Purification and characterization of a glycosyltransferase complex from the culture broth of Streptococcus mutans FA1. The extracellular glycosyltransferases from Streptococcus mutans FA1 were purified by using the following procedures: ammonium sulfate precipitation, poly-(acrylamide) gel filtration, DEAE-cellulose chromatography, and agarose-gel filtration. The dextransucrase and levansucrase activities were purified 350- and 500-fold, repsectively, and the ratio of the two activities remained almost constant throughout the purification. Both enzymes have a pH optimum of 6.0, a Km for sucrose of 55mM, and isoelectric points of 3.7 and 4.6. The enzymes are inactivated by repeated freezing and thawing, but retain partial activity even after heating at 100 degrees. The enzyme preparation contains a carbohydrate moiety which does not appear to be either bound levan or dextran.


Neural stimulation of mitotic activity in the crypts of lieberkühn in rat jejunum. The influence of nerve stimulation and sham-stimulation on the mitotic rate in epithelial cells lining the crypts of Lieberkühn in the jejunum of anaesthetized rats was studied. Administration of the anaesthetic and opening the abdominal cavity was without significant effect on the crypt cell mitotic rate. However, externalizing a loop of jejunum and applying sham-stimuli to its mesenteric nerves resulted in a significant decrease in the crypt cell mitotic rate in that loop. Application of electrical stimuli to the mesenteric nerves of another externalized jejunal loop resulted in a significant increase in the mitotic rate in the crypt cells of that segment. Similar acceleration of crypt cell proliferation by electrical stimuli applied to mesenteric nerves was also seen in chemically sympathectomized rats.


Toxicological implications of the mixed-function oxidase catalyzed metabolism of carbon disulfide. The results of these studies have indicated that the decrease in the activity of the hepatic mixed-function oxidase enzyme system and the concentration of cytochrome P-450 seen on incubation of carbon disulfide (CS2) with rat liver microsomes in the presence of NADPH is the result of the binding of the sulfur atom released in the mixed-function oxidase catalyzed metabolism of CS2 to carbonyl sulfide (COS). Moreover, it appears that COS is further metabolized by the mixed-function oxidase enzyme system to CO2 and that, analogous to the metabolism of CS2 to COS, the sulfur atom released in this reaction also binds to the microsomes and inhibits benzphetamine metabolism and decreases the concentration of cytochrome P-450 detectable as its carbon monoxide complex. The results of these studies also suggest that the decrease in the concentration of cytochrome P-450 and the liver damage seen on in vivo administration of CS2 to phenobarbital pretreated rats, is due to the mixed-function oxidase catalyzed release and binding of the sulfur atoms of CS2. The decrease in the concentration of cytochrome P-450 seen on incubation of CS2 with rat liver microsomes in the presence of NADPH  does not appear to be the result of destruction of the heme group or its dissociation from the apoenzyme since the total amount of protoheme is unchanged in microsomes which have been incubated with CS2 and NADPH as compared to those not incubated with these compounds.


The assay of pyruvate kinase activity in gastric mucosa. Pyruvate kinase activity in gastric mucosal supernatant preparations shows large responses to exogenous effectors. At pH 7.5, fructose 1,6-diphosphate can cause large stimulations in activity. Alanine inhibits the reaction but this effect is partially reversed by fructose 1,6-diphosphate. In the absence of these compounds, 4.0-5.0 mM phosphoenolpyruvate (PEP) is required to generate maximal activity in the assay system used. Electrophoresis reveals an isoenzyme pattern containing only one form of pyruvate kinase, an M isoenzyme, in both fundic and antral mucosa. The pyruvate kinase of gastric mucosa thus resembles the M-type enzymes of leucocytes and liver. Measurements of activity at both 1.0 mM PEP and 4.0--5.0 mM PEP, with and without additions of fructose 1,6-diphosphate, are recommended for the reliable estimation of pyruvate kinase activity in this tissue.


The radiometric assay of alkaline phosphatase activity with 125-I-labelled phenolphthalein monophosphate. The coupling of 125-I to phenolphthalein monophosphate is described. Hydrolytic cleavage of the phosphate group by alkaline phosphatases (EC 3.1.-3.1) releases labelled phenolphthalein which is extracted into ethyl acetate at pH 10. This forms the basis of a sensitive assay for the enzyme with the advantages of sensitivity and rapidity from the use of a gamma-emitter. Results with Escherichia coli and calf intestine alkaline phosphatases are presented.


Amino acid metabolism in the chronically uremic rat. The chronically uremic rat has been used as a model to study amino acid metabolism in uremia. Uremic rats fed low protein diets (6% casein) survived longer than uremic rats receiving either higher levels of dietary protein or a low protein diet supplemented with a mixture of nonessential amino acids. Alterations in plasma amino acid levels were observed in the uremic rats and were similar to those found in patients with renal failure. Plasma concentrations of citrulline, free tryptophan, glycine and the methylhistidines were increased and levels of serine, ornithine, lysine, total tryptophan, tyrosine, and the tyrosine-phenylalanine ratio were reduced. The metabolic basis of the altered tyrosine-phenylalanine ratio in plasma was studied. Tyrosine aminotransferase (TAT) and phenylalanine hydroxylase (PHL) activity were normal in the liver, but renal PHL activity of was decreased as compared to control rats. Tissue concentrations of citrulline were also found to be raised in liver and muscle of uremic rats. The activity of ornithine transcarbamoylase, was reduced in the liver and arginine synthetase activity was decreased in the kidneys of uremic rats. Thus elevated citrulline levels in uremic tissue appear to be caused by a decrease conversion of citrulline to arginine in the kidney. Preliminary studies of tryptophan metabolism in uremic rats have shown elevated brain levels of 5-hydroxyindoleacetic acid and increased hepatic tryptophan oxygenase activity. Increased plasma amine levels were associated with altered activities of monoamine oxidase and diamine oxidase in kidney and other tissues.


The effects of 2-thiophenecarboxylic acid on serum ionic calcium and magnesium levels in rats. 1. Administration of 2-thiophenecarboxylic acid to rats maintained on a low calcium diet resulted in a significant depression in the serum levels of total and ionized calcium and inorganic phosphate. 2. The serum magnesium levels were not altered by the drug.


Some actions of chandonium iodide, a new short-acting muscle relaxant, in anaesthetized cats and on isolated muscle preparations. 1. The actions of the new skeletal muscle relaxant chandonium (HS310) on the cardiovascular, respiratory and skeletal muscle systems of the cat under chloralose anaesthesia and on chick and rat isolated skeletal muscle preparations have been described. 2. In the cat chandonium exhibited a potent, competitive, non-depolarizing neuromuscular blocking action that was rapid in onset and of short duration. It possessed a selective atropine-like action at the cardiac vagus neuro-effector junction but little or no ganglion blocking activity. Neither adrenergic neurone noralpha-adrenoceptor blocking properties were evident. 3. Unlike tubocurarine, chandonium was without effect on the airways system of the anaesthetized cat. 4. Chandonium possessed weak anticholinesterase action. It is conceivable that this effect may contribute to its short duration of action. 5. The results from these studies suggest that chandonium may have possible clinical applications as a short-acting muscle relaxant.


Thromboembolic complications of cast immobilization for injuries of the lower extremities. Six cases of thromboembolic complications in patients being treated with plaster cast immobilization for a variety of injuries to the lower extremity were encountered over a four month period on an Air Force orthopaedic service. These injuries, consisting of three ruptures of the tendo achillis, two fractures of the tibia, and a sub-talar dislocation of the foot, resulted in one death from pulmonary emboli and three other serious complications of pulmonary emboli. The literature on thromboembolic complications following lower extremity injuries (including information of pathogenesis, clinical characteristics and epidemiology of venous thromboembolism and the hazards of lowere extremity cast immobilization in an individual susceptible to his complication) suggests that prophylaxis against thrombosis should be instituted in high risk individuals who must undergo case immobilization for injuries to the lower extremity.


Polymorphic acetylation procainamide in man. N-Acetylprocainamide (NAPA) and procainamide plasma and urine concentrations were determined by thin-layer chromatography (TLC) densitometry in people of known acetylator phenotype (dapsone phenotyping) taking procainamide for more than 3 days. The plasma NAPA/procainamide ratio 3 hr after the last dose for fast acetylators (mean plus or minus SD) is 1.8 plus or minus 0.59 (N equal to 8) and for slow acetylators, 0.61 plus or minus 0.09 (N equal to 6) P smaller than 0.001). The renal clearance of NAPA averaged 1.2 times the simultaneously measured endogenous creatinine clearance, whereas procainamide clearance was approximately double the creatinine clearance. There was no difference between slow and rapid acetylators in the renal clearance of either drug or the urine pH, indicating that the difference in plasma NAPA/procainamide ratios between these two groups is due to differences in their rates of acetylation. Therefore, procainamide is probably acetylated by the polymorphic N-acetyltransferase in man. Reflecting the blood level differences, the NAPA/procainamide ratio in urine (collected 99 to 180 min after last dose) was found to be higher in rapid than in slow acetylators. The plasma protein binding of NAa and of procainamide are similar. Since NAPA seems to have an antiarrhythmic potency similar to procainamide, NAPA probably contributes to the antiarrhythmic activity of procainamide therapy, especially in genetic rapid acetylators.


Changes in protein-bound calcium in the serum of haemodialysis patients. 1. Ionized calcium, protein-bound calcium and total calcium in serum were measured in patients receiving haemodialysis treatment. 2. During dialysis, the ionized calcium fraction decreased and the bound calcium increased. The blood pH also increased. 3. Studies in vitro suggested that the fall in ionized calcium and rise in protein-bound calcium were larger than could be explained by the redistribution of calcium fractions due to the pH change. 4. An explanation could be that haemodialysis increases calcium binding by serum proteins. Such an effect may be implicated in the aetiology of hyperparathyroidism and bone disease in patients undergoing haemodialysis.


The neurologic effects of noxious marine creatures. The concept of the sea as a source of noxious agents is perhaps not a familiar one to clinical neurologists, judging by the lack of reference to these agents in standard textbooks. Chemical, physiologic, and pharmacologic laboratories are increasingly investigating the properties of marine toxins, finding in them compounds with interesting and novel structures or unusual physiologic effects. Such substances are seen as possible agents for biologic and, more particularly, physiologic research, and as possible sources of new pharmaceuticals. These include hormone-like substances and antiviral or antitumor agents. Despite these specialized developments, which are in large measure a consequence of the technological advances of the present century, the clinician is at times directly concerned with the effects of marine toxic substances. For example, in Japan, puffer fish or tetrodotoxic poisoning is one of the major causes of deaths from food poisoning. Another marine toxin that has caused many explosive outbreaks of food poisoning. with many deaths in various parts of the world, comes from clams or mussels. This toxin, saxitoxin, is produced by species of marine protozoa including Gonyaulax, and is concentrated in filter-feeding molluscs. These two examples were of significant interest in medicine long before the technologic developments of the twentieth century. In the last few decades, entirely new problems of marine intoxication have arisen as a result of marine pollution from the disposal of industrial wastes in the sea. The most striking example of a man-made marine intoxication has been the outbreak of Minamata disease. In Minamata, Japan, the disposal of mercury-contaminated industrial wastes from a plastics factory into an enclosed bay, followed by human consumption of the contaminated fishes, crabs, or shellfish, led to many instances of acute cerebral degeneration. With the increasing exploration of the sea for both pleasure and economic exploitation, which is a feature of the second half of the twentieth century, it may be expected that the frequency and variety of human intoxications by marine creatures will be increased. This chapter reviews the neurologic effects of noxious substances of marine biologic origin. The subject is now developing so rapidly that overall surveys, such as this, of the general animal life of theocens will soon be beyond the scope of a single review. Nevertheless, it is hoped that the references given will enable the interested reader to pursue particular aspects further.


Human leptospirosis.

Human leptospirosis. Human leptospirosis is a subject of increasing interest. Although this disease was frequently associated with individuals whose occupation or geographic location placed them in close proximity to wild animals or farm animals, recent cases have been particularly prevalent in young children and adolescents in urban and suburban America. Many of the recent cases have been acquired from household pets, particularly from dogs or hamsters. In particular, healthy dogs who have been immunized with leptospiral organisms, thereby creating a significant risk for their owners. This article will review the pathophysiology, clincal manifestations, laboratory diagnosis, treatment, and prevention of leptospirosis in the modern era, with particular emphasis on a more complete understanding of the epidemiology of this disorder.


Alteration of Ringer pH and its effects on precursor incorporation into Drosophila salivary gland nuclei. Drosophila salivary glands were explanted and incubated with 3-H-uridine (or 3-H-thymidine) in Ringer's solution (Ephrussi-Beadle modified saline) adjusted to pH values in the integral range, 4 to 10. The results of autoradiographic investigations indicate a differential effect of altering Ringer pH on 3-H-uridine as opposed to 3-H-thymidine incorporation: a) Relatively uniform levels of chromosomal incorporation of 3-H-thymidine occured over the range of test pH. Some decrease of incorporation was noted at pH 5 and some increase at pH 9. b) Chromosomal incoroporation of 3H-uridine was severely depressed at pH 4 and 7 relative to the high incorporation levels observed at other pH values. Controlling pH of Ephrussi-Beadle Ringer's solution in such experiments a necessity. This appears especially important for studies involving 3-H-uridine incorporation.


[The effect of aspirin, antacids, alcohol, and bile acids on transmural potential difference of the human stomach (author's transl)]. The effect of orally administered buffered and unbuffered aspirin, antacids, alcohol and bile salts on functional changes in the gastric mucosal barrier was assessed by measuring gastric transmural potential differences (PD) in healthy subjects. In contrast to buffered aspirin, unbuffered aspirin caused a marked decrease in transmural PD due to its known effect of enhancing H+ back-diffusion. Simultaneous administration of antacids prevented the aspirin-induced drop of transmural PD. Ethanol (20% w/v), whisky (45% w/v), wine (6-7% ethanol), bile salts and ox-bile reduced transmural PD in healthy subjects. The measurement of transmural PD is a simple and sensitive method for detecting functional changes in the gastric mucosal barrier induced by drugs known to alter mucosal permeability.


Generation of hypophysiotropic activity in hypothalamic extracts in vitro. The luteinizing hormone (LH) and follicle stimulating hormone (FSH) releasing activity, as well as the prolactin (PRL) release-inhibiting activity were measured in both neutral aqueous, and acid ethanolic extracts of rat hypothalami. LH and FSH-releasing activities were detectable only in the latter type of extract, whereas PRL release-inhibiting activity appeared in both. Neutral ultrafiltrates of the neutral extracts contained no gonadotropin releasing activity, however, acidification of the filtration medium induced its appearance. PRL release was inhibited by both neutral and acid filtrates. These results suggest that LH and FSH releasing factor(s) may be stored in the hypothalamus in an inactive form from which the active peptide is generated in vitro under acid conditions; however, this does not appear to be true for the component(s) responsible for the inhibition of PRL release.


Guanyl cyclase activity of human blood lymphocytes. An enzyme, guanyl cyclase, which catalyzes formation of CYCLIC 3',5'-GMP from 5'-GTP, has been identified in human peripheral lymphocytes. The activity in lymphocyte homogenate is  14 pmol (min 10-7 lymphocytes). No activity is detected in red blood cells, and the amount found in platelets is very low. The properties of this enzyme are very close to those reported for other guanyl cyclases studied in other tissues: namely, its intracellular localization, its requirement for cation Mn-2+, its inhibition by Hg-2+, Zn-2+ and by nucleotides especially 5'-ATP. No change in enzyme activity occurs when phytohemagglutinin P is added to disrupted lymphocytes. However, when the mitogen is incubated with intact cells, guanyl cyclase activity increases in a few minutes.


Glutamine-dependent asparagine synthetase in fetal, adult and neoplastic rat tissues. Three enzyme reactions related to asparagine synthesis were studied in rat tissues: formation of aspartylhydroxamate, either from aspartate or by transfer from asparagine, and actual synthesis of asparagine from aspartate. Actual asparagine synthesis occurred at one-thousandth the rate of the other two reactions.  Optimal conditions for quantitative assay of asparagine synthesis were determined in fetal liver extract, which is a rich source of the enzyme. Demonstrable activity in liver fell 6 days after birth to 20% of the fetal value and decreased slowly thereafter to the low adult value.  Adult pancreas was the most active tissue found.  The asparagine synthetase of fetal liver extracts was significantly inhibited when combined with adult liver or tumor extracts. The inhibitor fractionated with ammonium sulfate in close association with the asparagine synthetase.  Therefore, demonstrable activities of asparagine synthetase in tissue extracts, measured in the presence of this inhibitor, do not necessarily parallel the concentrations of the enzyme present.


Relationship between steroids and pyridine nucleotides in the oxido-reduction catalyzed by the 17 beta-hydroxysteroid dehydrogenase purified from the porcine testicular microsomal fraction. The 17 beta-hydroxysteroid dehydrogenase which was purified from porcine testicular microsomal fraction [Inano, H. and Tamaoki, B (1974) Eur. J. Biochem. 44, 13-23] catalyzed the reduction of androstenedione to testosterone with the accompanying oxidation of equimolar NADPH. For the oxido-reduction of the steroids, the 17 beta-hydroxysteroid dehydrogenase preferred NADP(H) to NAD(h). Transhydrogenation from NADPH to NAD+ or NADH to NADP+ through the cyclic oxido-reduction of the steroids by the purified 17 beta hydroxysteroid dehydrogenase preparation was not spectrophotometrically detectable, because of selective preference of the testicular 17 beta-hydroxysteroid dehydrogenase against NADP(H). To examine stereospecific transfer of the hydrogen from NADPH to androstenedione by the purified 17 beta-hydroxysteroid dehydrogenase, the following tritiated cofactors were synthesized: [4-3-H]NADP+ was prepared by catalytic replacement from non-radioactive NADP+ and 3H2O in the presence of potassium cyanide. Then, [4-pro-R3H]NADPH was enzymatically synthesized from the [4-3H]NADP+ by glucose 6-phosphate and its dehydrogenase. On the other hand, [4-pro-S-3H]NADPH was prepared from the [4-3H]NADP+ by isocitrate and isocitrate dehydrogenase. When androstenedione was incubated with the 17 beta-hydroxysteroid dehydrogenase in the presence of these stereospecifically 3H-labeled cofactors, only the tritium located at 4-pro-S position of the nicotinamide moiety of NADPH was transferred to testosterone. The location of the tritium in the testosterone molecule produced, 17alpha-position of the steroid, was assigned by the fact that the tritium of the testosterone remained in its molecule after acetylation, but was completely lost by oxidation.


Inactivation of glyoxalase I from porcine erythrocytes and yeast by amino-group reagents. Glyoxalase I from porcine erythrocytes and from yeast is inactivated by the amino-group reagents 1-fluoro-2,4-dinitrobenzene, 5-dimethylaminonaphthalene-1-sulfonyl chloride, and 2,4,6-trinitrobenzenesulfonate (N-3ph-S). The inactivation follows pseudo-first-order kinetics, and the apparent first-order rate constant increases with pH, indicating that the basic form of a nucleophilic group is modified. The effect of increasing the inactivator concentration was tested with N-3PH-S, and it was found that the apparent rate constant increased to a limiting value. Such a result is consistent with a mechanism involving formation of a reversible inactivator x enzyme complex prior to the actual inactivation. Experiments with erythrocyte glyoxalase I and a variety of sulfhydryl-group reagents failed to show a dependence on sulfhydryl groups for catalytic activity, in contrast to previous results with the yeast enzyme. These experiments seem to exclude the possibility that essential sulfhydryl groups of the erythrocyte enzyme are modified by the amino-group reagents. Failure of reactivation of yeast glyoxalase I, and the similarities with the erythrocyte enzyme suggest that yeast glyoxalase I is not modified at essential sulfhydryl groups either by the latter reagents. This assumption has further support from experiments involving simultaneous inactivation with amino and sulfhydryl-group reagents. The results are consistent with the interpretation that amino groups of glyoxalase I are essential for catalytic activity. Glutathione derivatives, which are reversible competitive inhibitors of glyoxalase I, were found to protect the enzyme against inactivation by amino-group reagents. However, the concentration required for half-maximal protection was considerably higher than the inhibition constant of the reversible inhibition, which indicates that at least two molecules of the protector must be bound to the enzyme before full protection is obtained.


Characterization of an inducible amidase from Pseudomonas acidovorans AE1. The main molecular and catalytic properties of an acetanilide-hydrolyzing enzyme from Pseudomonas acidovorans AE 1, purified to a homogeneous state, were investigated. The molecular weight was 57 500 as determined by gel filtration and 55 300 as computed from the amino acid composition. By polyacrylamide gel electrophoresis in dodecylsulfate a polypeptide chain weight of 56 700 was obtained. Based on the reaction of the highly purufied enzyme with diethyl-4-nitrophenyl phosphate an equivalent weight of approximately 59 100 was found. From these results it was concluded that the enzyme consists of a single polypeptide chain and contains one active site per molecule. The enzyme hydrolyzed esters as well as certain aromatic amides. It also catalysed the transfer of acetyl groups to phenetidine yielding phenacetin. The activities towards aliphatic esters were much smaller. The enzyme was stable at pH values ranging from 7 to 9 and its pH-optimum was about 10. It was strongly inhibited by organophosphorous compounds, like diethyl-4-nitrophenyl phosphate or diisopropylphosphorofluoridate, as well as by physostigmine sulfate and -SH-blocking reagents, like HgCl-2 or 4-chloromercuribenzoic acid. o-Nitrophenol caused a competitive inhibition and phenetidine an uncompetitive inhibition.


Proton-magnetic-resonance spectroscopic study of the histidine residues of bovine alpha-lactalbumin. A study of the three histidine residues of bovine alpha-lactalbumin has been made using proton magnetic resonance (PMR) spectroscopy in order to obtain information on their environments in the protein and thereby to test in part the previously proposed structure. PMR titration curves are obtained for the H-4 resonances using difference spectroscopy and for the H-2 resonances and the 1-H-2-H exchange rates of the H-2 protons have been measured. The assignment of resonances to particular histidine residues is achieved by utilising their selective reaction with iodoacetate in conjunction with a PMR study of the carboxymethylation of alpha-N-acetyl-L-histidine. The H-2 and H-4 resonances labelled 1, 2 and 3 starting from the downfield end of the spectrum are assigned to histidine residues 107, 68 and 32 respectively. Their apparent pK values at low ionic strength and 20 degrees C are 5.78, 6.49 and 6.51 respectively. The experimental results on two histidine residues are consistent with the predictions of the proposed structure, which indicate that histidine-68 is an external residue and histidine-32 is partially buried and in the vicinity of aromatic residues. The experimental data on histidine 107 can also be rationalised with less certainty in terms of the proposed structure, which indicates a partially buried residue that may be involved in hydrogen bonding.


On the mechanism of malonyl-CoA-independent fatty-acid synthesis. Characterization of the mitochondrial chain-elongating system of rat liver and pig-kidney cortex. 1. Chain elongation of fatty acids by extracts of mitochondrial acetone powders from rat liver and pig kidney cortex are similar in their properties. The specific activity of the kidney system is about 30% as compared to the liver system 2. Different incorporation rates [1-14-C] acetate into fatty acids in the presence of NADH as the sole hydrogen donor that were reported in literature can be explained by different extraction methods. 3. In liver the incorporation into the saturated fatty acid, elongated by one C-2 unit, amounts to only 19% with NADH and 60% with NADPH in comparison with the incorporation in presence of both nucleotides. 4. Kinetics of the chain-elongating process favour the enoyl-CoA reductase to be the rate limiting step. 5. Long-chain saturated and unsaturated fatty acyl-CoA derivatives are very poor primers of the chain elongation. 6. Possion and the enoyl-CoA reductase may be the transfer of hydrogen from NADPH to the respiratory chain, and the conservation of reducing equivalents (NADH and NADPH) or acetate units during cellular anoxia.


The conformation of the RNA in cowpea chlorotic mottle virus: dye-binding studies. The binding of the dye acridine orange to cowpea chlorotic mottle virus (CCMV) and its purified RNA has been studied to obtain the number of dye-binding sites as a function of pH and, through further analysis, to estimate the degree of RNA secondary structure in situ. Acridine organe does not bind to CCMV protein and so the dye binding directly reflects the accessibility and structure of the RNA. The number of dye molecules per nucleotide which can be bound by native virus (pH 4.5, I = 0.1 buffer) is 0.13-0.18, the precise value depending upon the assumption of either heterogeneous binding sites or weak binding forces. The number of binding sites increases by a factor of about 2.7 to 0.34-0.48 when the pH is raised to pH 7.5 and "swelling" of the virus occurs. About 50% of the sites on the free RNA are available to bind dye in the swollen virus. The stacking coefficient, which is a measure of the degree of base pairing in a polynucleotide, has been calculated for the native  and swollen virus and for the isolated RNA. The values of the stacking coefficient for the RNA in the virus and following extraction are comparable, which suggests that the structure of the RNA in both cases is similar, and the values are low in magnitude, which indicates the existence of extensive regions of double-helix.


Content, synthesis and degradation of acetyl-coenzyme A carboxylase in the liver of growing chicks. Immunochemical techniques were used to study the mechanism underlying the marked increase in the level of acetyl-coenzyme A carboxylase activity in chick liver observed after hatching. The results of immunochemical titrations and Ouchterlony double-diffusion analysis indicated that this increase in the activity level of the enzyme was due to an elevation in the enzyme quantity. Isotopic leucine incorporation studies revealed that the rate of synthesis of the enzyme per liver was 18-fold higher in 9-day-old chicks than in 1-day-old chicks. In terms of the synthesis rate per gram of liver, this increase was 5-fold. The half-life for degradation of the enzyme in 9-day-old chicks was shown to be 46 h, whereas no apparent degradation of the enzyme as well as of total soluble liver protein was observed in 1-day-old chicks. These results indicate that the increase in the hepatic acetyl-CoA carboxylase content in growing chicks can be ascribed to accelerated synthesis of the enzyme.


Modulation of the activity of insulin-dependent enzymes of lipogenesis by glucocorticoids. Administration of triamcinolone or dexamethasone to rats led to a prompt, marked and persistent rise in liver acetyl-CoA carboxylase activity. The activity of fatty acid synthetase increased to a lesser extent and after a more prolonged glucocorticoid treatment, whereas the changes in that of NADP-malate dehydrogenase and ATP-citrate lyase were not appreciable. The overall channeling of [1-14-C]acetyl-CoA to fatty acids was enhanced. The triamcinolone effect on acetyl-CoA carboxylase activity appeared to be dependent on the coincident hyperinsulinemia since it was not obtained in alloxan-diabetic rats, whereas the alanine-aminotransferase-inducing effect of this hormone was additive to that of insulin deficiency. In adipose tissue triamcinolone treatment caused a reduction in the activity of all lipogenesis enzymes and blunted their response to insulin administration. The antagonism of glucocorticoids toward insulin, selectively modulating the responses of the insulin-sensitive enzymes in liver and adipose tissue is discussed. The rise in hepatic lipogenic capacity, through the retention of the ability of insulin to induce acetyl-CoA carboxylase, may be physiologically important in restraining the ketogenesis from acetyl-CoA despite the increased fat utilization during glucocorticoid excess.


Intermediates of the gamma-glutamyl cycle in mouse tissues. Influence of administration of amino acids on pyrrolidone carboxylate and gamma-glutamyl amino acids. GAMMA-Glutamyl transpeptidase, gamma-glutamyl cyclotransferase, L-pyrrolidone carboxylate hydrolase, gamma-glutamylcysteine synthetase and glutathione synthetase, the enzymes of the gamma-glutamyl cycle, were found in mouse brain, liver and kidney. The activity of L-pyrrolidone carboxylate hydrolase was many times lower than the activities of the other enzymes, and thus the conversion of L-pyrrolidone carboxylate to L-glutamate is likely to be the rate-limiting step of the cycle. The specificity of gamma-glutamyl cyclotransferase from mouse tissues was similar to that from rat tissues. The concentration of pyrrolidone carboxylate and gamma-glutamyl amino acids, intermediates of the gamma-glutamyl cycle, was determined by a gas chromatographic procedure coupled with electron capture detection. Administration of L-2-aminobutyrate, an amino acid that is utilized as substrate in the reaction catalyzed by gamma-glutamylcysteine synthetase, led to a large accumulation of gamma-glutamyl-2-aminobutyrate and pyrrolidone carboxylate in mouse tissues. L-Methionine-RS-sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase, abolished the increase in concentration of pyrrolidone carboxylate. No accumulation of pyrrolidone carboxylate was observed after L-cysteine. The separate administration of several protein amino acids had little effect on the concentration of pyrrolidone carboxylate; however formation of small amounts of the corresponding gamma-glutamyl derivatives (e.g. gamma-glutamylmethionine and gamma-glutamylphenylalanine) was detected. These intermediates are probably formed by transpeptidation between glutathione and the corresponding amino acid, catalyzed by gamma-glutamyl transpeptidase. The concentration of pyrrolidone carboxylate increased significantly after administration of a mixture containing all protein amino acids, the highest increase occurring in the kidney. The results suggest that two separate pathways for the formation of gamma-glutamyl amino acids and pyrrolidone carboxylate exist in vivo. One of these results from the function of gamma-glutamylcysteine synthetase in glutathione synthesis. The other pathway involves the amino-acid-dependent degradation of glutathione, mediatedby gamma-glutamyl transpeptidase. Only very small amounts of free intermediates are apparently derived from the latter pathway, suggesting that the gamma-glutamyl amino acids formed in this pathway are either enzyme-bound or are directly hydrolyzed to glutamate and free amino acid.


The ability of cell fractions of mouse spleen to repopulate the hemopoietic tissue and to mount graft-versus-host reaction. Mouse spleen cells were separated into seven fractions on a discontinuous gradient of dextran. The lightest, uppermost fraction contained colony-forming units and was capable of repopulating the hemopoietic tissues damaged by irradiation. The fraction below it also contained the colony-forming units, but its repopulating ability was limited. Heavy fractions contained immunocompetent cells capable of mounting graft-versus-host reaction, but the performance of the cells depended on the type of assay, i.e. on whether the cells were assayed in irradiated or in non-irradiated recipients. It is concluded, firstly, that the technique used resolved the colony-forming units into two classes - those capable of self-renewal and those committed to differentiation - which were concentrated in different portions of the light part of the gradient; and secondly, that the cells implementing graft-versus-host reaction were probably also resolved into two classes - the inciting and the proliferating cells - which were concentrated in different portions of the heavy part of the gradient.


Modification by calcium dobesilate of histamine effects on capillary ultrastructure. The ultrastructure of blood capillaries and venules are studied in rat skin. After i.v. injection of histamine the luminal surface of the endothelial cells show protrusions of variable size. Numerous gaps have been found in the capillary wall, specially in the venules. These alterations are not observed in the animals that were treated with calcium dobesilate before the administration of histamine. In these cases the capillary structure are indistinguishable of the controls. The possible effects of the histamine and calcium dobesilate on the cell coat and cell junctions of the endothelial capillary cells are discussed.


Hyperosmolar nature of diabetic coma.

Hyperosmolar nature of diabetic coma. Stupor in patients with nonketotic hyperglycemia has been ascribed to hyperosmolarity, but the cause of depressed consciousness in patients with ketoacidosis has been puzzling. In this study, blood pH, serum glucose and sodium concentrations, and serum osmolality were measured in eighty-five consecutive episodes of diabetic ketoacidosis and forty-seven of nonketotic hyperglycemia. In the acidotic patients, as in those with nonketotic hyperglycemia, stupor closely paralleled hyperosmolarity and not the severity of acidemia. Indeed, the mean elevations of serum osmolarity were almost the same in the ketotic and in the nonketotic patients who were deeply obtunded. It seems likely that depression of consciousness in patients with severely uncontrolled diabetes mellitus, if not due to a nonmetabolic disorder, such as acute stroke, is attributable to hyperosmolarity, whether or not ketoacidosis is present.


Amelioration of hypoglycemia in a patient with malignant insulinoma during the development of the ectopic ACTH syndrome. A patient with functioning islet cell carcinoma is described who had amelioration of her hypoglycemia during the development of ectopic ACTH syndrome. Moon facies and hyperkalemic metabolic acidosis were also present in this patient, features uncommonly seen in the actopic ACTH syndrome. At autopsy, she was found to have active tuberculosis. Prophylactic antituberculous therapy should be given to high-risk patients with the ectopic ACTH syndrome. High doses of ACTH may be palliative in refractory hypoglycemic states.


Rectal potential difference in the diagnosis of aldosterone excess. Rectal potential difference (pd) is directly related to the plasma aldosterone concentration, and rises when aldosterone is stimulated by sodium deprivation. However, when the measurement of rectal pd was tested at a screening test for hyperaldosteronism in 19 hypertensive subjects, four of the eight with primary hyperaldosteronism had a normal pd and four of the eight without aldosterone excess had an abnormally raised potential difference. The technique cannot therefore be recommended as a routine screening test for hyperaldosteronism. No relationship was found between rectal pd and hypertension associated with excess of deoxycorticosterone. Rectal pd rises in response to the mineralocorticoid-like agent carbenoxolone.


The effects of premedication drugs on the lower oesophageal high pressure zone and reflux status of rhesus monkeys and man. Thirty-five human  volunteers and eight Rhesus monkeys were studied with standard gastrooesophageal manometric techniqes and their  reflux status was evaluated witha pH probe placed in the lower oesophagus. morphine sulphate, pethidine hydrochloride, or idazepam was given intravenously until drowsiness was induced. The manometric and pH studies were repeated. All three drugs decreased the lower oesophageal high pressure zone and increased the probability of relux in both monkeys and man. Thes findings are relevant in the preparation of patients for surgery since gastrooesophageal reflux and pulmonary aspiration may be a problen in the pre-and postoperative phases.


Temporary, reversible binding of the competence factor to cellular receptors of Pneumococcus during induction of competence. The binding of the competence factor to cellular receptors of physiologically non-competent cells of Pneumococcus was followed as a function of time. A transformation medium without bovine serum albumin was used to study the binding of the competence factor. Control cells without the added factor remained completely non-competent under these conditions. The maximal binding of the factor to the cellular receptors took place already after 3 min of contact of the cells with the factor at 37 degrees C. After 10 min, when the maximum induction of competence occurs in the system used, the competence factor is fully released from the receptors to the medium. It follows that within the period between the 3rd and 10th min, when the cells are being modified for the irreversible binding of DNA, the presence of the competence factor on the cells is no longer necessary.


Biochemical and immunological properties of ribonucleic acid-rich extracts from Francisella tularensis. Ribonucleic acid (RNA)-rich extracts derived from the attenuated strain of Francisella tularensis (strain LVS) protected Swiss mice against lethal challenge with F. tularensis strain 425 but not against strain SCHU S4. No killed preparation, including an RNA-rich extract from SCHU S4 itself, offered protection against strain SCHU S4 in contrast to the high level of protection offered against this strain by vaccination with live strain LVS. The protective activity observed against strain 425 was sensitive to ribonuclease but not to Pronase. Protective activity is not a general property of bacterial RNA, since RNA-rich extracts from Staphylococcus aureus offered no protection against tularemia, although disc gel electrophoresis showed similar kinds and amounts of RNA in preparations form F. tularensis and S. aureus. Furthermore, inability to localize activity to a specific region in sucrose gradients suggests a structural rather than an informational role for the RNA in such extracts. RNA-rich extracts from F. tularensis but not from S. aureus were efficient inducers of F. tularensis opsonins in mouse serum, suggesting one mechanism by which such extracts confer protection.


Relationship of serum beta-glucuronidase and lysozyme to pathogenesis of tularemia in immune and nonimmune rats. A temporal study is reported of the febrile responses, tissue bacterial contents, and serum concentration of the lysosomal enzymes, beta-glucuronidase and lysozyme, in nonimmune rats inoculated with virulent or attenuated strains of Francisella tularensis, and in immune rats challenged with either a high or low dose of virulent organisms. The level of serum beta-glucuronidase appears to be an indicator of hepatocyte damage, whereas serum lysozyme correlates with the appearance, frequency, and severity of pyogranulomatous lesions. Survival of nonimmune rats after a challenge with either virulent or attenuated organisms appears to depend on a balance between dose of bacterial inoculum, celerity of irreversible pathologic events, and the ability of the reticuloendothelial and immune systems to collaboratively mount a response to limit or prevent dissemination of the infection. In immune rats, infection of parenchymal hepatic cells does not occur after a low dose (10-4) virulent challenge. Infection of parenchymal hepatic cells, however, does occur in immunized rats when the challenge dose is sufficiently large (10-8) so as to overcome the capacity of the reticuloendothelial to clear opsonized organisms.


Nature of the Kanagawa phenomenon of Vibrio parahaemolyticus. In a study of the Kanagawa phenomenon of Vibrio parahaemolyticus, both Kanagawa-positive and -negative strains were found to produce hemolytic factors that could not be differentiated on Wagatsuma blood agar. The presence of fermentable carbohydrates in media containing high concentrations of NaCl promoted the growth of V. parahaemolyticus and resulted in a marked decrease in medium pH and increased hemolysin production. The Kanagawa hemolysis of test strains differed according to the carbohydrates added. Clearly defined Kanagawa hemolysis was observed in blood agars of high salt content, but the distinction was lost in media containing 3% NaCl. From the results of this study, the Kanagawa hemolysis was interpreted as an expression of quantitative difference in hemolysin production, a conclusion that is clearly demonstrated on special blood agar of high salt content.


Intrinsic and chemically produced microheterogeneity of Staphylococcus aureus enterotoxin type C. Staphylococcus aureus enterotoxins C1 (SEC1) and C2 (SEC2) produced from 50-liter quantities of crude culture supernatants were purified chromatographically in a neutral or acid milieu. Microheterogenity of SEC1 was markedly increased by treatment of the purified toxin with alkali, and new more acidic charged species appeared. SEC2 was more heterogenous than any of the other S. aureus enterotoxins and was affected only slightly by treatment with alkali. Prolonged incubation of the organism during production of the SEC2 produced changes in charged species that may be related to a bacterial deamidase, since similar changes were not seen with alkaline treatment of the purified toxin. Although SEC1 and SEC2 showed complete identity immunologically, they are separate, distinct toxins, and alkali treatment of SEC1 did not produce SEC2.


Epstein-barr virus-induced cap formation in human lymphoblastoid cells. Redistribution and consequent cap formation of Epstein-Barr virus adsorbed to human lymphoblastoid cells were studied by indirect membrane immunofluorescence carried out at 0 degrees C. When EBV was adsorbed on cells at 0 degrees C, the cell surface fluorescence had a mostly r-ng-like pattern. However, the ring cells could be transformed into cap cells when warmed at 37 degrees C. This cap formation could be induced by EBV alone without participation of antibodies involved in the immunofluorescence procedure. The cap formation was temperature- and pH-dependent, and was reversibly inhibited by sodium azide or some sugars. Thus the EBV-induced cap formation was analogous to that induced by antibodies or ligands on other lymphoid cells.


Aminoglycosides from the clinical microbiologist's viewpoint. The author is discussing the aminoglycosides from the clinical microbiologists viewpoint. It is both an review of the literature and a personal attempt to put into perspective some of his own experience with these agents and reports in the literature. He is discussing the influence of the culture media composition of the outcome of susceptibility tests with these agents as well as the problems of bacterial resistance. Furthermore the topic of rapid and reasonably accurate serum drug level determination. Finally he gives some brief comments to the question of synergism or antagonism with other mainly penicillin type antibiotics.


Bovine serum albumin in lithium chloride solutions. The effects of li+ and H3O+ on the conformation of bovine serum albumin in azqueous solutions at room temperature are compared. At low pH (high concentration of H3O+) the change in conformation of the protein is demonstrated by an increase in effective volume, a decrease in helical content and a blue shift of tyrosyl residue. A similar change is observed for the protein in highly concentrated LiC1 solution (6.0-7.0M) at neutral pH. However, the H3O+ is 12,000 times more powerful than the Li+ in destabilizing the protein molecule. This is consistent with their thermodynamic and kinetic properties, since the H3O+ is often different from the Li+ in several orders of magnitude. While the changes in structural properties of the protein are almost identical in both the acidic solution and the highly concentrated LiC1 solution, further study using dioxane as a probe suggests different mechanisms under which the changes occur. The effect of H3O+ is related to electrostatic force, whereas the effect of Li+ is related to both the electrostatic hydrophobic forces. These two major forces are believed to be responsible for the conformation of protein molecules.


Digestion of insulin derivatives with subtilisin: a kinetic study. Native, denatured, performic acid-oxidized or S-sulfo insulin and S-sulfo or performic acid-oxidized A- and B-chains were digested with subtilisin type Carsberg. The proteolysis was followed by measuring the uptake of alkali through autotitration. The kinetic study shows the existence of 2 first-order reaction classes which differ markedly in rate constant. The number of bonds split with fast and with slow reactions has been calculated. Only one of a total of 12 cleavable bonds in native insulin is opened by fast reaction. In the denatured protein the number of bonds split by the fast reaction increases to 4 and in the oxidized and S-sulfo protein 3 bonds are cleaved, while the slow cleavable bonds number 2 and 7, respectively, The kinetic study of the proteolysis of S-sulfo A-chain and of oxidized or S-sulfo B-chain shows that two bonds are split in A-chain with the fast and slow reactions, while in B-chain only one of the six cleavable bonds is susceptible to fast attack.


Carbamylation of alkaline mesentericopeptidas. The effects of carbamylation with potassium cyanate, and methylation with methyl p-nitrobenzene sulphonate on the mesentericopeptidase activity are studies. The treatment with potassium cyanate causes the enzyme to lose its activity towards ester substrates and casein. The specific reagent N-trans-cinnamoylimidazole does not acylate the active site in the  carbamylated enzyme. The pH dependence of the rate of inactivation indicates that an ionizing group of pK = 7.3, probably the protonated imidazole group of the active site histidine, is involved in the reaction. The competitive inhibitor boric acid protects mesentericopeptidase against inactivation with potassium cyanate. These suggest that the active site residues are modified in the unprotected enzyme. Sixty per cent of the enzyme activity toward N-acetyl-L-tyrosine ethyl ester was restored after treatment of the carbamylated mesentericopeptidase with 1 M hydroxylamine hydrochloride. Circular dichroism spectra show that the carbamylation does not change markedly the native protein conformation.


A study of the unfolding of the inhibited subtilisin in guanidine hydrochloride. Inhibited subtilisin (Subtilism Carlsberg; Subtilopeptidase A) is unfolded in the presence of 7 M guanidine hydrochloride. The unfolding reaches a maximum in approximately 6 min at 20C at pH 8.0. This is demonstrated by an increase of the mean residue ellipticity at 222 nm from -8.02 x 10-3 to -1.72 x 10-3 deg. cm-2/decimole. The unfolding is partially reversible and this reversibility is favoured by lower concentrations of enzyme. The fact that the refolding process is not complete may be attributed to either the demonstrated self association of the denatured enzyme or to interference of non-covalently bound autolysis peptides.


Flavin nucleotides, glutathione reductase and assessment of riboflavin status. The in vitro effects of flavin nucleotides on human erythrocyte glutathione reductase were studied. Flavin mononucleotide was found to be a potent inhibitor of glutathione reductase in haemolysates, having an effect at a concentration of 1 muM. Flavin adenine dinucleotide inhibited at a concentration at least ten fold this, lower levels producing the expected stimulation of enzyme activity. Different assay conditions also altered the activity of the enzyme, although the flavin mononucleotide inhibition remained. The implications of these findings in our understanding of glutatione reductase and in using the enzyme to assess riboflavin status are discussed.


A comparison between bromazepam (Ro 5-3350, Lexotan) and diazepam (Valium) in anxiety neurosis. A controlled, double-blind clinical trial. The new benzodiazepine bromazepam (Ro 5-3350, Lexotan) was compared with diazepam in a double-blind, crossover trial on 30 patients with prolonged anxiety symptoms. Each drug was given for 3 weeks in a dose of 5 mg t.d.s. Bromazepam proved to be statistically significantly superior to diazepam when evaluated through patients' preferences. Fourteen patients preferred bromazepam to diazepam and four the opposite. Three cases were tied. Nine patients had to be withdrawn from the evaluation, which may create bias. A plea is made for further controlled clinical trials on bromazepam, in particular in obsessive and phobic neurosis.


The stability constants of magnesium oxalate complexes. The stability constants of MgC204 and Mg2C204-2nCOmplexes were estimated at 37--38 C by four different techniques; there was good agreement among the results of the different techniques. The stability constant of MgC204 is (4019 plus or minus 76 M-minus 1). The stability constant of Mg2C204-2nWAS FOund to be less than 5 M-minus 1. These stability constant are needed in the evaluation of the stone-forming potential of urine.


The effect of thyrotrophin on oxidative activity in thyroid follicle cells. Biochemical evidence, based on supra-physiological concentrations of thyrotrophin (TSH), has indicated that this hormone increases the NADP concentration in thyroid follicle cells. The hormone is believed to act both to enhance the reoxidation of the reduced coenzyme and to generate NADP from NAD. The latter effect, mediated by NAD-+-kinase, could be a major control mechanism in thyroid metabolism. In the present study increased sensitivity has been obtained by the use of maintenance culture and of microdensitometry for measuring enzyme activity in the follicle cells. Using low physiological (0.1 muU/ml), and sub-physiological concentrations of the hormone as are used in the cytochemical bioassay systems, it has been shown that the rate of reoxidation of NADPH is enhanced by TSH. Moreover the NAD-+-kinase activity is also greatly increased, and shows a direct relationship to the concentration of the hormone acting on the segments of the gland in vitro. It is possible that this phenomenon could be used to assay TSH. The results indicate that NAD-+-kinase activity may play a significant part in the control of pentose-shunt oxidation in thyroid follicle cells.


Cytochemical discrimination between catalases and peroxidases using diaminobenzidine. The influence on diaminobenzidine staining of four variables: prefixation in aldehyde, temperature and pH of incubation, and H2O2 concentration, was investigated in catalase-, as well as in peroxydase-containing material. Catalase from five different sources and five types of peroxidase were examined. It is concluded: (a) when cells are incubated without prior fixation, in a DAB medium at room temperature and pH 7.3 with 0.003% H2O2, peroxidases produce a visible cytochemical stain, while catalases do not; (b) the cytochemical reaction elicited by catalases is stimulated by prior aldehyde fixation in specified conditions, and incubation at 45 degrees C and pH 9.7 with 0.06% H2O2; (c) under the latter circumstances several peroxidases also stain. Ultrastructural preservation is satisfactory in tissues incubated prior to fixation.


Cytoplasmic protein network in HeLa cells. Monolayer HeLa culture nuclei isolated in situ with nonionic detergents remained attached to the glass as a "nuclear monolayer". Searching for nuclear fixing structures a fine continuous protein network was revealed around the isolated nuclei with fluorescence microscope after acridine orange staining.


Enzyme histochemical observations on the segmentation of the proximal tubules in the kidney of the female rat. The segmentation of the proximal tubules in the kidney of the female rat was studied by means of enzyme histochemical reactions and the results compared with those observed in male and recently described by Jacobsen and J0rgensen (1973 a). Reactions were performed for the following soluble, coezyme-dependent oxido-reductases: glucose 6-phosphate dehydrogenase, alpha-glycerophosphate dehydrogenase, 3 alpha-hydroxysteroid dehydrogenase, NAD-as well as NADP-dependent isocitrate dehydrogenases, NAD-dependent malate dehydrogenase, NADP-dependent, decarboxylating malate dehydrogenase, uridine diphosphate glucose dehydrogenase. Measures were taken to reduce enzyme diffusion and eliminate interference from tissue tetrazolium reductases. Furthermore, reactions were performed for a number of less soluble or insoluble enzymes: glucose 6-phosphatase, mitochondrial alpha-glycerophosphate dehydrogenase, beta-hydroxybutyrate dehydrogenase, succinate dehydrogenase and tetrazolium reductases. In the proximal tubules of the female rat all enzymes studied--except beta-hydroxybutyrate dehydrogenase--showed segmental differences, most of them clearly revealing three segments. Sex differences were found concerning all enzymes except uridine diphosphate glucose dehydrogenase and NADP-dependent isocitrate dehydrogenase. The most pronounced sex-related differences were seen in the third segment in which part the male rat showed highest activity in respect to tetrazolium reductases, NAD-dependent isocitrate dehydrogenase, succinate dehydrogenase, beta-hydroxybutyrate dehydrogenase, 3 alpha-hydroxysteroid dehydrogenase and glucose 6-phosphate dehydrogenase and the female in respect to glucose 6-phosphatase, alpha-glycerophosphate dehydrogenases, and NADP-dependent, decarboxylating malate dehydrogenase. A few of the enzymes exhibited minor sex differences in the first two segments.


Mitochondrial enzymatic adaptation of skeletal muscle to endurance training. Some mitochondrial enzymatic activities (succinate dehydrogenase, NADH cytochrome reductase, cytochrome oxidase) were studied in the gastrocnemius and soleus muscle of the rat. The modifications of the enzyme activity, induced by endurance training, were found to be functions of 1) daily work load and 2) total training time. The treatment with an effective dose of vasodilating substances (papaverine, nicergoline, dipyridamole, and bamethan) showed that 1) nicergoline, bamethan, and dipyridamole were differently able to shorten the time of appearance of the increase in the enzymatic activities; 2) however, long-term treatments with these drugs did not prove able to modify the plateau level of the enzymatic activity increase, for a given amount of endurance training; 3) the pharmacodynamic effect on enzymatic activities was in no way related to the vasodilating effect of these drugs, since the effect was not observed with papaverine. The transition from a given level of endurance training to a lower one led to a proportional decrease of the mitochondrial enzymatic activities, thus pointing out the relation between amount of training and enzymatic activity. The drugs studied were unable to modify the decrease of enzymatic activity induced by lower work load.


Arterial blood gases in conscious rats exposed to hypoxia, hypercapnia, or both. Adult male rats were anesthetized and catheters were implanted in the caudal artery. Soon after recovery from short-lasting anesthesia, a total of 20 groups of six each were individually exposed to five different oxygen levels varying from 21.0 to 9.0% combined with four CO2 levels ranging from 0 to 12.9% at a mean barometric pressure of 744 Torr. Arterial blood samples were collected and analyzed for pH, Po2, and Pco2 before and near the end of 20-min exposures. During an air-breathing control period, pH averaged 7.466 plus or minus 0.020 SD, Paco2 41.2 plus or minus 1.9 Torr and Pao2 91.8 plus or minus 3.5 Torr. During hypoxia, Pao2 levels were similar to that of acutely hypoxic humans. Rats apparently differ from man in that blood buffering is greater, resulting in a higher pH during air breathing and a smaller [H-+] increase with increasing Paco2. Differences between arterial and inspired CO2 were about 10 Torr at 60 and 90 Torr Plco2 and were not influenced by Plo2.


Regulation of arterial PCO2 during intravenous CO2 loading. Increased CO2 flow to the lung produced by increasing cardiac output (with constant PVCO2) results in hyperpnea with arterial PCO2 maintained at its control value (J. Appl. Physiol. 36: 457, 1974). To study if arterial PCO2 could be similarly regulated when CO2 flow was elevated by increasing PVCO2 (without changing cardiac output), we produced graded increases in PVCO2 (up to a mean of 69 mmHg) using an extracorporeal gas exchanger in five chloralose-urethan-anesthetized dogs. CO2 output increased up to fourfold. Ventilation increased in proportion to the additional CO2 flow to the lung with consequent regulation of arterial PCO2 at its control value. Comparable increases in VE produced by "conventional" airway loading resulted in arterial hypercapnia. The resulting CO2 response curve was similar to that found in unanesthetized dogs. We conclude that intravenous delivery of CO2 to the lung results in infinite "sensitivity" when computed as Delta VE/Delta paco2. These results provide evidence for a CO2-linked hyperpnea which is not mediated by measurable increases in mean arterial PCO2.


Renal response to secretin.

Renal response to secretin. The effect of Jorpes secretin on the urinary volume, pH, and excretion of sodium, potassium, chloride, bicarbonate, titratable acidity, ammonia and phosphate was studied in five healthy male volunteers with and without simultaneous aspiration of duodenal fluids. A three- to fourfold increase in urinary volume and sodium excretion occurred within the first 30 min after secretin injection and this was accompanied by a significant rise in urinary pH in each instance. Urinary bicarbonate excretion increased from 55 plus or minus 13 to 395 plus or minus 33 mueq/30 min after secretin injection. Aspiration of alkaline duodenal contents was accompanied by an even greater postsecretin increase in urinary bicarbonate excretion. No significant changes in arterial pH or blood gases were detected throughout the study. These observations are compatible with a direct effect of secretin upon the renal tubular reabsorption of water, bicarbonate, and other ions, and could account for the transient alterations in urinary pH occurring in response to a meal.


Effects of moderate hypoxemia and hypocapnia on CSF [H+] and ventilation in man. The effects of 26 h of normoxic hypocapnia (PaCO2, 31 MMHg) vs. 26 h of hypocapnia plus hypobaric hypoxia (PaCO2 32, PaO2 57 mmHg) were compared with respect to: a) CSF acid-base status; and b) the spontaneous ventilation (at PIO2 145 mmHg) which followed the imposed (voluntary) hyperventilation. For each condition of prolonged hypocapnia, PaCO2 was held constant throughout and pHa and [HCO3-]a were constant over the final 6-10 h. We assumed that measured changes in lumbar CSF acid-base status paralleled those in cisternal CSF. Spontaneous hyperventilation followed both normoxic and hypoxic hypocapnia but was significantly greater following hypoxic hypocapnia. In the CSF, pH compensation after 26 h of hyperventilation was incomplete (similar to 45-50%), was similar to that in arterial blood, and was unaffected by a superimposed hypoxemia. These data were inconsistent with current theory which proposes the regulation of CSF [HCO2] via local mechanisms and, in turn, the mediation of ventilatory acclimatization to hypoxemia and/or hypocapnia via CSF [H+]. Alternative mediators of ventilatory acclimatization were postulated, including mechanisms both dependent on and independent of "chemoreceptor" stimuli.


Effects of CO-2 and extracellular H+ iontophoresis on single cell activity in the cat brainstem. The activity of single cells in deep regions of the medulla oblongata was observed both during CO2 inhalation and during the extracellular iontophoresis of hydrogen ions in peripheral chemoreceptor-denervated cats. All 53 neurons that fired in synchrony with some part of the ventilatory cycle showed increased firing during CO2 inhalation; yet none responded in a graded fashion to the extracellular application of hydrogen ions. Seventy-one of the 74 nonperiodic cells studied showed no response to CO2 inhalation. Of the 3 nonperiodic cells that did respond to CO2, 2 also responded in a graded fashion to the extracellular iontophoresis of hydrogen ions. It is concluded that the cell bodies of medullary neurons with respiratory periodicity are relatively insensitive to hydrogen ions. Further the paucity of hydrogen ion-sensitive cells found in deep areas of the medulla does not support the notion that medullary hydrogen ion chemoreception is largely achieved by structures located deep in the lower brainstem.


Time course of exchanges between red cells and extracellular fluid during CO2 uptake. A stopped-flow rapid-reaction apparatus was used to follow the time course of extracellular pH in a human red cell suspension following a sudden increase in PCO2. The extracellular pH change was slow (t1/2 similar to 3.5 s) considering the presence of carbonic anhydrase in the cells. When carbonic anhydrase was added to the extracellular fluid, the half-time was reduced to less than 20 ms. The explanation for these phenomena is that the equilibration of H+ across the red cell membrane is rate-limited by the uncatalyzed reaction CO2 plus H2O formed from H2CO3 outside the cells. A theoretical model was developed which successfully reproduced the experimental results. When the model was used to simulate CO2 exchange in vivo, it was determined that blood PCO2 and pH require long times (greater than 50 s) to approach equilibrium between cells and plasma after leaving an exchange capillary. We conclude that cell-plasma equilibrium may never be reached in vivo, and that in vitro measurements of these quantities may not represent their true values at the site of sampling.


Incomplete compensation of CSF [H+] in man during acclimatization to high altitude (48300 M). This study has assessed the regulation of arterial blood and cerebrospinal fluid acid-base status in seven healthy men, at 250 m altitude and after 5 and 10-11 days sojourn at 4,300 m altitude (PaO2 = 39 mmHg day 1 to 48 mmHg day 11). We assumed that observed changes in lumbar spinal fluid acid-base status paralleled those in cisternal CSF, under these relatively steady-state conditions. Ventilatory acclimatization during the sojourn (-14 mmHg PaCO2 at day 11) was accompanied by: 1) reductions in [HCO3-] (-5 to -7 meq/1) which were similar in arterial blood and CSF; 2) substantial, yet incomplete, compensation (70-75%) of both CSF and blood pH; and 3) a level of CSF pH which was maintained significantly alkaline (+0.05 +/- 0.01) to normoxic control values. These data at 4,300 m confirmed and extended our previous findings for more moderate conditions of chronic hypoxia. It was postulated that the magnitude and time course of pH compensation in the CSF during chronic hypoxia and/or hypocapnia are determined by corresponding changes in plasma [HCO2-].


Distribution of ventilation-perfusion ratios in dogs with normal and abnormal lungs. We have recently described a new method for measuring distributions of ventilation-perfusion ratios (VA/Q) based on inert gas elimination. Here we report the initial application of the method in normal dogs and in dogs with pulmonary embolism, pulmonary edema, and pneumonia. Characteristic distributions appropriate to the known effects of each lesion were observed. Comparison with traditional indices of gas exchange revealed that the arterial PO2 calculated from the distributions agreed  well with measured values, as did the shunts indicated by the method and by the arterial PO2 while breathing 100 per cent 02. Also the Bohr dead space closely matched the dispersion of ventilation in realtion to VA/Q. Assumptions made in the method were critically evaluated and appear justified. These include the existence of a steady state of gas exchange, an alveolar-end-capillary diffusion equilibration, and the fact that all of the observered VA/Q inequality occurs between gas exchange units in parallel. However, theoretical analysis suggests that the method can detect failure of diffusion equilbration across the blood-gas barrier should it exist. These results suggest that the method is well-suited to clinical investigation of patients with pulmonary disease.


Saturation dependency of the Bohr effect: interactions among H-+, CO2, and DPG. The Bohr effect was measured in normal whole blood and in blood with low DPG concentration as a function of oxygen saturation. pH was changed by varying CO2 concentration (CO2 Bohr effect) or by addition of isotonic NaOH or HC1 at constant PCO2 (fixed acid Bohr effect). At nornal DPG concentration CO2 Bohr effect was -0.52 at 50% blood oxygen saturation, increasing in magnitude at lower saturation and decreasing in magnitude at higher saturation. In DPG depleted blood with base excess (BE) similar to 0 meq/1, there was similar dependence of CO2 Bohr effect on oxygen saturation. At BE similar to -10 meq/1, influence of saturation was comparable, but the magnitude of the Bohr effect was markedly increased at all saturations. Fixed acid Bohr effect at normal DPG concentration was -0.45 at saturations of 50-90% but decreased at lower saturations. In DPG-depleted blood fixed acid Bohr effect averaged about -0.33 with minimal variation with saturation. Influence of DPG on oxygen affinity was greater at intermediate saturations and less at saturations below 20% and above 80%. Effect of CO2, independent of pH, was many fold greater at lower oxygen saturations than at higher saturations. These results support the suggestion that the alpha chain of hemoglobin is the site of the initial oxygenation reaction. Physiologically they indicate that the relative contribution of CO2 and fixed acid, as well as the level of oxygen saturation and DPG concentration, may be important in determining PO2 of capillary blood and resulting oxygen delivery.


A simplified method for determining the P50 of blood. A method  to determine the P50 of whole blood is described using a modified American Optical reflectance oximeter, pump, and membrane tonometer, together with PO2, PCO2, and pH measurements in a standard blood gas machine. Determinations of P50 were made in 66 patients and normal subjects and in two situations where P50 was very low and very high. The results were compared to oxygen saturations  calculated from measured oxygen content. The directly determined oxygen saturation agreed with the assumed saturation of 50 per cent in the oximeter within 0.5 per cent. The apparatus appears to be a simple and relatively inexpensive method to obtain P50 as long as blood carboxyhemoglobin or methemoglobin contents are not elevated.


Colorimetric assay of strychnine and brucine in nux vomica. Two colorimetric methods are described for the estimation of strychnine and brucine in nux vomica. The first is a modification of the Karawya and Ghourab method for the determination of strychnine, in which the sensitivity of the color is increased by changing certain conditions of the method. The second was developed for the determination of brucine and is based on measuring the intensity of the violet color produced by treating brucine with nitric acid and methanolic stannous chloride. In the presence of large amounts of strychnine, brucine is isolated prior to colorimetric analysis by a quantitative thin layer chromatographic technique.


Determination of procaine and related local anesthetics. I. Partition chromatographic separation and assay of mixtures of procaine with tetracaine and with propoxycaine. Determination of ionization and extraction constants for procaine, tetracaine, and propoxycaine led to selection of a simple partition chromatographic system for separation and assay of mixtures of these anesthetics. A 65% solution of chloroform in isooctane elutes tetracaine or propoxycaine from a pH 4:sodium bromide column; procaine is retained and subsequently eluted by chloroform as the bromide ion-pair. The anesthetics are then determined spectrophotometrically. Results of assay of standard and commercial formulations are presented.


Determination of potassium guaiacolsulfonate by ion-pairing partition chromatography. Potassium guaiacolsulfonate, a highly polar, acidic substance, is readily eluted by chloroform from a pH 4.5 Celite column in the form of its ion-pair with trihexylamine, thereby effecting facile separation from other pharmaceuticals. The sulfonic acid is then back-extracted into aqueous alkali and determined spectrophotometrically. Assay of standard solutions by this procedre averaged 100.44 plus or minus 0.81%. The method was applied successfully to 4 commercial cough preparations containing a variety of other drugs.


Mycotoxin bioassay, using Bacillus stearothermophilus. Spores of Bacillus stearothermophilus in standardized spore strips are pretreated with solutions of the mycotoxins aflatoxin B1, patulin, rubratoxin B, and diacetoxyscirpenol and subsequently incubated in a nutrient solution containing bromocresol purple as pH indicator. After 16.5 hr of inclubation the color of the indicator medium inoculated with untreated spore strips of B. stearothermophilus changes from purple to yellow; no color change occurs in the indicator medium inoculated with spore strips treated 15 min with 0.01 mug of any of the 4 mycotoxins/ml during a 60 hr incubation.


Factors in the diagnosis and treatment of chronic pain. Symptoms are the verbal and nonverbal communicative expressions of experience. At any one time, the symptom is dependent on structural changes, genetic variations, metabolic abnormalities, immunopathic and other disorders, and the totality of social, economic, and situational experience. Complaints of pain may vary with time depending on the interaction of these features in a manner not directly related to the intensity of a noxious stimulus.


Purification and properties of the glutathione reductase of Chromatium vinosum. The Chromatium vinosum glutathione reductase [NAD(P)H: glutathione disulfide oxidoreductase, EC 1.6.4.2] was purified to apparent homogeneity. The enzyme was found to require reduced nicotinamide adenine dinucleotide (NADH) as a reductant and to be specific for oxidized glutathione (GSSG). The polypeptide molecular weight in sodium dodecyl sulfate was found to be 52,000. Incubation of enzyme with NADH in the absence of GSSG resulted in a significant loss in activity. The enzyme was stimulated by phosphate and sulfate ion, but was inhibited by chloride ion, heavy metals, and sulfhydryl reagents. Adenylate nucleotides were inhibitory, and the data suggested that they were acting as competitive inhibitors of flavin adenine dinucleotide (FAD). The Km values of 7 X 10-3 for GSSG and 6 X 10-5 M for NADH were the highest reported of any previously investigated glutathione reductase. The order of addition of components markedly affected the response of the enzyme to FAD. A requirement for FAD (Km 5.2 X 10-7 M) was seen if the enzyme was incubated with NADH prior to GSSG addition, whereas no FAD was required if the order was reversed.


Identification of a deoxyribonuclease implicated in genetic transformation of Diplococcus pneumoniae. A mutation of Diplococcus pneumoniae, end-1, reduces the major deoxyribonuclease activity of the cell, an endonuclease, to 10% of its normal value without impairing transformation. Further mutations, called noz, abolish the residual endonuclease activity and block transformation. The residual endonuclease is similar to the wild-type enzyme in size, charge, divalent cation dependence, inhibition by ribonucleic acid, and formation of oligonucleotide products. However, the mutant endonuclease is more temperature sensitive, which suggests that the end-1 mutation occurred in a structural gene for the enzyme. Genetic analysis showed that the noz mutations occur at the same genetic locus. A number of new end mutants were analyzed. Those that retained more than 1.4% of the normal endonuclease activity were essentially normal in transformation; those with less than 1% were defective. The transformation-defective end mutants appear to be blocked in the entry of deoxyribonucleic acid (DNA) since they carry out the prior step of binding DNA to the outside of the cell. The major endonuclease of the cell may act as a DNA translocase by attacking and degrading one strand of DNA, thereby facilitating entry of the complementary strand into the cell.


Effect of pH and biotin on a circadian rhyythm of conidiation in Neurospora crassa. Expression of a circadian rhythm of conidiation in Neurospora crassa is enhanced in cultures grown on medium that is neutral or deficient in biotin.


Glucose transport in Acholeplasma laidlawii B: dependence on the fluidity and physical state of membrane lipids. The uptake of D-glucose by Acholeplasma laidlawii B occurs via a mediated transport process, as shown by the following observations: (i) glucose permeates A. laidlawii B cells at a rate at least 100 times greater than would be expected if its entry occurred only by simple passive diffusion; (ii) the apparent activation energy for glucose uptake in A. laidlawii is significantly lower than that expected and observed for the passive permeation of this sugar; (iii) glucose uptake appears to be a saturable process; (iv) glucose uptake can be completely inhibited by low concentrations of phloretin and phlorizin; and (v) glucose uptake is markedly inhibited at temperatures above 45 C, whereas the passive entry of erythritol continues to increase logarithmically until at least 60 C. The metabolism of D-glucose by this organism is rapid and, at low glucose concentrations, the intracellular radioactivity derived from D-[14-C]glucose is at any given time a reflection of the net effect of glucose transport, glucose metabolism, and loss from the cell of radioactive metabolic products. Care must thus be taken when attempting to determine the rate of glucose transport by measuring the accumulation by the cells of the total radioactivity derived from D-[14-C]glucose. The rate of uptake of D-glucose by A. laidlawii B cells is markedly dependent on the fatty acid composition and cholesterol content of the plasma membrane and exhibits a direct dependence on the fluidity of the membrane lipids as measured by their reversible, thermotropic gel to liquie-crystalline phase transition temperatures. In contrast to the transport rates, the apparent activation energy for glucose uptake above the phase transition temperature is not dependent on membrane lipid composition. At the temperature range within the membrane lipid phase transition region, the apparent activation energy of glucose uptake is different from the activation energy observed at temperatures above the phase transition. This may reflect the superimposed operation within the phase transition region of more than one temperature-dependent process.


Phosphorylation of solubilized sarcoplasmic reticulum by orthophosphate and its thermodynamic characteristics. The dominant role of entropy in the phosphorylation. A large fraction of the Ca-2plus- and Mg-2plus-dependent ATPase (EC 3.6.1.3) in sarcoplasmic reticulum membranes solubilized with Triton X-100 was phosphorylated with Pi. The phosphorylation required Mg-2plus but was strongly inhibited by low concentrations of Ca-2plus. A Ca-2plus ion concentration of 30 muM caused half-maximum inhibition in the presence of 50 mM MgCl2. The phosphorylated enzyme showed a rapid turnover and was in dynamic equilibrium with Pi in the medium. At equilibrium the amount of the phosphorylated enzyme increased markedly with increased in the reaction temperature. The apparent standard free energy change, the apparent standard enthalpy change, and the apparent standard entropy change in the formation of the phosphorylated enzyme from the enzyme-phosphate complex in the presence of excess Mg-2plus at 37 degrees and pH 7.0 were, respectively, 0.35 Cal per mol, 15.9 Cal per mol, and 50.2 e.u. per mol. The susceptibility of the acid-denatured phosphorylated enzyme to hydroxylamine showed that the phosphorylated enzyme is of an acyl phosphate type. The present results are consistent with the probability that the phosphorylation results from reversal of late steps in the Ca-2plus transport process. The results clearly show that the phosphorylated enzyme is stabilized by a great increase in entropy upon its formation from the enzyme-phosphate complex.


Purification and characterization of mouse hypoxanthine-guanine phosphoribosyltransferase. Hypoxanthine-guanine phosphoribosyltransferase (HGPR transferase) (EC 2.4.2.8) has been purified approximately 4500-fold to apparent homogeneity from mouse liver. The procedure involves the use of affinity chromatography and was designed to be readily adaptable to small scale isolations. The enzyme appears to be composed of 3 subunits of identical molecular weight (27,000 per subunit). The subunit molecular weight has also been determined by the analysis of radioactively labeled HGPR transferase immunoprecipitated from wild type and mutant (HGPR transferase) mouse tissue culture cell lines.


Threonine deaminase from Escherichia coli. II. Maturation and physical properties of the enzyme from a mutant altered in its regulation of gene expression. The biosynthetic L-threonine deaminase (L-threonine hydrolase deaminating, EC 4.2.1.16) has been purified from Escherichia coli K12 regulatory mutant CU18. This mutant has properties that follow the predictions of the autogregulatory model previously proposed for the control of synthesis of the isoleucine-valine biosynthetic enzymes. The autoregulatory model specifies that L-threonine deaminase participates in the control of the expression of the ilv ADE gene cluster as well as the ilv B gene and ilv C gene, which constitute three separate units of regulation. The single mutation in strain CU18 results in altered regulation of ilv gene expression and in the production of an altered L-threonine deaminase. The immature form of the enzyme purified from mutant CU18 exhibits an altered response to L-valine, a maturation-inducing ligand. The native form of the mutant is altered in its apparent Km for L-threonine and in its response to the effects of L-valine and L-isoleucine upon catalytic activity. The mutant and wild type L-threonine deaminases differ in the apoenzyme formed as a consequence of alkaline dialysis. Dialysis of the mutant enzyme yields an apoenzyme mixture, apparently of dimers and monomers, while the wild type enzyme yields only dimers. The CU18 L-threonine deaminase, is however, indistinguishable from the wild type enzyme in molecular weight and subunit composition.


Nicotinamide adenine dinucleotide phosphate-malic enzyme of rat liver. Purification, properties, and immunochemical studies. Rat liver malic enzyme (EC 1.1.1.40) was purified from livers of rats fasted and refed a high sucrose diet containing 1% desiccated thyroid powder. The purification was accomplished by a six-step procedure. The specific activity of the purified enzyme was increased 181-fold above that of the initial high speed supernatant of liver extracts. Slight additional purification of malic enzyme was achieved with preparative disc electrophoresis. The specific activities of the purified rat liver malic enzyme from the least two steps were between 28.0 and 30.5 units per mg of protein. Homogeneity of the purified enzyme was determined by disc and starch gel electrophoresis as well as sedimentation velocity and sedimentation equilibrium studies. The molecular weight and S20, w values of rat liver malic enzyme are 268,000  and 10.2, respectively. Amino acid analysis based on milligram of protein hydrolyzed yielded higher amounts of leucine and glutamic acid but lower quantities of alanine and voline per subunit than the corresponding Escherichia coli enzyme...


A deoxyribonucleic acid ligase from nuclei of rat liver. Purification and properties. A DNA ligase has been extensively purified from nuclei of rat livers. The ligase seals single strand nicks in DNA with any of the four usual bases on either the 3 or 5 sides. It requires ATP and a divalent cation (Mg-2plus or Mn-2plus) for activity. At low Mg-2plus concentrations the activity is greatly stimulated by a variety of monovalent cations. Relatively small excesses of either monovalent or divalent cation above the amounts which give maximal activity lead to inhibition of activity. Poly(G) and poly(I) inhibit ligase activity; several other polyribonucleotides are not inhibitory. Low concentrations of inorganic pyrophosphate are inhibitory. The molecular weight of the ligase is estimated from gel filtration to be about 10 times 10-4.


Effects of 6- and 8-substituted analogs of adenosine 3':5'-monophosphate on phosphoenolpyruvate carboxykinase and tyrosine aminotransferase in hepatoma cell cultures. A variety of 6- and 8-substituted analogs of cAMP (cyclic adenosine 3:5-monophosphate) have been tested for their ability to increase activity of tyrosine aminotransferase (EC 2.6.1.5) in cultured Reuber H35 hepatoma cells. Some analogs, particularly the 8-thio-substituted ones, produced effects approximately equivalent to those generated by N-6, O2'-dibutyryl cAMP. In contrast, cAMP and its O-2-monobutyryl derivative were relatively ineffective even at very high concentrations, whereas three other analogs actually depressed the activity of the aminotransferase. Changes in enzyme activity generated by the various analogs were paralleled closely by changes in the relative rate of aminotransferase synthesis. An excellent correlation was found to exist between the ability of any given analog to influence the activity of tyrosine aminotransferase and that of phosphoenolpyruvate carboxykinase (EC 4.1.1.32). A similar correlation was found to exist between the ability of various analogs to evelate the activity of these enzymes and to inhibit reversibly the growth of H35 cells. Only one of five inhibitors of cAMP phosphodiesterase activity tested produce any increase in aminotransferase activity when added alone. All of the 6- and 8-substituted analogs tested, including noniducers, stimulated f1 histone phosphorylation in crude rat liver extracts with approximately equal potencies. On the other hand, dibutyryl cAMP was only a weak activator of protein kinase in vitro, even though it is a potent enzyme inducer. A possible resolution of this apparent discrepancy has been provided by preliminary analyses of site-specific f1 histone phosphorylation in whole cells. Only compounds active as aminotransferase inducers are capable of stimulating phosphorylation of the serine-37 residue of endogenous f1 histone (3- to 10-fold).


Nuclear relaxation studies on human methemoglobin. Observation of cooperativity and alkaline Bohr effect with inositol hexaphosphate. Ehanced spin-lattice relaxation (1/t1) of water protons induced by the heme iron of human aquomethemoglobin is exchanged-limited (koff = 1.4 times 10-4 per s at 30 degrees, H+ =7.5 Cal per mol) as indicated by the temperature and frequencey dependencies. A comparison of deuteron and proton relaxation rates revealed an order of magnitude primary isotope effect and a small inverse secondary isotope effect on the escape rate of protons from the heme iron into bulk water establishing the exchange of protons and not the exchange of the entire water molecule to be the chemical mechanism of the entire water molecule to be the chemical mechanism of the exchange process. With fluoromethemoglobin, the relaxation rate is in the fast exchange region. The results can be understood in terms of a water molecule interacting with the heme iron at an iron to proton distance less than 3.4 A in aquomethemoglobin and a single proton at a distance of 4.11 A assignable to the NH proton of the distal histidine imidazole group in fluoromethemoglobin. The relaxation rates are pH-dependent and normal titrations with Hill coefficients n = 1 are observed. The pKa is less than or equal to 6. 7 with aquomethemoglobin and 8.5 with fluoromethemoglobin at 30 degrees C. The binding of inositol hexaphosphate in stoichiometric amounts has no significant effect on the magnetic susceptibility of solutions of aquomethemoglobin and fluoromethemoglobin, but in the former case it increases koff to 3.8 times 10-4 per s by lowering the H+ barrier to 6.8 Cal per mol. In fluoromethemoglobin, inositol hexaphosphate decreases the iron to distal histidine NH distance by 0.17 A and the electron relaxation time taus by 10% as determined by the frequency dependence of 1/T1. In the aquomethemoglobin system, inositol hexaphosphate induces a Bohr effect, raising the pKa of the ionization responsible for the 1/T1 titration to 7.2, and induces cooperativity in the pH titration with a Hill coeffocoemt n = 2.8 plus or minus 0.1. With fluoromethemoglobin, the normal pH titration curve is unaffected by inositol hexaphosphate (n approximately equal to 1). Further, relaxivity titrations with varying amounts of azide and fluoride near neutral pH show normal behavior (n = 1) with and without inositol hexaphosphate. These results indicated that inositol hexaphosphate alters the quaternary structure of methemoglobin to the deoxy conformation without causing a change in the spin state of the heme iron...


Effect of protein concentration on the molecular weight of delta5-3-ketosteroid isomerase. The molecular weight of delta-5-3-ketosteroid isomerase from Pseudomonas testosteroni was determined by means of sedimentation equilibrium and exclusion chromatography over a wide range of enzyme concentrations in 0.2 M potassium phosphate buffer, pH 7.0. In addition, the sedimentation constant of the enzyme was determinded over an extended range of concentrations. The enzyme was found to have a molecular weight of 26,000 plus or equal to 1,000, suggesting that it is a dimer of identical or similar 13,400 molecular weight polypeptide chains. In the ultracentrifuge this dimeric species was found to undergo aggregation at enzyme concentrations above 2 mg per ml and dissociation at enzyme concentrations below 0.05 mg per ml. Exclusion chromatography studies indicate that under the conditions of chromatography the oligomeric enzyme is partially dissociated at enzyme concentrations in the range 0.2 to 0.002 mug per ml. These results suggest that under conditions of enzyme assay in 0.2 M potassium phosphate buffer, pH 7.0, isomerase is in a monomeric state of aggregation.


Kinetic study by pulse radiolysis of the lactate dehydrogenase-catalyzed chain oxidation of nicotinamide adenine dinucleotide by HO2 and O2-RADICALS. The lactate dehydrogenase-catalyzed chain oxidation of NADH (LDH-NADH) by the superoxide radicals, HO2 and O2, has been studied with pulse radiolysis in the pH range between 4.5 and 9.0. The rate constants for the oxidation of the LDH-NADH by HO2 and O2 determined at 23 degrees are 1.2 times 10-6 M(-1) s(-1) and 3.6 times 10-4 M(-1) s(-1), respectively. The latter represents an activation of over 1000-fold by the enzyme. A chain reaction mechanism consistent with the results from these kinetic studies has been proposed.


Effect of N-bromosuccinimide modification on dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli. Activity, spectrophotometric, fluorescence and circular dichroism studies. When dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli B, MB 1428, is treated with approximately a 5 mol ratio of N-bromosuccinimide (NBS) to enzyme at pH 7.2 and assayed at the same pH, there is a 40% loss of activity due to the modification of 1 histidine residue and possibly 1 methionine residue before oxidation of tryptophan occurs. The initial modification is accompanied by a shift of the pH for maximal enzymatic activity from pH 7.2 to pH 5.5 Upon further treatment with N-bromosuccinimide, the activity is gradually reduced from 60 to 0% as tryptophan residues become oxidized. An NBS to enzyme mole ratio of approximately 20 results in 90% inactivation of the enzyme. When the enzyme is titrated with NBS in 6 M guanidine HCl, 5 mol of tryptophan react per mol of enzyme, a result in agreement with the total tryptophan content as determined by magnetic circular dichroism. The 40% NBS-inactivated sample posses full binding capacity for methotrexate and reduced triphosphopyridine nucleotide, and the Km values for dihydrofolate and TPNH are the same as for the native enzyme. After 90% inactivation, only half of the enzyme molecules bind methotrexate, and the dissociation constant for methotrexate is 40 nM as compared to 4 nM for native enzyme in solutions of 0.1 M ionic strength, pH 7.2 Also, TPNH is not bound as tightly to the modified enzyme-methotrexate complex as to the unmodified enzyme-methotrexate complex. Circular dichroism studies indicate the 90% NBS-inactivated enzyme has the same alpha helix content as the native enzyme but less beta structure, while the 40% inactivated enzyme is essentially the same as the native enzyme. Protection experiments were complicated by the fact that NBS reacts with the substrates and cofactors of the enzyme. Although protection of specific residues was not determined, it was clear that TPNH was partially protected from NBS reaction when bound to the enzyme, and the enzyme, and the enzyme was not inactivated by NBS until the TPNH had reacted.


Coordinated incorporation of nascent peptidoglycan and teichoic acid into pneumococcal cell walls and conservation of peptidoglycan during growth. Choline-containing pneumococcal cell wals are sensitive to autolysin, whereas ethanolamine-containing walls are not. Bacteria were labeled with radioactive peptidoglycan precursors while growing either in choline- or in ethanolaminecontaining media. Subsequently, the labeled cells were allowed to grow for four to five generations in nonradioactive medium supplemented with the alternative amino alcohol source (i.e. cells labeled in choline medium yields ethanolamine; cells labeled in ethanolamine medium yields choline). The autolysin sensitivity of the isotope label in cell walls prepared from such bacteria indicates that nascent peptidoglycan and teichoic acid units that are synthesized at the same time are attached to one another, incorporated into the cell surface at the cellular equator, and remain conserved during growth the division of the bacteria.


Altered ganglioside biosynthesis in mouse cell cultures following transformation with chemical carcinogens and x-irradiation. Chemicaly and x-ray-transformed subclones of BALB/c 3T3 mouse embryo cells were found to have reduced amounts of the mono- and disialogangliosides galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (Gm1) and N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (Gd1a), and increased amounts of N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide (Gm2). The activity of the enzyme UDP-Gal:Gm2 galactosyltransferase was reduced to between 2.7 and 14.3% of normal in the transformed clones. Other ganglioside glycosyltransferase activities were unaffected. This enzymatic change was consistent with the observed alteration in ganglioside pattern in the transformed cells. The residual galactosyltransferase activity in the transformed cells was kinetically similar to the normal enzyme, suggesting that transformation alters ganglioside biosynthesis by blocking enzyme synthesis at the translational or transcriptional levels.


A novel diazonium-sulfhydryl reaction in the inactivation of yeast alcohol dehydrogenase by diazotized 3-aminopyridine adenine dinucleotide. Diazotized 3-aminopyridine adenine dinucleotide has been found to modify four sulfhydryl groups per molecule of enzyme during the complete inactivation of yeast alcohol dehydrogenase. The reaction of sulfhydryl groups was indicated by titration studies with 5,5-dithiobis(2-nitrobenzoic acid) as well as isolation and quantitation of the cysteinyl derivative released by acid hydrolysis of the modified enzyme. The cysteinyl derivative was identified as S-(3-pyridyl)cysteine. Authentic S-(3-pyridyl)cystein was synthesized and structurally characterized for these studies. Diazonium-sulfhydryl reactions were demonstrated for a number of diazonium derivatives with cysteine, homocysteine, glutathione, and mercaptoethanol at 0-4 degrees and neutral pH. Second order rate constants were determined in reactions of these sulfhydryl compounds with diazotized 1-methyl-3-aminopyridinium chloride, diazotized 3-aminopyridine adenine dinucleotide, and diazotized 3-aminopyridine adenine dinucleotide phosphate.


Purification and properties of hyaluronidase from bull sperm. Hyaluronidase from bull sperm was fractionated by ammonium sulfate and further purified by DEAE-cellulose and Sephadex chromatography. The highly purified hyaluronidase preparation showed 2,370 units per mg of protein (68,730 N.F. units per mg of protein), i.e. 182-fold purification. Disc gel electrophoresis showed one major component. The molecular weight of bull sperm hyaluronidase was 62,000 by sodium dodecyl sulfate gel electrophoresis. Hyaluronidase from bull sperm has optimum activity at pH 3.8 and an absolute requirement for cations. Kplus and Naplus have a greater effect than Ca2plus, Mg2plus, and Mn2plus, whereas Co2plus, Cu2plus, and Zn2plus do not affect the enzyme activity. Purified preparations are less stable than crude extracts stored frozen at minus 15 degrees. Km of hyaluronidase with hyaluronic acid as substrate is 3.7 mg per ml and Vmax is 2.4 mumol per min by Hofstee plot.


Postillumination adenosine triphosphate synthesis in Rhodospirillum rubrum chromatophores. I. Conditions for maximal yields. The very low level of postillumination ATP synthesis in chromatophores was markedly stimulated when permeant anions (thiocyanate or perchlorate) or permeant cations (potassium in the presence of valinomycin) were added to the light stage. Although these compounds stimulated also light-induced proton uptake in chromatophores the pH dependence of both photoreactions was different. Proton uptake peaked at pH 6.5 while the amount of postillumination ATP was maximal when the light stage was carried out around pH 7.7. The increased yield of ATP at the more alkaline pH could not be explained by a slower decay of the high energy state at this pH, since the decay rate was faster at pH 7.7 than at pH 6.5. The proton concentration gradient which is maintained across the chromatophore membrane in the light was also found to increase when the external pH was raised from 6.0 to 8.0. Only a minimal amount of postillumination ATP was formed when this gradient was below 2.1 pH units, but above this value the ATP yield rose steeply as a function of the increasing pH gradient. In light of these results it is suggested that in order to obtain a high yield of postillumination ATP synthesis in chromatophores two conditions are required: the particles have to be loaded with a sufficient number of protons and a light-induced pH gradient above a certain threshold value has to be maintained across their membrane. The low yield of postillumination ATP in chromatophores and the increase obtained by adding permeating ions, is thus explained by similar variations in the extent of the pH gradient, which exceeded the threshold value only in the presence of the permeating ions.