogenase. A derivative of the flavoprotein pig heart lipoamide dehydrogenase has been described recently (Thorpe, C., and Williams, C.H. (1976) J. Biol. Chem. 251, 3553-3557), in which 1 of the 2 cysteine residues generated on reduction of the intrachain active center disulfide bridge is selectively alkylated with iodoacetamide. This monolabeled enzyme exhibits a spectrum of oxidized bound flavin. The addition of 1 mM NAD+ to this derivative at pH 8.3 causes a decrease in absorbance of approximately 50% at 448 nm, with a concomitant increase at 380 nm. These spectral changes are complete within 3 ms and are reversible. NAD+ titrations generate isosbestic points at 408, 374, and 327 nm; allowing values for the apparent dissociation constant for NAD+ and the extent of bleaching at infinite ligand to be obtained from double reciprocal plots. Between pH 6.1 and 8.8, the apparent KD decreases from 320 to 35 muM, whereas the extrapolated delta epsilon 448 values remain approximately constant at 1/2 epsilon 448. Direct measurement of NAD+ binding by gel filtration at pH 8.8 indicates that the spectral changes are associated with a stoichiometry of 1.2 mol of NAD+ bound/2 mol of FAD. The modified protein is a dimer containing 1 FAD and 1 alkylated cysteine residue/subunit; the native enzyme is also dimeric. The visible spectrum of the species absorbing at 380 nm, approximated by correction for the residual oxidized FAD, shows a single maximum at 384 nm, epsilon 384 = 8.7 mM-1cm-1. Comparison of this spectrum with that of model compounds of known structure suggests that it may represent a reversible covalent flavin adduct induced on binding NAD+.


Gonadotropin-induced loss of hormone receptors and desensitization of adenylate cyclase in the ovary. Gonadotropin receptor sites and adenylate cyclase activity were analyzed in luteinized rat ovaries following injection of human chorionic gonadotropin (hCG). Gonadotropin binding capacity and hormonal stimulation of adenylate cyclase declined rapidly to a minimum at 6 to 12 h, remained depressed for 4 days, and returned to the control level between 5 and 7 days. Total adenylate cyclase activity measured in the presence of fluoride fell by 50% within a few hours but returned to normal by 24 h. A close correlation was observed between the number of gonadotropin receptors and the ability of adenylate cyclase to be stimulated by hormone. Assay of tissue-bound hormone showed that the initial loss of hormone sensitivity and binding capacity was associated with occupancy of luteinizing hormone receptor sites, but that the prolonged changes in these activities were not attributable to receptor occupancy. These studies have demonstrated that induction of a refractory or desensitized state in ovarian adenylate cyclase by gonadotropin results from the loss of specific hormone receptor sites.


Purification and properties of an endonuclease from nuclei of uninfected and polyoma-infected 3T3 cells. An endonuclease activity has been purified approximately 800-fold from nuclei of 3T3 cells infected with polyoma virus. The purfied enzyme catalyzes an endonucleoytic cleavage of single- and double-stranded DNA and single-stranded RNA. Evidence that the activity towards these substrates resides in the same protein molecule is provided by the finding that they co-sediment in sucrose gradients and have identical rates of heat inactivation. Studies on the DNase activity shows that the rate of hydrolysis of single-stranded T7 DNA is 100-fold greater than that for double-stranded T7 DNA. Single-stranded DNA is extensively hydrolyzed to low molecular weight acid-insoluble products. With duplex DNA as substrate, only a limited number of single strand breaks are introduced. A limit digest with polyoma DNA (component I) as substrate results in the introduction of four breaks per strand. The phosphdiester bond interruptions can be repaired by polynucleotide ligase. Approximately 80% of the 5' termini present at the point of phosphodiester bond cleavage are purine nucleotides. Additional studies have demonstrated that a similar endonuclease is present in nuclei of uninfected cells and that this enzyme purified 400-fold has catalytic properties identical with those of the endonuclease from infected cells.


Hormonal control of [14C]glucose synthesis from [U-14C]dihydroxyacetone and glycerol in isolated rat hepatocytes. The hormonal control of [14C]glucose synthesis from [U-14C-A1dihydroxyacetone was studied in hepatocytes from fed and starved rats. In cells from fed rats, glucagon lowered the concentration of substrate giving half-half-maximal rates of incorporation while it had little or no effect on the maximal rate. Inhibitors of gluconeogenesis from pyruvate had no effect on the ability of the hormone to stimulate the synthesis of [14C]glucose from dihydroxyacetone. The concentrations of glucagon and epinephrine giving half-maximal stimulation from dihydroxacetone were 0.3 to 0.4 mM and 0.3 to 0.5 muM, respectively. The meaximal catecholamine stimulation was much less than the maximal stimulation by glucagon and was mediated largely by the alpha receptor. Insulin had no effect on the basal rate of [14C]clucose synthesis but inhibited the effect of submaximal concentration of glucagon or of any concentration of catecholamine. Glucagon had no effect on the uptake of dihydroxyacetone but suppressed its conversion to lactate and pyruvate. This suppression accounted for most of the increase in glucose synthesis. In cells from gasted rats, where lactate production is greatly reduced and the rate of glucose synthesis is elevated, glucagon did not stimulate gluconeogenesis from dihydroxyacetone. Findings with glycerol as substrate were similar to those with dihyroxyacetone. Ethanol also stimulated glucose production from dihydroxyacetone while reducing proportionately the production of lactate. Ethanol  is known to generate reducing equivalents fro clyceraldehyde-3-phosphate dehydrogenase and presumably thereby inhibits carbon flux to lactate at this site. Its effect was additive with that of glucagon. Estimates of the steady state levels of intermediary metabolites and flux rates suggested that glucagon activated conversion of fructose diphosphate to fructose 6-phosphate and suppressed conversion of phosphoenolpyruvate to pyruvate. More direct evidence for an inhibition of pyruvate kinase was the observation that brief exposure of cells to glucagon caused up to 70% inhibition of the enzyme activity in homogenates of these cells. The inhibition was not seen when the enzyme was assayed with 20 muM fructose diphosphate. The effect of glucagon to lower fructose diphosphate levels in intact cells may promote the inhibition of pyruvate kinase. The inhibition of pyruvate kinase may reduce recycling in the pathway of gluconeogenesis from major physiological substrates and probably accounts fromsome but not all the stimulatory effect of glucagon.


Endgoenous protein kinase in outer plasma membrane of cultured 3T3 cells. Nature of the membrane-bound substrate and effect of cell density, serum addition, and oncogenic transformation. Endogenous protein kinase activity was detected in the outer plasma membrane of 373 and SV40 transformed 3T3 cells. When intact cells were incubated with [gamma-32P]ATP, there was a transfer of [32P]phosphate into an acid-insoluble product. The reaction was: (a) linear as a function of time (up to 30 min), (b) proportional to the number of cells present and (c) dependent on temperature and Mg2+ concentration. The acid-insoluble product was susceptible to pronase but not RNase or DNase. More specifically, phosphomonoester bonds to serine and threonine were identified. There was less than 3% hydrolysis of the [gamma-32P]ATP during the reaction; moreover, free [32P]phosphate failed to substitute for the ATP. The reaction product was located on the cell surface, as evidenced by the fact that it could be removed by mild trypsin treatment of intact 3T3 cells. Further evidence for the surface location of the kinase was shown by its activity in phosphorlating exogenous substrate, histone, and phosvitin. The level of phosphorylation increased by 2- to 4-fold prior to the start of S phase when quiescent 3T3 cells were stimulated to reinitiate growth by the addition of serum. The SV40 3T3 cells had from 5- to 10-fold more activity per cell than the quiescent 3T3 cells. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioautography show at least 25 phosphorylated proteins; the surface label pattern of 3T3 cells differs from that of SV40-transformed 3T3 cells.


Electronic state of heme in cytochrome oxidase. I. Magnetic circular dichroism of the isolated enzyme and its derivatives. Magnetic circular dichroism (MCD) spectra have been recorded for beef heart cytochrome oxidase and a number of its inhibitor complexes. The resting enzyme exhibits a derivate shape Faraday C term in the Soret region, characteristic of low spin ferric heme, which accounts for 50% of the total oxidase heme a. The remaining heme a (50%) is assigned to the high spin state. MCD temperature studies, comparison with the MCD spectra of heme a-imidazole model compounds, and ligand binding (cyanide, formate) studies are consistent with these spin state assignments in the oxidized enzyme. Furthermore, the ligand binding properties and correlations between optical and MCD parameters indicate that in the resting enzyme the low spin heme a is due solely to cytochrome a3+ and the high spin heme a to cytochrome a33+. The Soret MCD of the reduced protein is interpreted as th sum of two MCD curves: an intense, asymmetric MCD band very similar to that exhibited by deoxymyoglobin which we assign to paramagnetic high spin cytochrome a3(2+) and a weaker, more symmetric MCD contribution, which is attributed to diamagnetic low spin cytochrome a2+. Temperature studies of the Soret MCD intensity support this proposed spin state heterogeneity. Ligand binding (CO, CN-) to the reduced protein eliminates the intense MCD associated with high spin cytochrome a3(2+); however, the band associated with cytochrome a2+ is observed under these conditions as well as in a number of inhibitor complexes (cyanide, formate, sulfide, azide) of the partially reduced protein. The MCD spectra of oxidized, reduced, and inhibitor-complexed cytochrome oxidase show no evidence for heme-heme interaction via spectral parameters. This conclusion is used in conjunction with the fact that ferric, high spin heme exhibits weak MCD intensity to calculate the MCD spectra for the individual cytochromes of the oxidase as well as the spectra for some inhibitor complexes of cytochrome a3. The results are most simply interpreted using the model we have recently proposed to account for the electronic and magnetic properties of cytochrome (Palmer, G., Babcock, F.T., and Vcikery, L.E. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2206-2210).


Steady state and exchange kinetics of pyruvate, phosphate dikinase from Propionibacterium shermanii. Evidence is presented based on requirements for exchange in the partial reactions, initial velocity and exchange kinetics and product inhibition, that the pyruvate, phosphate dikinase reaction of propionibacteria occurs by a nonclassical Tri Uni Uni Ping Pong mechanism. The mechanism involves a pyrophosphoryl enzyme, a phosphoryl enzyme, and the free enzyme, and three functionally distinct and independent substrate sites. On the first site, there is pyrophosphorylation of the enzyme by ATP with subsequent release of AMP. The pyrophosphoryl moiety then reacts at the second site with Pi yielding the product PPi and the phosphoryl from of the enzyme. At the third site pyruvate is phosphorylated yielding P-enolpyruvate and the free enzyme. The three catalytic sites are proposed to be linked by a histidyl residue which functions as a pyrophosphoyrl- and phosphoryl-carrier between the three sites. This proposal is based on the following observations. (A) The patterns of the double reciprocal plots of the initial velocities were all parallel; (b) product inhibition between each pair of substrates and products of the three partial reactions were competitive, i.e. ATP against AMP, Pi against PPi, and pyruvate against P-enolpyruvate; (c) the other product inhibitions, with one exception, were noncompetitive as required by the nonclassical ping-pong mechanism; (d) ATP or P-enolpyruvate was required for the Pi in equilibrium PPi exchange reaction which is in accord with the participation of a pyrosphosphoryl or phosphoryl form of the enzyme in this exchange; (e) the ATP in equilibrium AMP exchange and pyruvate in equilibrium P-enolpyruvate exchange did not require additional substrates. In addition, the inhibition and participation in the exchange reactions of the alpha,beta and beta,gamma-methylene analogues of ATP and of the methylene analogue of inorganic pyrophosphate were investigated and the results were in accord with the proposed mechanism. The combined evidence provides a well documented example of a three site nonclassical Tri Uni Uni Ping Pong mechanism.


Properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450. Procedures are described for the isolation of two forms of rabbit liver microsomal liver microsomal cytochrome P-450 (P-450LM) in homogeneous state. They are designated by their relative electrophoretic mobilities on polyacrylamide gel in the presence of sodium dodecyl sulfate as P-450LM2 and P-450LM4. P-450LM2, which was isolated from phenobarbital-induced animals, has a subunit molecular weight of 48,700. The best preparations contain 20 nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. P-450LM4, which is induced by beta-naphthoflavone but is also present in phenobarbital-induced and untreated animals, was isolated from all three sources and found to have a subunit molecular weight of 55,300. The best preparations contain 17nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. Some of the purified preparations of the cytochromes, although electrophoretically homogeneous, contain apoenzyme due to heme loss during purification. The purified proteins contain no detectable NADPH-cytochrome P-450 reductase, cytochrome b5, or NADH-cytochrome b5 reductase, and only low levels of phospholipid (about 1 molecule per subunit). Amino acid analysis indicated that P-450LM2 and P-450LM4 are similar in composition, but the latter protein has about 60 additional residues. The COOH-terminal amino acid of P-450LM2 is arginine, as shown by carboxypeptidase treatment, whereas that of P-450LM4 is lysine. NH2-terminal amino acid residues could not be detected. Carbohydrate analysis indicated that both cytochromes contain 1 residue of glucosamine and 2 of mannose per polypeptide subunit. The optical spectra of the oxidized and reduced cytochromes and carbon monoxide complexes were determined. Oxidized P-450LM2 has maxima at 568, 535, and 418 nm characteristic of a low spin hemeprotein, and P450LM4 from beta-naphthoflavone-induced, phenobarbital-induced, or control microsomes has maxima at 645 and 394 nm, characteristic of the high spin state. The spectrum of -450lm4 becomes similar to that of P-450LM2 at high protein concentrations or upon the addition of detergent (Renex), whereas the spectrum of P-450LM2 is unaffected by the protein concentration or the presence of detergent. Electron paramagnetic resonance spectrometry of the purified cytochromes indicated that oxidized -450lm2 is in the low spin state, whereas P-450LM4 is largely, but not entirely, in the high spin state.


Solubilization of adenylate cyclase from normal and Rous sarcoma-transformed chicken embryo fibroblasts. Adenylate cyclase activities in membranes prepared from Rous sarcoma-transformed chicken embryo fibroblasts are 2 to 4 times lower than in membranes prepared from normal chicken embryo fibroblasts. Adenylate cyclase activities were solubilized from normal and transformed membranes with five different nonionic detergents. In all cases, the specific activities of the enzyme solubilized from normal and transformed preparations were essentially identical. These data suggest that the microenvironment of adenylate cyclase in transformed membranes may be wholly or partially responsible for the decreased activities of this enzyme.


Stopped flow kinetics of pyrene transfer between human high density lipoproteins. The transfer of pyrene between high density lipoproteins was studied as a model of lipid exchange. When high density lipoprotein containing pyrene was mixed with unlabeled lipoprotein, pyrene excimer fluorescence decreased with a half-time of approximately 3 ms. The rate of pyrene transfer was invariant over a 100-fold range of unlabeled lipoprotein concentrations. Since a decrease in excimer fluorescence indicates a decrease in the microscopic concentrations of pyrene, the observed fluorescence change relfects pyrene transfer to unlabeled lipoproteins, and, therefore, dilution of the pyrene molecules. When high density lipoprotein labeled with pyrene was rapidly diluted 1:14 into buffer, a small decrease in excimer fluorescence was observed.  The half-time of this fluorescence change was also about 3 ms and represents the half-time for the dissociation of pyrene from high density lipoprotein into water. The latter observation, coupled with the invariant exchange rate with lipoprotein concentration suggests strongly that the limiting step in the transfer of pyrene between high density lipoproteins is the dissociation of pyrene into solvent. Finally, regardless of mechanism, the exchange of pyrene, and presumably other hydrophobic aromatic compounds, among serum high density lipoproteins is extremely fast. This result indicates that these types of compounds can be rapidly assimilated and transported through the body by plasma lipoproteins.


Ultrastructural and biochemical evidence for a steroid-containing secretory organelle in the perfused cat adrenal gland. A correlative study of the ultrastructural and biochemical effects of ACTH on fasciculata cells was carried out on the isolated cat adrenal gland perfused in situ with Locke's solution. The outstanding morphologic feature of cortical cells exposed to microunit ACTH concentrations for 40 min was the abundance of electron-dense granules (0.2-0.4 mum). These organelles were observed in small groups in close proximity to the Golgi region and to the cell membrane. Morphometric and biochemical analysis of control and ACTH-treated glands demonstrated that ACTH stimulation was associated with a fourfold increase in the number of these granules and a comparable increase in the corticosteroid content of the gland. By contrast, ACTH failed to augment cortical lysosomal enzyme activity. These findings, which link steroid release to the appearance of intracellular granules, extend further the parallels between the mechanism of release of newly synthesized steroid and the release of preformed hormones stored in secretory organelles. These results also lend support to the concept that a process related to exocytosis may be the underlying mechanism for extruding steroid from the cortical cell.


Intranuclear membranous inclusions in oocytes of a viviparous teleost (Xiphophorus helleri). Intranuclear inclusions were observed in oocytes of Xiphophorus helleri during prophase I. In osmium-fixed leptotene nuclei, the inclusions were made up of groups of membrane-limited vesicles or tubules with pale contents, situated near the inner nuclear membrane with which some of them exhibited apparent continuities. In zygotene nuclei, larger vesicles also appeared bounded by two or three membranes and containing tubules apparently invaginated from their walls. In pachytene-dictyate nuclei most vesicular bodies had a wall formed by stratified membranes, or were entirely made up of membranous whorls. In glutaraldehyde-osmium fixed material some of these myeline-like bodies showed a peculiar arrangement, consisting of concentric bands each containing thick inner dense lamellae 2-0-3-0 nm thick and a 5-0 nm outer lamella. It is suggested that these inclusion bodies arise from the inner nuclear membrane of oocytes when cells start to grow intensely during prophase I. The bodies seem to become more complex at late prophase, probably by association of individual vesicles and the occurrence of multiple membrane invaginations, which may be related to active metabolic phenomena taking place at this stage in oocytes.


Comparison of batch and chromatographic assays of cyclic nucleiotide phosphodiesterases. An improved method for the assay of cyclic nucleotide phosphodiesterases (CNPases) is described in which residual 3H-cAMP or 3H-cGMP is separated from products by chromatography on thin layers of PEI-cellulose. Comparison of CNPase activity in crude or purified kidney enzyme assayed by the PEI-cellulose method with a batch anion-exchange resin method showed serious underestimation of activity by the latter method. The batch method underestimated CNPase activity by 34-82%. The error was due to substantial binding of nucleosides (adenosine and guanosine) to the resin, which is assumed not to occur by those using this method.


Preparative high-performance liquid chromatography applied to peptide synthesis. A variety of protected synthetic peptides were purified by high-performance liquid chromatography on pre-packed silica gel columns. The compounds varied in protecting groups, amino acid composition and molecular weight. Flow-rates of two to four column volumes per hour were employed, with resultant back pressure of less than 150 p.s.i. A typical column load of 0.2-5 g was purified in 2-5 h.


Liquid chromatography of neohesperidin dihydrochalcone. The determination of a number of impurities in neohesperidin dihydrochalcone is described, as well as the determination of neohesperidin dihydrochalcone in food products. A reversed-phase chromatographic system is used, with octadecyltrichloro-silane-treated silica gel as chemically bonded stationary phase and methanol-water as mobile phase; a detailed procedure for preparing the packing material is given, and the dependence of the amount of bonded stationary phase on the humidity of the silica gel before the bonding reaction is shown. The glass column was packed at 350 atm in a steel pressure vessel, with use of a slurry-packing technique; details are given of the pressure vessel and of the packing method. Chromatograms are reproduced to show the performance of such columns in this type of analysis.


Presence of H-Y antigen and testis in 46, XX true hermaphroditism, evidence for Y-chromosomal function. Endocrinologic and serologic studies of a 2-year-old child with the chromosomal complement 46,XX and ambiguous genitalia suggested the preoperative diagnosis of true hermaphroditism. Urinary and serum androgen production in response to human chorionic gonadotrophin was in the range expected for normal males, implying presence of cryptic testicular tissue. Moreover, detection of H-Y antigen, a cell surface component associated with testicular differentiation and coded or regulated by a Y-chromosomal gene, indicated presence of Y-chromosomal material. The diagnosis of true hermaphroditism was confirmed at surgery. Assuming a constant association of H-Y antigen and testicular differentiation is established, human H-Y serology may be an important adjunct to the endocrinologic evaluation of intersex patients. Our studies support the interpretation that a Y-chromosomal translocation too small for cytologic detection accounts for testicular differentiation in 46,XX true hermaphroditism. Expression of H-Y antigen remained positive after castration.


Evidence for an unidentified ACTH-induced steroid hormone causing hypertension. The hypothesis that hyperaldosteronism is not the sole cause of hypertension in dexamethasone-suppressible hyperaldosteronsim was tested in an 18-year-old male. After six years of little or no treatment, the hypertension and mild hyperaldosteronism were promptly decreased by a small dose of dexamethasone. During dexamethasone treatment, when aldosterone secretion was suppressed to less than normal and he was normotensive, steroids were given by constant infusion in an attempt to reproduce the hypertension of the dexamethasone-free state. Neither five days of aldosterone or 18-hydroxydesoxycorticosterone (18-OH-DOC) at 1 mg/day, nor desoxycorticosterone (DOC) at 30 mg/day caused hypertension. However, sodium retention and potassium loss was observed during aldosterone and DOC infusion. Hypertension was produced within five days during infusion with ACTH or oral metyrapone. The hypertensive effect of the latter was abolished by addition of aminoglutethimide treatment. These studies suggest that a steroid other than aldosterone, 18-OH-DOC, or DOC may be the cause of the ACTH-induced hypertension in this patient. The aminoglutethimide data suggest that the ACTH effect on blood pressure is due to a steroid, and the metyrapone studies suggest that the steroid may be an 11-desoxysteroid. Urine and blood collected under ACTH stimulation and metyrapone treatment may be a rich source from which we may characterize this hormone.


Nonautonomous function of a pancreatic insulinoma. A 56-year-old woman with symptoms of weakness, visual blurring, and sweating underwent diagnostic studies to evaluate the etiology of her hypoglycemia. Fasting hypoglycemia was never documented; in diagnostic studies performed during her two hospitalizations and several outpatient glucose tolerance tests, the lowest fasting plasma glucose recorded was 56 mg/dl. The patient displayed exaggerated plasma insulin responses following oral glucose (peak response: 447 muU/ml at 30 min) and following 1 gm of iv tolbutamide (peak response: 719 muU/ml at 5 min) with symptomatic profound hypoglyceria during both tests. Basal per cent proinsulin was elevated at 49% (normal range 5-22%). Throughout a 72 h fast, values for plasma glucose, insulin, and glucose/insulin ratios were all within the normal range. During the infusion of exogenous insulin (0.1 U/kg for 60 min) serum C-peptide reactivity suppressed to less than 1.3 ng/ml when the plasma glucose fell below 40 mg/dl representing normal suppression. At surgery, a pancreatic beta cell adenoma was found and removed. This patient represents the uncommon circumstances in which stimulation tests with tolbutamide and glucose were more helpful in establishing a preoperative diagnosis than were the suppression tests.


Urinary cyclic nucleotide levels in patients with hyper- and hypothyroidism. Urinary cyclic AMP, cyclic GMP and creatinine excretion were measured in 38 patients with hyperthyroidism, 32 patients with hypothyroidism and in 57 normal subjects. The excretion of both cyclic nucleotides was significantly increased in hyperthyroid females, but not in hyperthyroid males. The cyclic nucleotides/creatinine ratios, however, were uniformly elevated in both male and female hyperthyroid subjects and this was due, in part, to decreased creatinine excretion. Cyclic AMP excretion was significantly decreased in the hypothyroid subjects, but the cyclic AMP/creatinine ratios were not significantly different from normal. There were no significant alterations in cyclic GMP and cyclic GMP/creatinine ratios in the male hypothyroid patients, but ratios in the female patients were slightly greater than in the normals. These results demonstrate that hyper- and hypothyroidism may be associated with appreciable alterations in urinary cyclic nucleotide levels and that there may be sex-related differences in the patterns of urinary excretion of these nucleotides. The cyclic nucleotide levels herein described in patients with hyper- and hypothyroidism are qualitatively and, in some instances, quantitatively similar to those found in patients with hyper- and hypoparathyroidism, respectively.


Lithium does not inhibit the parathyroid hormone-mediated rise in urinary cyclic AMP and phosphate in humans. To test the hypothesis that lithium is a general inhibitor of hormone-activated adenylate cyclase, we infuse parathyroid hormone (PTH) into human subjects prior to and during lithium carbonate administration. PTH infusion caused a significant increase in urinary cyclic AMP and urinary phosphate excretion. There was no significant difference in these responses in the lithium compared to the control period. In four patients with primary hyperparathyroidism, lithium had no significant effect on serum calcium or phosphate or on tubular reabsorption of phosphate. The data do not substantiate the hypothesis that lithium (at therapeutic concentrations) is a general inhibitor of hormonally-activated adenylate cyclase, nor do they support its potential clinical utility in primary hyperparthyroidism.


A low molecular weight substance obtained from serum which has luteinizing hormone like activity ("mini LH"). A low molecular weight substance of 1000-2000 daltons has been isolated in dialysates of normal human serum which, on testing in a radio-immunoassay, a radioligand receptorassay and an in-vitro bioassay exhibited LH activity comparable to the range of LH activity found in normal serum. The substance was heat labile. Its reactivity in a RIA suggests peptide nature. No "mini LH" activity could be derived from LH-free serum samples of hypophysectomized patients.


Improved rubella hemagglutination inhibtion test: inactivation of non-immunoglobulin hemagglutination inhibitors by phospholipase C. A method using phospholipase C (PL-C) for removing nonspecific inhibitors (NSI) of rubella virus hemagglutinin is described. PL-C was found to hydrolyze NSI without altering the hemagglutination inhibition (HI) activity of the specific antibody and could be used to remove NSI in the rubella HI test by using formalinized erythrocytes, which resisted the enzymatic action; fresh erythrocytes were lysed by PL-C. The HI test using PL-C treated sera gave true measurements of actual rubella antibody content, and HI titers of PL-C treated sera were identical or equivalent (+/-1 dilution) to those of sera treated with dextran sulfate and CaCl2 (DS-C). Thus, the PL-C method gave results as reproducible and reliable as the DS-C method and was more convenient.