Serum corticosterone as a quantitative test of the phlogistic potency of various agents topically applied in the rat. The application into the rat conjunctiva of various phlogistic agents, such as croton oil, mustard oil and formaldehyde, elicits an increase of serum corticosterone linearly related to the log of the applied concentrations, so that from their parallelized regression lines it is possible to calculate the phlogistic potency of each tested agent in reference to croton oil. The time kinetic of such an increase (elicited by croton oil) is compared with that of two other parameters previously adopted as indirect quantitative indices of the phlogosis: the adrenal ascorbic acid depletion and the liver tyrosine-alpha-ketoglutarate transaminase increase. Serum corticosterone is shown to be the quickest and the most sensitive of the adopted indices, even if the phlogistic potency of the tested agents and the precision of these evaluations substantially coincides whatsoever the index adopted. Finally the pathways of adrenocortical activation are investigated and it is shown that the activation may be peripherally blocked by topical application of corticosteroids (but not of local anesthetics) and centrally by hypophysectomy or parenteral administration of pentobarbital plus morphine.


Photoproduction of ammonium ion from N2 in Rhodospirillum rubrum. NH+4 excretion was undetectable in N2-fixing cultures of Rhodospirillum rubrum (S-1) and nitrogenase activity in these cultures was repressed by the addition of 10 mM NH+4 to the medium. The glutamate analog, L-methionine-DL-sulfoximine (MSX), derepressed N2 fixation even in the presence of 10 mM extracellular NH+4. When 10 mg MSX/ml was added to cultures just prior to nitrogenase induction they developed nitrogenase activity (20% of the control activities) and excreted most of their fixed N2 as NH+4. Nitrogenase activities and NH+4 production from fixed N2 were increased considerably when a combined nitrogen source, NH+4 (greater than 40 mumoles NH+4/mg cell protein in 6 days) or L-glutamate (greater than 60 mumoles NH+4/ mg cell protein in 6 days) was added to the cultures together with MSX. Biochemical analysis revealed that R. rubrum produced glutamine synthetase and glutamate synthase (NADP-dependent) but no detectable NADP-dependent glutamate dehydrogenase. The specific activity of glutamine synthetase was observed to be maximal when nitrogenase activity was also maximal. Nitrogenase and glutamine synthetase activities were repressed by NH+4 as well as by glutamate. The results demonstrate that utilization of solar energy to photoproduce large quantities of NH+4 from N2 is possible with photosynthetic bacteria by interfering with their regulatory control of N2 fixation.


Energy coupling and respiration in Nitrosomonas europaea. Intact cells of Nitrosomonas europaea grown in an ammonium salts medium will oxidise ammonium ions, hydroxylamine and ascorbate-TMPD; there is no oxidation of carbon monoxide, methane or methanol. The Km value for ammonia oxidation is highly pH dependent with a minimum value of 0.5 mM above pH 8.0. This suggests that free ammonia is the species crossing the cytoplasmic membrane(s). The measurement of respiration driven proton translocation indicates that there is probably only one proton translocating loop (loop 3) association with hydroxylamine oxidation. The oxidation of "endogenous" substrates is sometimes associated with more than one proton-translocating loop. These results indicate that during growth hydroxylamine oxidation is probably associated with a maximum P/O ratio of 1.


D-Alanine dehydrogenase. Its role in the utilisation of alanine isomers as growth substrates by Pseudomonas aeruginosa PA01. Pseudomonas aeruginosa PA01 was found to utilise both the D- and L-isomers of alpha-alanine and also beta-alanine as sole sources of carbon and energy for growth. Enzymological studies of wild-type cultures and comparison with mutants deficient in growth upon one or more isomers of alanine led to the following conclusions: (i) utilisation of D-alanine involved its direct oxidation by an inducible, membrane-bound, cytochrome-linked dehydrogenase; (ii) utilisation of L-alanine required its conversion to the directly oxidisable D-form by a soluble racemase; (iii) utilisation of beta-alanine, like L-alanine, involves both the racemase and D-alanine dehydrogenase enzymes, but in addition must involve other enzymes the identity of which is still speculative; (iv) P. aeruginosa, like Escherichia coli, appears to take up D-alanine and L-alanine by means of two specific permeases.


Monosaccharide transport systems in the yeast Rhodotorula glutinis. By using D-glucose, D-xylose, D-galactose and D-fructose in the strictly aerobic yeast Rhodotorula glutinis and by comparing the half-saturation constants with inhibition constants the yeast was shown to possess a single common system for D-xylose and D-galactose (Km's and Ki's all between 0.5 and 1.1 mM) but another distinct transport system for D-fructose. The transport of D-glucose has a special position in that glucose blocks apparently allotopically all the other systems observed although it uses at least one of them for its own transport. The different character of D-glucose uptake is underlined by its relative independence of pH (its "Km" is completely pH-insensitive) in contrast with all other sugars. At low concentrations, all sugars show mutual positive cooperativity in uptake, suggesting at least two transport sites plus possibly a modifier site on the carrier.


Energetic aspects of anaerobic growth of Aerobacter aerogenes in complex medium. Molar growth yields for anaerobic growth of Aerobacter aerogenes in complex medium were much higher than for growth in minimal medium. In batch cultures the molar growth yield for glucose varied from 44 to 50 and YATP from 17.1 to 18.8. For glucose-limited chemostat cultures a value of 17.5 g/mole was found for Y max ATP and a value of 2.3 mmoles ATP/g dry weight h for the maintenance coeficient. Growth-dependent pH changes were used to control the addition of fresh medium, containing excess of glucose to a continuous culture. The specific growth rate and the population density were dependent on the pH difference between the inflowing medium and the culture. At a mu value of 1.44 h-1 the molar growth yield for glucose was about 70 and Y ATP about 28.5. An equation is presented, which gives the relation between theoretical and experimental Y max ATP values.


Proton-motive force and the motile behavior of Bacillus subtilis. Changes in the proton-motive force cause a transient change in the motile behavior of Bacillus subtilis cells. Both an increase and a decrease in the proton-motive force caused transient tumbling. Simultaneous decrease of proton-motive force and increase of attractant concentration lessens the response toward the attractant. A simultaneous increase of proton-motive force and increase of attractant concentration prolonges the response toward attractant. A hypothesis explaining the various effects is given.


Ammonium uptake and metabolism by mitrogen fixing bacteria. II. Klebsiella pneumoniae. The primary steps of N2, ammonia and nitrate metabolism in Klebsiella pneumoniae grown in a continuous culture are regulated by the kind and supply of the nitrogenous compound. Cultures growing on N2 as the only nitrogen source have high activities of nitrogenase, unadenylated glutamine synthetase and glutamate synthase and low levels of glutamate dehydrogenase. If small amounts of ammonium salts are added continuously, initially only part of it is absorbed by the organisms. After 2-3 h complete absorption of ammonia against an ammonium gradient coinciding with an increased growth rate of the bacteria is observed. The change in the extracellular ammonium level is paralleled by the intracellular glutamine concentration which in turn regulates the glutamine synthesis and an induction of glutamate dehydrogenase synthesis. Upon deadenylation these events are reversed.--Addition of dinitrophenol causes transient leakage of intracellular ammonium into the medium.


Ammonia assimilation in the fission yeast Schizosaccharomyces pombe 972. Glutamine synthetase (GS) activity of Schizosaccharomyces pombe 972 was high in ammonia-limited cultures, low in phosphate- and sulphate-limited cultures and not detected in glucose-limited cultures. When ammonia was 'pulsed' into an ammonia-limited culture then GS activity decreased at a rate faster than that calculated if enzyme synthesis ceased and enzyme was diluted out by growth. Enzyme activity increased in ammonia-starved, phosphate-limited cultures and in the ammonia 'pulse' system when the added ammonia had been utilised. These increases in enzyme activity were prevented by the presence of 100 mug/ml cycloheximide. GS activity was inversely related to the intracellular concentration of glutamate.


Monoamine metabolism in human brain.

Monoamine metabolism in human brain. Norepinephrine (NE), dopamine (DA), tyrosine hydroxylase (TH), catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) levels were measured in human brain tissue obtained at autopsy from a series of 39 patients dying of various medical and accidental causes. The nine following brain areas were studied: globus pallidus, thalamus, hypothalamus, hippocampus, substantia nigra, floor of the fourth ventricle, orbital cortex, caudate nucleus, and mammillary bodies. Enzyme activity correlated positively with age in all brain areas for MAO (with both benzylamine and tryptamine substrates) but no consistent pattern of correlation was found for COMT and TH. Mean MAO activity was significantly higher in women than men. There is increased brain MAO activity during late childhood and adolescence. These data are consistent with previous evidence suggesting that age and sex are important determinants of amine metabolism in the human central nervous system.


[Management of second stage of labour: observations, reflections, advices (author's transl)]. All vaginal deliveries of the Department of Obstetrics and Gynecology of the University Basel (N = 4081) during the year 74/73 and of the University Tübingen (N = 3249) 75/74 were analysed using an IBM-system 370/135 Only alive singletons beyond the 28th week of gestation were analysed. Clinical management was quite different in the two departments; the incidence of vaginal operations (Basal 11.2%, Tübingen 12.6%), however, as well as the distribution of pH-values and Apgar-scores after 1 min were quite similar. Basel: Acidotic risk (i.e. pHUA less than 7.200) 13.5%, severe acidotic risk (i.e. pHUA less than 7.100) 1.55%, low Apgar-scores (1--3) 0.7%. Tübingen: :12.3%, 2.11%, 1.6%. 3.5% of all parturients (Basel) had duration of second stage of labour with  active maternal pressure support lasting more than 30 min. In two highly selected samples differing only with regard to the occurrence of cord-entanglements at birth (N1 = 1755, N2 = 1098) the association (rank correlation method according to Kendall) between the parameters of the fetal acid-base balance and the duration of second stage of labour as well as duration of the period with "active bearing down" was studied. Without cord encirclements pH in the umbilical artery fall --0.087 and in the umbilical vein --0.115 units and with cord complications the values amounted to --0.062 (UA) and --0.120 (UV) respectively pro 60 min duration of second stage with "bearing down efforts". Analogous computations for pCO2, pO2 and HbO2 are presented. Apgar-scores in these samples showed a very loose connection with the time variables. From these data the conclusion is drawn that the indication to perform vaginal operations for termination of delivery should not primarily be governed by the factor time but rather by the whole obstetrical situation i.e. the possible fetal risk of the intervention. This holds only if maternal welfare is established and fetal well being is monitored continuously.


Antibiotics produced by Streptomyces olivaceus 142. I. Characterization of the FPG mutant and conditions of production of antibiotic WR 142-FPG. By treating the Streptomyces olivaceus 142 strain simultaneously with ethyleneimine and UV radiation, the FPG mutant was isolated, which was characterized by the fact that in submerged cultures it produces a cytotoxic substance for fibroblasts and tumor cells and inhibits growth of pathogenic fungi. The mutant differs from other strains not only in having a different spectrum of antimicrobial activity, but also by taxonomic properties such as color of the aerial mycelium, liquefaction of gelatin, growth on cellulose, production of ammonia and nitrate reduction. An optimal culture medium and conditions of biosynthesis of the antibiotic in submerged cultures on the shaking machine and in 20-liter fermentation tanks were elaborated. The active substance was designated by the symbol WR 142-FPG.


Search for new aminoguanidine derivatives with immunosuppressive and cytostatic properties. I. Reactions of amino-, nitroamino- and diaminoguanidine with acetylpyruvic acid ethyl ester. Reactions of amino-, nitroamino- and diaminoguanidine (I, II, III) with acetylpyruvic acid ethyl ester (IV) at varying pH of the medium were studied. It was found that the nucleophilic hydrazine NH2 group of compounds I, II, and III) attacks the keto group of ester IV in position gamma or alpha. The newly synthesized compounds were submitted to biological evaluation.


Sustrate-specificity of glucomylase (E.C. 3.2.1.3) exemplified by p-nitroaniline n-glucosides. The present paper supplements the previous investigations on the mechanism of reactions catalyzed by amylolytic enzymes. The performed experiments corroborated the data on transglucosylative properties of animal glucoamylases which were found to depend on the structure and concentration of substrate. The p-nitroanilines were first introduced in this type of research enabling to find the differences of activity mechanism of animal glucoamylases and Aspergillus niger glucoamylase. In complement to the data reported by Japanese students the transglucosylative properties of glucoamylases were shown to depend not only on the enzyme origin and substrate concentration but also on the chemical composition of applied substrate and concentration of hydrogen ions in the reaction medium.


Effect of acid pH, salts, and temperature on the infectivity and physical integrity of enteroviruses. At 2 degrees and 30 degrees C, enteroviruses are more stable on the acid than on the alkaline side of neutrality. In the range from pH 3 to 9, temperature is so influential that the fastest inactivation rate at 2 degrees C is slower than the slowest inactivation rate at 30 degrees C. Specific ions or salts also affect the rate of inactivation of enteroviruses. NaCl and other chloride salts enhance the inactivation of poliovirus at pH 3. NaCl is considerably less effective against poliovirus in the range of pH 4.5 to 7.0 than at pH less than 4.5. Loss of RNA infectivity of the virus particle proceeds as rapidly as the loss of infectivity of the particle itself, except at pH 3 in the presence of MgCl2. Inactivation results in alterations to the physical integrity of enteroviruses. At pH 5 and 7, RNA hydrolysis of poliovirus particles occurs; and at pH3, 5,6, and 7 the nucleic acid becomes susceptible to ribonuclease. Only virus particles inactivated at pH 3 show a sensitivity to chymotrypsin. The hemagglutinins of echovirus type 7 are destroyed during inactivation at pH 3,4,5, and 6; but at pH 6 this alteration precedes the loss of infectivity. The pH of the suspension is a primary determinant of the mechanism of virus destruction and possibly of the loss of infectivity at these temperatures.


Investigation on the infection of cucumber mesophyll protoplasts with cucumber mosaic virus. Isolated protoplasts from the first leaf mesophyll of cucumber plants have been successfully infected in vitro with cucumber mosaic virus (CMV). Virus instability before, during and after inoculation of the protoplasts resulted in low infectivities when extracts were assayed on cowpea; however, viral RNA extraction improved the bioassay technique. Attempts to optimize inoculation and incubation of protoplasts are outlined, incorporating the improved assay.


Thymidine-kinase in cytomegalovirus infected cells. In human diploid fibroblast LEP cells infected with AD169 strain of human cytomegalovirus (CMV) a sharp increase of cytosol thymidine kinase activity was observed. The properties of the cytosol enzymes from infected and non-infected cells were compared. No significant differences between the enzymes from infected and control cells were observed in substrate specificity, pH dependence, thermostability and relative electrophoretic mobility. Human sera containing high titres of CMV complement-fixing antibodies did not neutralize the enzyme from infected cells. It is concluded from these results that the increase of cytosol thymidinekinase activity in CMV-infected cells was due to an enhancement of cellular thymidine kinase.


[Role of viral-bacterial associations in meningitis in children]. A total of 50 section cases of meningitis in children were investigated. All patients revealed acute viral respiratory infections with generalization, including lesions of the central nervous system (CNS). In 14 children moreover meningococcal infection was diagnosed. In 30 children lesions of the CNS were due to mixed bacterial microflora. In 6 children along with acute viral respiratory infections (AVRI) mycoplasmosis was also revealed. Etiology of the process was determined on the basis of characteristic structural changes in the CNS and other organs, findings of virological and bacteriological investigations. In the majority of children the intravascular blood coagulation was observed. Waterhouse-Friderichsen's syndrome was revealed mainly in meningococcal infection. In order to ascertain the data obtained case records of 120 children, who had undergone treatment in connection with meningococcal infection, were analysed. It turned out that all 42 children, who had developed this disease independently, recovered. Combination of the disease with AVRI led to fatal outcomes in 12 cases out of 72.


[Structural and functional characteristics of the spleen in systemic and local allogenic graft versus host reaction]. Morphological and histochemical characteristics of the spleen in the systemic and locally induced in the spleen transplant host reaction were compared. In both forms there were observed infiltration of the organ with macrophages and pyroninophilial blasts characterized by a high level of the activity of enzymes of lysosomas and a hexosemonophosphate cycle. In the process of interaction between genetically heterogenous mononuclear cells the disturbance of enzymatic processes in them and in the adjacent cellular elements took place, and susequently they underwent necrosis. Most frequently this process was observed in lymphoid follicles. The destructive component in the spleen tissue was more pronounced in the systemic transplant against host reaction.


Murray Valley encephalitis virus infection in mosquitoes and domestic fowls in Queensland, 1974. Field studies during an epidemic of Murray Valley encephalitis (MVE) led to the isolation of MVE virus from a pool of mosquitoes (Culex annulirostris) and a sentinel chicken from Charleville, south-west Queensland. A high proportion of domestic fowls at Charleville had antibody to MVE virus at the beginning of February 1974, in advance of the first case recognized in Queensland and allowing early warning from health authorities. A survey of antibody in domestic fowls in mid-1974 suggested widespread activity of MVE virus in western and east-central Queensland. Virus isolation and serological studies showed activity in south-west Queensland of three other viruses known to infect man, Ross River, Sindbis and Kunjin viruses.


Clinical evaluation of a dialysate regeneration system for maintenance haemodialysis. A commercially available sorbent-based dialysate regeneration system has been compared to conventional single-pass dialysate delivery systems for treatment periods of six weeks in 13 patients on maintenance dialysis. The results of treatment were virtually identical in comparing sorbent and conventional systems except that seven of the eight patients using 2-5 M2 dialysers for 3--4 hours thrice per week developed asymptomatic metabolic acidosis with the dialysate regeneration system. This complication was not seen in the five patients using 1-3 M2 dialysers and having a 6--7 hour treatment thrice weekly. Dialysate regeneration systems are particularly suited for use when water supplies are limited or of insufficient purity for single-pass dialysis, and when a portable artificial kidney is required. To avoid metabolic acidosis with this system, using currently available disposable cartridges, each dialysis treatment should be of at least 4-5 hours duration.


Effects of induced cryptorchidism in bulls. Ninety bull calves were made cryptorchid by forcing the testes into the abdominal cavity and ablating the scrotum with elastrator rings. Normal spermatogenesis was seen following histological examination of some of the testes approximately 17 months after the operation. No effect on bull behaviour was noted in the cryptorchids although plasma testosterone levels were lower than those reported for bulls. It is possible that this method may leave some of the cryptorchids fertile.


[Tubular structure and germ cell distribution of cryptorchid or normal testes in early childhood (author's transl)]. Many recent publications have demonstrated that the cryptorchid testicle (and, to a lesser extent, the descended partner) are progressively injured from the second year of life onwards. Do these injuries occur in an organ which has been healthy up to this time or are they superimposed on a structurally abnormal testicle? In order to answer this, parts of cryptorchid testicles, of the descended partners, and of normal testicles were compared by histological examination of serial sections. Parts of four testes from children aged 4-7 months (2 specimens obtained by biopsy and 2 from autoptic material) and parts of four testes from children 1 1/2 years old (2 obtained by biopsy and 2 from autoptic material) were examined. The biopsies were fixed in Stieve's fixative. Tissue samples from clinically healthy children who had died suddenly were fixed in 4% formalin. The tissue was embedded in paraffin and sectioned serially; 6 mum sections were stained with HE. The spermatogonia in each cross-section and in each oblique section of a same tubule were counted and the counts of the latter were adjusted to a cross-section 50-60 mum in diameter. This counting technique did not alter the density of spermatogonia. The graphs present data on the density of spermatogonia through the lengths of the tubules examined and demonstrate tubular branching and blind ends. In the first year of life the cryptorchid testis and its descended partner showed repeated long sections lacking spermatogonia in the same tubule, whereas in normal testes the spermatogonia were more evenly distributed. The cryptorchid testis showed increased tubule branching in the areas examined. In the second year of life the tubules of the cryptorchid testis and its descended partner manifest areas free of germ cells, increased branching, and blind ends. The cryptorchid testis also had a tubule completely free of spermatogonia. The germ cell-free parts were always associated with a smaller tubule diameter than normal. The normal testes did not disclose increased branching or spermatogonium-free areas within similar lengths of tubules and showed an even distribution of spermatogonia. The different distribution of spermatogonia within the tubules and the increased branching of the tubules in cryptorchid testes indicate a previous disturbance of testis development.


Genetics of peroxisomal enzymes in the mouse: nonlinkage of D-amino acid oxidase locus (Dao) to catalase (Cs) and L-alpha-hydroxyacid oxidase (Hao-1) loci on chromosome 2. An electrophoretic polymorphism of the peroxisomal enzyme D-amino acid oxidase was observed in NZC strain Mus musculus. F1 (NZC X BALB/c) mice showed a codominant allcle two-banded phenotype which is inconsistent with the dimeric subunit structure reported for this enzyme in other species. The enzyme locus (Dao) was shown to segregate independently of Hao-1, encoding the peroxisomal enzyme hydroxyacid oxidase (liver on A4 isozyme). Thus Dao is not linked to previously mapped peroxisomal enzyme loci, Hao-1 and Cs, closely localized on chromosome 2.


Myocardial function during acute hypoxia in the calf. Hemodynamics and myocardial contractility were evaluated in five unanesthetized calves during acute hypocapnic and isocapnic hypoxia and during acute hypocapnic hypoxia with beta-adrenergic blockade. Both hypocapnic and isocapnic hypoxia, with mean PaO2 levels of 33.1 and 39.1 mm Hg respectively, produced a decline in stroke volume and index, while cardiac output and index were maintained at normoxic control levels by an increase in heart rate. Evaluation of myocardial contractility indices suggested an augmentation of left ventricular contractility during both hypocapnic and isocapnic hypoxia. Beta-adrenergic blockade effectively eliminated the increase in left ventricular contractility during hypocapnic hypoxia, suggesting an important role of the adrenergic nervous system in the genesis of the cardiovascular response of the calf to acute hypoxia. Right ventricular contractility indices failed to demonstrate a clear-cut augmentation of contractility during hypocapnic and isocapnic hypoxia when the concurrent increase in afterload was considered. Mean pulmonary arterial blood pressure rose significantly during hypocapnic and isocapnic hypoxia. The pulmonary pressor response to hypocapnic hypoxia was significantly augmented by beta blockade, indicating that the autonomic nervous system is capable of modifying the hypoxic pulmonary pressor response in this species.


The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenases, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates. Comparison of the activities of hexokinase, phosphorylase and phosphofructokinase in muscles from marine invertebrates indicates that they can be divided into three groups. First, the activities of the three enzymes are low in coelenterate muscles, catch muscles of molluscs and muscles of echinoderms; this indicates a low rate of carbohydrate (and energy) utilization by these muscles. Secondly, high activities of phosphorylase and phosphofructokinase relative to those of hexokinase are found in, for example, lobster abdominal and scallop snap muscles; this indicates that these muscles depend largely on anaerobic degradation of glycogen for energy production. Thirdly, high activities of hexokinase are found in the radular muscles of prosobranch molluscs and the fin muscles of squids; this indicates a high capacity for glucose utilization, which is consistent with the high activities of enzymes of the tricarboxylic acid cycle in these muscles [Alp, Newsholme & Zammit (1976) Biochem. J. 154, 689-700]. 2. The activities of lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, cytosolic and mitochondrial glycerol 3-phosphate dehydrogenase and glutamate-oxaloacetate transaminase were measured in order to provide a qualitative indication of the importance of different processes for oxidation of glycolytically formed NADH. The muscles are divided into four groups: those that have a high activity of lactate dehydrogenase relative to the activities of phosphofructokinase (e.g. crustacean muscles); those that have high activities of octopine dehydrogenase but low activities of lactate dehydrogenase (e.g. scallop snap muscle); those that have moderate activities of both lactate dehydrogenase and octopine dehydrogenase (radular muscles of prosobranchs), and those that have low activities of both lactate dehydrogenase and octopine dehydrogenase, but which possess activities of phosphoenolpyruvate carboxykinase (oyster adductor muscles). It is suggested that, under anaerobic conditions, muscles of marine invertebrates form lactate and/or octopine or succinate (or similar end product) according to the activities of the enzymes present in the muscles (see above). The muscles investigated possess low activities of cytosolic glycerol 3-phosphate dehydrogenase, which indicates that glycerol phosphate formation is quantitatively unimportant under anaerobic conditions, and low activities of mitochondrial glycerol phosphate dehydrogenase, which indicates that the glycerol phosphate cycle is unimportant in the re-oxidation of glycolytically produced NADH in these muscles under aerobic conditions. Conversely, high activities of glutamate-oxaloacetate transaminase are present in some muscles, which indicates that the malate-aspartate cycle may be important in oxidation of glycolytically produced NADH under aerobic conditions. 3. High activities of nucleoside diphosphate kinase were found in muscles that function for prolonged periods under anaerobic conditions (e.g...


Phosphate transport into brush-border membrane vesicles isolated from rat small intestine. Uptake of Pi into brush-border membrane vesicles isolated from rat small intestine was investigated by a rapid filtration technique. The following results were obtained. 1. At pH 7.4 in the presence of a NaCl gradient across the membrane (sodium concentration in the medium higher than sodium concentration in the vesicles), phosphate was taken up by a saturable transport system, which was competitively inhibited by arsenate. Phosphate entered the same osmotically reactive space as D-glucose, which indicates that transport into the vesicles rather than binding to the membranes was determined. 2. The amount of phosphate taken up initially was increased about fourfold by lowering the pH from 7.4 to 6.0.3. When Na+ was replaced by K+, Rb+ or Cs+, the initial rate of uptake decreased at pH 7.4 but was not altered at pH 6.0.4. Experiments with different anions (SCN-,Cl-, SO42-) and with ionophores (valinomycin, monactin) showed that at pH 7.4 phosphate transport in the presence of a Na+ gradient is almost independent of the electrical potential across the vesicle membrane, whereas at pH 6.0 phosphate transport involves the transfer of negative charge. It is concluded that intestinal brush-border membranes contain a Na+/phosphate co-transport system, which catalyses under physiological conditions an electroneutral entry of Pi and Na+ into the intestinal epithelial cell. In contrast with the kidney, probably univalent phosphate and one Na+ ion instead of bivalent phosphate and two Na+ ions are transported together.


Validation of a simple radiochemical assay measuring hydrolysis of choline-labelled microsomal phosphatidylcholine by phospholipase C. pH-dependence. Selective hydrolysis of phosphatidylcholine species, which are selectively radioactively labelled in vivo, does not appear to interfere with a radiochemical assay for hydrolysis of microsomal phosphatidylcholine by C-type phospholipases from Bacillus cereus or Clostridium perfringens. Both phospholipases substantially hydrolysed phosphatidylcholine over the pH range 4.0-10.0.


Transannular dioxygenation of 9,10-dimethyl-1,2-benzanthracene by cytochrome P-450 oxygenase of rat liver. 9,10-Dimethyl-1,2-benzanthracene is oxygenated by rat liver microsomal cytochrome P-450 oxygenase to its 9,10-epidioxide. This transannular 1,4-peroxide is converted further into the diol by the microsomal preparation and NADPH. These two products constitute the majority of the metabolites found under the conditions described.


Fatty acid synthesis in aorta. Isolation of fatty acid synthetase from chicken aorta. Fatty acid synthesis by subcellular fractions of human aorta was studied by measuring the incorporation of either radioactive acetyl-CoA or malonyl-CoA into long chain fatty acids. The high speed supernatant fraction contained fatty acid synthetase and was capable of de novo fatty acid synthesis. The fatty acid synthetase from chicken aorta was purified 800-fold from the high speed supernatant and was judged to be 10% pure at this level. Its molecular weight was estimated to be 450,000 on the basis of agarose gel filtration chromatography, while under dissociating conditions a molecular weight of 220,000 was obtained on sodium dodecyl sulphate disc gel electrophoresis. Fatty acid synthesis was dependent on acetyl-CoA, malonyl-CoA and NADPH. The major product was free palmitic acid. In enzymatic and physical characteristics the chicken aorta fatty acid synthetase strongly resembles the synthetase isolated from chicken liver. The two enzymes cross-react immuno-chemically and this homology provides the possibility of studying the synthesis and degradation of the aorta synthetase during the development of atherosclerosis.


Effect of altered lymphocyte function on immunologic disorders in NZB/NZW mice. NZB/NZW F1 female mice were treated with the immunosuppresive enzyme L-asparaginar antibodies, diminished deposition of gamma-globulins in kidneys, significantly delayed the onset of proteinuria, and reduced deaths from nephritis. These effects were associated with reduction of cellular IgM antibody synthesis to both T-dependent and T-independent antigens, but the graft-versus-host reaction was not affected. After several weeks of therapy, antibodies against Asnase appeared in the circulation, the effect on antibody synthesis was lost, ANA and anti-DNA appeared, followed by proteinuria and deaths from nephritis. Therefore Asnase proved to be an effective therapy in NZB/NZW mice, but its usefulness was limited by the appearance of inactivating antibodies.


Acute-phase reactant protein profiles: an aid to monitoring large bowel cancer by CEA and serum enzymes. The profiles of 4 acute-phase reactant proteins (APRPs) (haptoglobin (HPT), alpha1 antitrypsin (AAT), alpha1 acid glycoprotein (AGP) and prealbumin (PALB)) have been studied during the evolution of bowel cancer. Serial measurements of these APRPs can add to the information obtained from measurements of the level of CEA and hepatic enzymes during the monitoring of postoperative patients. There is considerable stability in the profile in a given individual in health, Rises of AAT and AGP are associated with metastases. High levels of HPT may suggest involvement of the bowel wall by recurrent cancer. PALB levels tend to reflect the nutritional status. A discriminant function based on the log CEA, AAT and AGP preoperative blood levels can considerably improve on the predictive value attained using CEA levels alone.


Amino acid sequence of the Anthopleura xanthogrammica heart stimulant, anthopleurin A. A highly potent heart stimulant, anthopleurin A, from Anthopleura xanthogrammica was shown to exist as a single polypeptide chain consisting of 49 amino acid residues. The sequence of the peptide was shown to be: Gly-Val-Ser-Cys-Leu-Cys-Asp-Ser-Asp-Gly-Pro-Ser-Val-Arg-Gly-Asn-Thr-Leu-Ser-Gly-Thr-Leu-Trp-Leu-Tyr-Pro-Ser-Gly-Cys-Pro-Ser-Gly-Trp-His-Asn-Cys-Lys-Ala-His-Gly-Pro-Thr-Ile-Gly-Trp-Cys-Cys-Lys-Gln as judged by Edman degradation of the carboxymethylcysteine derivative and the tryptic peptides obtained from the derivative. Although six carboxymethylcysteine residues were present in the polypeptide, no cysteine residues were detectable in the native protein, indicating that there are three cystine residues in anthopleurin A.


Magnetic resonance studies of the binding of 13C-labeled carbon monoxide to myoglobins and hemoglobins containing modified hemes. The effects of changes in the groups attached to the periphery of the porphyrin ring of the heme of various hemoglobin and myoglobins on the environment experienced by the ligand, carbon monoxide, have been studied by observation of the chemical shift of the bound 13CO. The results indicate that the major interaction between bound ligands and substituents around the porphyrin is that transmitted electronically from substituent to ligand. The nature of the protein environment around the ligand and the interaction between the proximal histidine (F8) and the ligand (through the iron atom) impose differences between subunits of hemoglobin and between myoglobins and hemoglobins which are largely, but not entirely, independent of these substituent effects. To assess the influence of protein structure on the chemical shifts of bound ligand, the shifts of 13CO bound to myoglobin and hemoglobins from a wide range of species have also been measured.


Correlation between the exposure of aromatic chromophores at the surface of the Fc domains of immunoglobulin G and their ability to bind complement. The recognition that certain biological effector functions associated with the Fc region of human IgG are mediated exclusively by either the Cgamma2 or Cgamma3 domains prompted a study of some of the physical properties of the isolated domains in an attempt to correlate these with functional differentiation. The degree of aromatic chromophore exposure of intact Fc and fragments corresponding to the Cgamma2 and Cgamma3 domains were determined by solvent perturbation difference spectroscopy using 20% ethylene glycol. For the monomeric Cgamma2 fragment one of the two tryptophans and all four of the tyrosines were exposed to solvent. In the pFc' fragment, which represented a dimer of two intact Cgamma3 domains, an average of 0.4 of the two tryptophans of 3.3 of the five tyrosines per chain were exposed. These data were consistent with the suggested involvement of tryptophan in complement fixation since Cgamma2 binds C1q but pFc' does not. Several fragments derived from the Cgamma3 region had previously been shown to have differing environments for their aromatic side chains from circular dichroism studies. These fragments have now been shown to exhibit different degrees of chromophore exposure to solvent. Removal of the carboxy-termimal heptapeptide from the intact, Cgamma3 domain resulted in a fragment not only showing a greater exposure of aromatic residues but also having the ability to bind Clq. Our data suggest that the structural requirements for C1Q binding may be quite commonplace within Fc, but tertiary folding limits their expression except in Cgamma2 in the native molecule. The solvent perturbation observed with Fc was somewhat lower than would have been expected from the results with the isolated domains, suggesting that interdomain interactions may result in burial of aromatic residues.


[Further remarks on histochemistry applied to myodiagnosis: findings of "type predominance" (author's transl)]. Data concerning muscular biopsies (histochemically examined) of three patients affected by Charcot-Marie-Tooth disease, neurogenic atrophy of spondilosic origin and benign congenital hypotonia, are described. The common finding was a histochemical appearence of "type predominance". This point and the possible "neurogenic" origin of benign congenital hypotonia, are discussed.


Purification and properties of debranching enzyme from dogfish muscle. Glycogen debranching enzyme (4-alpha-glucanotransferase amylo-1,6-glucosidase, EC 2.4.1.25 + 3.2.1.33) was purified 140-fold from dogfish muscle in a rapid, high-yield procedure that takes advantage of a strong binding of the enzyme to glycogen, and its quantitative adsorption to concanavalin A-Sepharose only when the polysaccharide is present. The final product was hrophoresis in the presence and absence of dodecyl sulfate. A molecular weight of 162,000 +/- 5000 was determined by sedimentation equilibrium analysis in good agreement with the value of 160,000 estimated by gel electrophoresis, but a low-sedimentation constant of 6.5 S suggests that the enzyme is asymmetric. The molecule appears to be made up of a single polypeptide chain with no evidence for multiple repeating sequences: it could not be dissociated into smaller fragments by dodecyl sulfate even after complete carboxymethylation; tryptic cleavage of the native protein yielded only two fragments of molecular weight 20,000 and 140,000 without loss of enzymatic activity. The amino acid composition of the enzyme is reported; no covalently bound phosphate or carbohydrate could be detected. All 32 sulfhydryl groups present were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) under denaturing conditions; eight reacted readily in the native enzyme without loss of catalytic activity, while substitution of eight additional ones lowered the activity by 50%. Inactivation was greatly reduced by glycogen; the polysaccharide also influenced markedly the electrophoretic behavior of the enzyme and large filamentous aggregates were formed when solutions of both were mixed. Purified debranching enzyme releases 3 mumol of glucose min-1 mg-1 at 19 degrees C, pH 6.0, from a glycogen limit dextrin and one-tenth this amount when the native polysaccharide is used as substrate; glycogen is quantitatively degraded in the presence of phosphorylase. None of the usual sugar phosphates or nucleotide effectors of glycolysis affected enzymatic activity. No phosphorylation by either dogfish or rabbit skeletal muscle protein kinase or phosphorylase kinase could be demonstrated, nor any direct interaction with phosphorylase as measured by SH-group reactivity, enzymatic activity, or rate of phosphorylase b to a conversion. Purification of the 160,000 molecular weight M-line protein of skeletal muscle resulted in the quantitative removal of debranching enzyme, indicating that the two proteins are different.


Mechanism of pigeon liver malic enzyme: kinetics, specificity, and half-site stoichiometry of the alkylation of a cysteinyl residue by the substrate-inhibitor bromopyruvate. Malic enzyme from pigeon liver is alkylated by the substrate analogue bromopyruvate, resulting in the concomitant loss of its oxidative decarboxylase and oxalacetate decarboxylase activities, but not its ability to reduce alpha-keto acids. The inactivation of oxidative decarboxylase activity follows saturation kinetics, indicating the formation of an enzyme-bromopyruvate complex (K congruent to 8 mM) prior to alkylation. The inactivation is inhibited by metal ions and pyridine nucleotide cofactors. Protection of malic enzyme by the substrates L-malate and pyruvate and the inhibitors tartronate and oxalate requires the presence of the above cofactors, which tighten the binding of these carboxylic acids in accord with the ordered kinetic scheme (Hsu, R. Y., Lardy, H. A., and Cleland, W. W. (1967), J. Biol. Chem. 242, 5315-5322). Bromopyruvate is reduced to L-bromolactate by malic enzyme and is an effective inhibitor of L-malate and pyruvate in the overall reaction. The apparent kinetic constants (90 muM-0.8 mM) are one to two orders of magnitude lower than the half-saturation constant (K) of inactivation, indicating a similar tightening of bromopyruvate binding in the E-NADP+ (NADPH)-Mn2+ (Mg2+)-BP complexes. During alkylation, bromopyruvate interacts initially at the carboxylic acid substrate pocket of the active site, as indicated by the protective effect of substrates and the ability of this compound to form kinetically viable complexes with malic enzyme, particularly as a competitive inhibitor of pyruvate carboxylation with a Ki (90 muM) in the same order as its apparent Michaelis constant of 98 muM. Subsequent alkylation of a cysteinyl residue blocks the C-C bond cleavage step. The incorporation of radioactivity from [14C]bromopyruvate gives a half-site stoichiometry of two carboxyketomethyl residues per tetramer, indicating strong negative cooperativity between the four subunits of equal size, or alternatively the presence of structurally dissimilar active sites.


Mechanism of mitochondrial carbamoyl-phosphate synthetase: synthesis and properties of active CO2, precursor of carbamoyl phosphate. This paper demonstrates the formation of "active CO2" (CO2-P), a precursor of carbamoyl phosphate (CP), with frog liver carbamoyl-phosphate synthetase. Absence of ammonia is essential for the demonstration by pulse incubation with H14CO3- of CO2-P. Adenosine triphosphate (ATP) and acetylglutamate are required for the synthesis of CO2-P, which is highly unstable in aqueous solutions (t1/2 = 0.75 s at 24 degrees C at neutral pH). In the absence of ammonia, CO2-P attains rapidly a steady-state level, which depends on the concentration of ATP and HCO3-. The "apparent KM'S" are approximately equal to those found for the adenosine triphosphate (ATPase) activity of the enzyme. The maximum level of CO2-P is limited by the amount of enzyme, and approximates 4 mol of intermediate/mol of enzyme. The unprotonated form of ammonia seems to be the species reacting with CO2-P to produce CP. The reaction of CO2-P and NH3 is very fast (rate constant kn = 8 x 10(4) M-1 S-1) and does not consume free ATP. Therefore, the 2 mol of ATP necessary for CP synthesis binds or reacts with the enzyme and/or CO2 prior to reaction with NH3. The reaction of CO2-P with NH3 also takes place in acetone under conditions at which the enzyme is not active, suggesting little or no assistance from enzyme catalysis or that a part of the catalytic site is "frozen" by the solvent in the active conformation. In the light of these and other findings, a new scheme is proposed for the mechanism of frog liver carbamoyl-phosphate synthetase and some considerations are made on the chemical nature of the intermediate and on the possible evolutionary significance of the reaction of CO2-P with NH3 in acetone.


PH-induced changes in the reactions controlled by the low- and high-affinity Ca2+-binding sites in sarcoplasmic reticulum. The effect of pH on the Ca2+-binding sites of high and low affinity, located respectively on the outer and inner surfaces of the sarcoplasmic reticulum membrane, was investigated using intact and leaky sarcoplasmic reticulum vesicles. With the use of intact vesicles, different pH profiles of membrane phosphorylation and rates of nucleoside triphosphate hydrolysis were obtained depending on the assay temperature, on the Ca2+ concentration, and on whether ATP or ITP was used as substrate. The different pH profiles were related to the amount of Ca2+ accumualted by the vesicles, i.e., to different degrees of saturation of the inner, low-affinity Ca2+-binding site. With the use of leaky vesicles, the saturation of the two Ca2+-binding sites could be controlled more precisely since the Ca2+ concentration on both sides of the membrane was equal to the Ca2+ concentration of the assay medium. Using leaky vesicles and measuring the rates of nucleotide hydrolysis, nucleotide-phosphate exchange and membrane phosphorylation by nucleotide as an indication of the degree of saturation of the Ca2+-binding sites, we observed that the affinity of both the high- and low-affinity sites increased three to four orders of magnitude when the pH of the assay medium was increased from 6.1 to 8.65.


Investigation of the open ring form of nicotinamide adenine dinucleotide. In strong alkali, nicotinamide adenine dinucleotide (NAD+) undergoes a ring opening of the nicotinamide ring. The open form of NAD+, ONAD, has two pKa values at--1.9 and 11.2 and absorbs maximally at 350 nm in its acidic form, at 372 nm in its neutral form, and at 340 nm in its aniomic form. ONAD has the chemical properties expected for a Schiff base of 2-carboxamideglutacondialdehyde (CGDA) and adenosine diphosphate ribosylamine. The decomposition of ONAD has been studied over a wide range of pH. A final product of ONAD hydrolysis is the base fluorescent compound 2-hydroxynicotinaldehyde. In the pH range 10--13, CGDA can be trapped as an intermediate, which absorbs maximally at 345 nm in its anionic form and at 320 nm in its neutral form, pKa = 2.9. The yield of 2-hydroxynicotinaldehyde from ONAD has been estimated as 95% at NaOH concentrations of 5 N and above, and is postulated to result from ring closure of CGDA. The pseudobase hydroxide ring addition adduct of NAD+, psiNAD-OH, is reversibly formed from NAD+ and is the 370-nm precursor of ONAD.


Metal site conformational states of vanadyl(IV) human serotransferrin complexes. This study was undertaken to investigate the conformational states of the two metal sites in the human serum transferrin molecule. The 9.2 GHz electron paramagnetic resonance spectra of frozen solutions of divanadyl(IV) transferrin consist of a superposition of two sets of resonances, A and B, due to the magnetically nonequivalent binding environments of the VO2+ ion. Examination of the intensities of the A and B resonances as a function of pH from 6.0 to 10.7 reveals that they arise from two conformational states of the metal sites in which the geometrical arrangement and/or identity of one or more ligands in the first coordination sphere are different. From pH 7.5 to 9.0, the metal sites exist in A and B conformations but above pH 9.0 the A conformation. This transformation is coupled to the ionization of an apparently noncoordinating protein functional group with a pK - 10.0 +/- 0.1. Below pH 7.0, binding in the B conformation is rapidly lost, driven in part by the protonation of a functional group, possibly the anion, with a pK - 6.6 +/- 0.1. In 90% D2O, this pK is elevated to 7.8 +/- 0.1. At pH 6.0 in H2O, essentially one VO2+ ion remains bound to the protein with the metal site in the A conformation. Experiments with mixed VO2+ -Fe3+ transferrin complexes indicate that the same may be true of Fe3+. At pH 10.7, a new set of VO2+ resonances, labeled C, are observed; they possibly arise from a third conformation of the metal site. One bicarbonate or corbonate is required per VO2+ ion bound to the protein. 2.7 H+ are released per VO2+ bound in either the A or B conformations. The above results are discussed in terms of the "equivalence" and "nonequivalence" of the metal sites.


Isolation, characterization, and activation of the magnesium dependent endodeoxyribonuclease from Bacillus subtilis. A major endodeoxyribonulcease was isolated from a mutant of the transformable Bacillus subtilis 168. The magnesium-dependent endonuclease was purified approximately 750-fold to electrophoretic homogeneity. The enzyme had a molecular weight of about 31 000, as determined by gel filtration and polyacrylamide gel electrophoresis. The protein appears to be composed of two subunits. The nuclease was dependent on magnesium or maganese ions for hydrolytic activity. The purified nuclease degraded DNA from several  species of Bacillus, as well as Escherichia coli DNA, alkylated, depurinated, and thymine-dimer containing B. subtilis DNA, and hydroxymethyluracil-containing phage DNA. The enzyme also hydrolyzed single-stranded DNA, although native DNA was the preferred substrate. However, the nuclease was unable to degrade ribosomal RNA. The cleavage products of the DNA hydrolysis have 5'-phosphate and 3'-hydroxyl ends. The enzyme could be activated in crude extracts by heat treatment or treatment with guanidine hydrochloride. The nuclease activity was inhibited by phosphate and by high concentrations of NaCl. A possible function for this endonuclease in bacterial transformation is discussed.


Mechanism of lysozyme catalysis: role of ground-state strain in subsite D in hen egg-white and human lysozymes. The association constants for the binding of various saccharides to hen egg-white lysozyme and human lysozyme have been measured by fluorescence titration. Among these are the oligosaccharides GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-GlcNAc, GlcNAc-beta(1 leads to 4)-MurNAc-beta(1 leads to 4)-GlcNAc-beta(1 leads to 4)-N-acetyl-D-xylosamine, and GlcNAc-beta(1 leads to 4-GlcNAc-beta(1 leads to 4)-MurNAc, prepared here for the first time. The binding constants for saccharides which must have N-acetylmuramic acid, N-acetyl-D-glucosamine, or N-acetyl-D-xylosamine bound in subsite D indicate that there is no strain involved in the binding of N-acetyl-D-glycosamine in this site, and that the lactyl group of N-acetylmuramic acid (rather than the hydroxymethyl group) is responsible for the apparent strain previously reported for binding at this subsite. For hen egg-white lysozyme, the dependence of saccharide binding on pH or on a saturating concentration of Gd(III) suggests that the conformation of several of the complexes are different from one another and from that proposed for a productive complex. This is supported by fluorescence difference spectra of the various hen egg-white lysozyme-saccharide complexes. Human lysozyme binds most saccharides studied more weakly than the hen egg-white enzyme, but binds GlcNAc-beta(1 leads to 4)-MurNAc-beta(1leads to 4)-GlcNAc-beta(1 leads to 4)-MurNAc more strongly. It is suggested that subsite C of the human enzyme is "looser" than the equivalent site in the hen egg enzyme, so that the rearrangement of a saccharide in this subsite in response to introduction of an N-acetylmuramic acid residue into subsite D destabilizes the saccharide complexes of human lysozyme less than it does the corresponding hen egg-white lysozyme complexes. This difference and the differences in the fluorescence difference spectra of hen egg-white lysozyme and human lysozyme are ascribed mainly to the replacement of Trp-62 in hen egg-white lysozyme by Tyr-63 in the human enzyme. The implications of our findings for the assumption of superposition and additivity of energies of binding in individual subsites, and for the estimation of the role of strain in lysozyme catalysis, are discussed.


Energetics of the cooperative and noncooperative binding of nicotinamide adenine dinucleotide to yeast glyceraldehyde-3-phosphate dehydrogenase at pH 6.5 and pH 8.5. Equilibrium and calorimetric analysis over a range of temperature. The binding of nicotinamide adenine dinucleotide (NAD+) to yeast glyceraldehyde-3-phosphate dehydrogenase (GPDH) has been studied at pH 6.5 and 8.5, at 5,25, and 40 degrees C, by calorimetry, fluorometry, spectrophotometry, equilibrium dialysis, and flow dialysis. As reported earlier for pH 7.3 (Velick S.F., Baggott, J.P., and Sturtevant, J.M. (1971), Biochemistry 10, 779), the binding is accompanied by enthalpy changes which become rapidly more negative as the temperature increases, with delta Cp = -500 to -750 cal deg-1 (mole of NAD+ bound)-1, and by entropy changes which also, as required by the large negative delta Cp, become rapidly more negative with increasing temperature. The binding data at pH 6.5 can be fitted on the basis of either four identical noninteracting sites, or of four sites showing a small degree of negative cooperativity. The data at pH 8.5, particularly at 40 degrees C, require the introduction of positive cooperativity, as was previously shown by Kirschner et al. (Kirschner, K., Eigen, M., Bittman, R., and Voigt, B. (1966), Proc. Natl. Acad. Sci. U.S.A. 56, 1661), and can be equally well fitted on the basis of a sequential model (Adair, G.S. (1925), J. Biol. Chem. 63, 529) or a concerted model (Monod, J., Wyman, J., and Changeux, J.P. (1965), J. Mol. Biol. 12, 88). It is proposed that the observed thermodynamic changes are largely the result of a hydrophobic effect due to a decrease in the exposure of nonpolar groups to the solvent, and of a tightening of the protein structure when the coenzyme is bound with concomitant decrease in the number of easily excitable internal degrees of freedom.


Interaction of acetazolamide and 4-nitrothiophenolate ion with bivalent metal ion derivatives of bovine carbonic anhydrase. The stability and rate constants for the interaction of acetazolamide (diamox) and 4-nitrothiophenolate ion (NTP) with the bivalent Mn, Co, Ni, Cu and Cd forms of bovine carbonic anhydrase have been measured by utilizing the distinct visible spectra of each metalloenzyme-NTP adduct. Differing stabilities of the various NTP and (particularly) diamox complexes reside mainly in varying values for the dissociation rate constants (kd). Intrinsic formation rate constants (for the acid form of the enzyme reacting with the basic form of the ligand) are uniformly high (greater than or equal 2 X 10(7) M-1 s-1 at 25 degrees C). Invariance of kd with pH and a bell-shaped log K-pH profile with the Cu-enzyme adducts are features observed previously with the native enzyme. Binding of NTP with the Cu and Cd metalloenzymes is stronger than to the native form.


Thermodynamic and conformational studies on an immunoglobulin light chain which reversibly precipitates at low temperatures. A lambda light chain, isolated from an immunoglobulin G molecule, was found to reversibly precipitate at low temperatures. This cryoprecipitation was a function of pH, ionic strength, protein concentration, and time as well as temperature. The lambda chain underwent a cooperative conformational change as the temperature was lowered from 26 to 0 degrees C as judged by ultraviolet difference spectroscopy and circular dichroism. Normal lambda chains showed no conformational change. By difference spectroscopy it was possible to calculate the equilibrium constant governing the conformational change. The  change was strongly exothermic (delta H approximately -80 kcal mol-1) and accompanied by a large decrease in entropy (delta S approximately -280 eu). The midpoint of the transition was dependent on the initial protein concentration, suggesting that only the noncovalent dimer of the lambda chain exhibited the conformational change. The existence of a monomer-dimer eqiulibrium (KA approximately 4 X 10(5) M-1) was confirmed by sedimentation velocity. No conformational change was observed by circular dichroism at concentrations where greater than 95% of lambda chain was in the form of a monomer. Although high ionic strength inhibited cryoprecipitation, it had no effect on the conformational change. Stabilization of the dimer by forming an interchain disulfide bond between two monomers abolished both the conformational change and cryoprecipitation. A fragment corresponding to the constant region was isolated from both peptic and tryptic digests of the lambda chain. This fragment neither cryoprecipitated nor showed temperature dependence conformational changes. It proved impossible to isolate a fragment corresponding to the variable region. Both qualitative and quantitative models are presented to account for the behavior of the lambda chain at low temperatures.


Determination of the rate-limiting steps for malic enzyme by the use of isotope effects and other kinetic studies. Isotope effects have been measured with Mg2+ as the activator and L-malate labeled with deuterium or tritium at carbon 2 as the substrate over the pH range 4-10. Comparison of the nearly pH-independent deuterium-isotope effect on V/Kmalate of 1.5 with the tritium effect of 2.0 by the method of Northrop (Northrop, D.B. (1975), Biochemistry 14, 2644) gives limits on the true effect of deuterium substitution on the bond-breaking step of 5-8 in the forward reaction and 4-6.5 in the reverse direction. Comparison of the deuterium effect on V/K with the 13C-isotope effect of 1.031 reported by Schimerlik et al. (Schimerlik, M.I., Rife, J.E., and Cleland, W.W. (1975), Biochemistry 14, 5347) allows the deduction that at pH 8 reverse hydride transfer is six to eight times faster than decarboxylation, which is thus largely rate limiting for the catalytic reaction. The absence of a deuterium-isotope effect on V at pH 7-8 and comparison of the Ki of pyruvate as an uncompetitive inhibitor of the forward reaction and a substrate for the reverse reaction indicate that at neutral pH the release of TPNH from enzyme-reduced triphosphopyridine nucleotide (E-TPNH) is the rate-limiting step in the forward direction. The observation of a deuterium effect on V that approaches 3 at pH 4 and 10 shows, however, that, at very low and very high pH, hydride transfer may become partly rate limiting. In the reverse reaction, the probable rate-limiting step at pH 7 is the isomerization of E-TPNH, while at pH 8.5 and above V becomes too large to measure and appears infinite. Substitution of Co2+, Ni2+, or low levels of Mn2+ for Mg2+ gives similar deuterium-isotope effects, although the values of V and Kmalate vary considerably with metal. The kinetics of Mn2+ show pronounced negative cooperativity, with Km values of 7 muM and 5 mM for concentration ranges from 4 to 100 muM and over 1 mM.


pH variation of the kinetic parameters and the catalytic mechanism of malic enzyme. The pH variation of the kinetic parameters for the oxidative decarboxylation of L-malate and decarboxylation of oxalacetate catalyzed by malic enzyme has been used to gain information on the catalytic mechanism of this enzyme. With Mn2+ as the activator, an active-site residue with a pK of 5.4 must be protonated for oxalacetate decarboxylation and ionized for the oxidative decarboxylation of L-malate. With Mg2+ as the metal, this pK is 6, and, at high pH, V/K for L-malate decreases when groups with pKs of 7.8 and 9 are deprotonated. The group at 7.8 is a neutral acid (thought to be water coordinated to Mg2+), while the group at 9 is a cationic acid such as lysine. The V profile for reaction of malate shows these pKs displaced outward by 1.4 pH units, since the rate-limiting step is normally TPNH release, and the chemical reaction, which is pH sensitive, is 25 times faster. TPN binding is decreased by ionization of a group with pK 9.3 or protonation of a group with pK 5.3. The pH variation of the Km for Mg shows that protonation of a group with pK 8.7 (possibly SH) decreases metal binding in the presence of malate by a factor of 1400, and in the absence of malate by a factor of 20. A catalytic mechanism is proposed in which hydride transfer is accompanied by transfer of a proton to the group with pK 5.4-6, and enolpyruvate is protonated by water coordinated to the Mg2+ (pK 7.8) after decarboxylation and release of CO2.


Effect of magnesium on the properties of zinc alkaline phosphatase. Alkaline phosphatase of Escherichia coli, isolated by procedures which do not alter its intrinsic metal content, contains 4.0 +/- 0.3 g-atoms of tightly bound zinc per mole (Kd less than 1 muM) and 1.3 +/- 0.2 g-atoms of magnesium per mole (Bosron, W.F., Kennedy, F.S., and Vallee, B.L. (1975), Biochemistry 14, 2275-2282). Importantly, the binding of magnesium is dependent both upon pH and zinc content. Hence, the failure to assign the maximal magnesium stoichiometry to enzyme isolated by conventional procedures may be considered a consequence of the conditions chosen for optimal bacterial growth and purification of the enzyme which are not the conditions for optimal binding of magnesium to alkaline phosphatase. Under the conditions employed for the present experimental studies, a maximum of six metal sites are available to bind zinc and magnesium, i.e., four for zinc and two for magnesium. Magnesium alone does not activate the apoenzyme, but it regulates the nature of the zinc-dependent restoration of catalytic activity to apophosphatase, increasing the activity of enzyme containing 2-g-atoms of zinc five-fold and that of enzyme containing 4-g-atoms of zinc 1.4-fold. Moreover, hydrogen-tritium exchange reveals the stabilizing effects of magnesium on the structural properties of phosphatase. However, neither the KM for substrate nor the phosphate binding stoichiometry and Ki are significantly altered by magnesium. Hence, magnesium, which is specificially bound to the enzyme, both stabilizes the dynamic protein structure and regulates the expression of catalytic activity by zinc in alkaline phosphatase.


Dissociation and reconstitution of human ferroxidase II. The ferroxidase II protein from human serum is large and structurally complex. It possesses protein-bound lipid and copper components which are essential for the maintenance of its catalytic activity. Treatment of ferroxidase II with 8 M urea, 6 M guanidine hydrochloride, or 6 M guanidine hydrochloride and alkylation does not result in the dissociation of the enzyme into subunits. However, treatment with sodium dodecyl sulfate results in the dissociation of ferroxidase II into two nonidentical subunits, designated S-I and S-II. S-I contains little phospholipid, cholesterol, or copper and has a molecular weight of 3.8-3.9 X 10(5). In contrast, S-II contains bound phospholipid, cholesterol, and copper and has a molecular weight of 2.2-2.4 X 10(5). The lipid compositon of S-II is identical with the native enzyme. Sodium dodecyl sulfate-free S-I exhibits no ferroxidase activity. Immediately following removal of sodium dodecyl sulfate, S-II exhibits ferroxidase activity but S-II rapidly loses its activity in the absence of S-I. The separated subunits spontaneously reassociate upon removal of the sodium dodecyl sulfate to yield a fully active enzyme which chemically appears identical with native ferroxidase II. Furthermore, the reconstituted enzyme is stable. Both native and reconstituted ferroxidase II may be stored at 4 degrees C for 6 weeks without any loss in activity. This suggests that S-II, the copper and lipid-containing subunit, is the catalytic subunit and that S-I is essential for the stabilization of the enzymic activity of S-II. These results provide insight into the molecular structure and chemical composition of ferroxidase II and suggest that the complete native structure of ferroxidase II is required for the maintenance of i-s functional integrity.


Determination of ionization state by resonance Raman spectroscopy Sulfonamide binding to carbonic anhydrase. Resonance Raman (RR) spectroscopy has been used to study the ionization state of the sulfonamide, 4'-sulfamylphenyl-2-azo-7-acetamido-1-hydroxynaphthalene-3,-6-disulfonate (Neoprontosil), bound to carbonic anhydrase. The correlation of effects of pH and deuteration on the spectra of model compounds with these effects on the Neoprotosil spectrum allows us to assign spectral bands in the 900-1000 and 100-1200 cm-1 regions to the SO2NH2 group. Large shifts in these bands occur upon ionization of the sulfonamide. On the basis of the positions of bands in the enzyme complex, it was determined that the sulfonamide was bound to the enzyme as SO2NH2, rather than as SO2NH-. Rates of association and dissociation and the dissociation equilibrium constant were measured as a function of pH. The rate behavior for Neoprontosil is consistent with that observed for other sulfonamides and kdissoc/kassoc = kdissoc, suggesting a one-step binding mechanism. Since RR spectroscopy establishes that the final ionization state of the sulfonamide in the enzyme complex is SO2NH2, protonated sulfonamide must bind directly to basic form of the enzyme. These conclusions suggest that sulfonamides form "outer-space" complexes with metal at the enzyme active site.


Circular dichroism studies of angiotensin II and analogues: effects of primary sequence, solvent, and pH on the side-chain conformation. Conformational aspects of the pressor hormone angiotensin II and 11 of its structural analogues were studied by circular dichroism. Each position of the peptide was singly substituted with an aliphatic residue and alterations of the CD spectra of the resulting analogues in the peptide and aromatic spectral regions (320-250 nm, 250-190 nm) were examined. The spectra of these peptides in 2,2,2-trifluoroethanol solution permit estimation of the relative importance of the various side chains in maintaining the backbone conformation of the hormone. The evolution of the CD spectra in both spectral regions of the peptides in aqueous solution during a titration from pH 1 to pH 12 makes it possible to elucidate further the role of ionizable groups and their interaction with aromatic amino acids such as tyrosine. The results obtained indicate that substitutions in aspartic acid 1, proline 7, and phenylalanine 8 of angiotensin II entail changes in the backbone conformation. On the other hand, the side chains of valine 3, isoleucine 5, and the biologically essential histidine 6 serve mainly to correctly align the phenolic ring of tyrosine in position 4.


Mechanism of the reaction of hydrated electrons with ferrocytochrome c. 1. The hydrated electron reacts with ferrocytochrome c to form an unstable intermediate. This intermediate decays in a first-order manner to give, in the first instance, a product which has a similar absorption spectrum in the range 400-610 nm as normal ferricytochrome c. 2. At 21 degrees C the rate constant for the reaction of hydrated electrons with ferrocytochrome c at pH 7.4 (2 mM phosphate buffer) is (3.0 +/- 0.3) = 10(10) M-1 - S-1. As the pH is increased above pH 8.0 the rate constant steadily decreases. The dependence of the rate constant on pH can be explained if ferrocytochrome c has a pK of around 9.2. 3. At 21 degrees C and pH 7.4, the rate constant for the decay of the intermediate is (1.40 +/- 0.15) - 10(5) S-1. This reaction shows no pH dependence in the range 6-2-11.0. 4. A mechanism is proposed whereby the central metal atom of the ferrocytochrome c is oxidased and a thioether bond is reduced. The resulting ferricytochrome c species then slowly develops an absorbance at 606 nm due to the attack of the sulfhydryl group on the haem.


Cation transport in cytochrome oxidase reconstituted vesicles. Cation translocation across the membrane of cytochrome oxidase reconstituted vesicles may be followed with a simple spectrophotometric method. Cytochrome oxidase reconstituted vesicles, supplemented with ascorbate and cytochrome c. induce large spectral changes of the positive dye safranine, reversed by uncouplers and inhibitors of respiration. The dye is probably accumulated in the inner space of the vesicles, where it reaches high concentrations and aggregates. The spectral shifts and the absorbance changes, due to aggregation, are proportional to the amount of the dye taken up and depend on the respiratory control. In the presence of potassium, valinomycin causes an inhibition, whereas nigericin stimulates the dye uptake. The data are discussed in terms of electrical potential dependent fluxes.


Energy-linked protonation of quinacrine in beef heart submitochondrial membranes. 1. The absorption spectrum of quinacrine in aqueous solution, in the visible region, changes with the pH of the medium in the pH range from 6.0 to 9.0 with an isosbestic point at 353 nm. This indicates that the monoprotonated (quinacrine - H+) and the diprotonated (quinacrine - 2H+) forms of quinacrine at equilibrium in this pH range have a 1 to 1 stoichiometry. 2. The monoprotonated and the dipronated forms to quinacrine exhibit similar fluorescence emission spectra, but distinctive fluorescence excitation spectra. 3. The relative fluorescence quantum yields of quinacrine in aqueous media of various pH values are estimated. The relative fluorescence quantum yield of quinacrine at pH 9.0 is more than 3 fold of that at pH 6.0. 4. The fluorescence excitation and emission spectra, as well as the relative fluorescence quantum yield of quinacrine associated with non-energized submitochondrial membranes, are similar to those of quinacrine alone. 5. Analyses of the absorption spectra, the fluorescence excitation spectra and the relative fluorescence quantum yield indicate that the energy-linked fluorescence decrease of quinacrine associated with the energized submitochondrial membranes results from the protonation of quinacrine - H+ to form quinacrine - 2H+. 6. Quantitative data are provided indicating that the maximal efficiency of protonation of quinacrine - H+ to form quinacrine - 2H+ depends on the concentration of H+ in the membranes generated through energy coupling, and the concentration of quinacrine - H+ initially present in the reaction medium. Under optimal conditions virtually complete conversion of quinacrine - H+ into quinacrine - 2H+ is observed. 7. The fluorescence intensity of quinacrine, either alone or associated with non-energized submitochondrial membranes, decreases with increasing temperature. When quinacrine is associated with the energized membranes, however, its fluorescence intensity increases slightly with increasing temperature. This unusual fluorescence behavior towards temperature, together with the fact that under optimal conditions virtually all the quinacrine molecules associated with the energized membranes are in the diprotonated form, further substantiate our earlier conclusion that the diprotonated quinacrine molecules are tightly bound to the energized membranes in a fashion which does not permit ready equilibration with the external medium.


Binding of [3H]ctyochalasin B and [3H]colchicine to isolated liver plasma membranes. The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([3H]cytochalasin B and [3H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [3H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB3H4. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insensitive to changes of pH or ionic strength. At 10(-6) M [3H]cytochalasin B, glucose of p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 A) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10(-5) M [3H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [3H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes. [3H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [3H]cytochalasin B binding and cytochalasin B stimulated 3H colchicine binding. [3H]Colchicine also bound to intracellular membranes, especially smooth microsomes.


Membrane-bound ATPase of intact vacuoles and tonoplasts isolated from mature plant tissue. Intact vacuoles were isolated from petals of Hippeastrum and Tulipa (Wagner G.J. and Siegelman, H.W. (1975) Science 190, 1298--1299). The ATPase activity of fresh vacuole suspensions was found to be 2--3 times that of protoplasts from the same tissue. 70--80% of the ATPase activity of intact vacuoles was recovered in tonoplast preparations. The antibiotic Dio-9 at 6mug/10(6) vacuoles or protoplasts causes 40% inhibition. However, only the protoplast ATPase is sensitive to oligomycin. N,N'-dicyclohexylcarbodiimide (DCCD) slightly stimulates ATPase activity in both vacuole and protoplast suspensions, whereas ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDAC) strongly inhibits. Spectrophotometric studies show that in the petal the vacuolar contents have a pH of 4.0 for Tuplipa and 4.3 for Hippeastrum, whereas the intact isolated vacuole has an internal pH of 7.0 (in pH 8.0 buffer) for (Tulipa and about 7.3 for Hippeastrum. Internal ion concentrations of 150, 46, 30, 30 and 6 mM were found for K+, Na+, Mg2+, Cl-, and Ca2+ respectively, which are about the same as those in protoplasts.


Ureidosuccinic acid permeation in Saccharomyces cerevisiae. Some strains of Saccharomyces cerevisiae exhibit a specific transport system for ureidosuccinic acid, which is regulated by nitrogen metabolism. Ureidosuccinic acid uptake occurs with proline but with ammonium sulfate as nitrogen source it is inhibited. The V for transport is 20-25 mumol/ml cell water per min. The apparent Km is 3-10(-5) M. For the urep1 mutant (ureidosuccinic acid permease less) the internal concentration never exceeds the external one. In the permease plus strain ureidosuccinic acid can be concentrated up to 10 000 fold and the accumulated compound remains unchanged in the cells. Energy poisons such as dinitrophenol, carbonyl cyanide-m-chlorophenyldrazone (CCCP) or NaN3 inhibit the uptake. No significant efflux of the accumulated compound occurs even in the presence of these drugs. The specificity of the permease is very strict, only amino acids carrying an alpha-N-carbamyl group are strongly competitive inhibitors. The high concentration capacity of the cells and lack of active exit of the accumulated compound support the hypothesis of a carrier mediated active transport system.


The reactivities of tyrosine and tryptophan residues in lipid-bound cytochrome b5. Purified cytochrome b5 from rabbit liver microsomes was bound to liposomes prepared from microsomal lipids. Tyrosyl and tryptophyl side chains of the protein were modified by water-soluble reagents and the reactivities of these amino acid residues in the liposome-bound cytochrome b5 were compared to those of the free protein. At pH 13, 80% of the tyrosines in lipid-free cytochrome b5 ionized immediately, whereas in the lipid-bound protein only 65% ionized within the first minute. In contrast, acetylation with acetylimidazole resulted in the conversion of all 5 tyrosine groups of lipid-free as well as lipid-bound cytochrome b5 into O-acetylated derivatives, which upon treatment with hydroxylamine were completely deacetylated. Reaction with N-bromosuccinimide revealed that only 60% of the 4 tryptophan residues present in cytochrome b5 were accessible to the reagent in the lipid-bound protein, although all tryptophans could be modified in lipid free cytochrome b5. It was concluded that the two tyrosines in the region linking the protein to the membrane are not shielded by lipid bilayer but that of the three tryptophans in the same region one is completely buried in the membrane, whereas the remaining two tryptophans may be both partly exposed to the solvent or alternatively, one may be partially and the other completely exposed.


Calcium ion-flux across phosphatidylcholine membranes mediated by ionophore A23187. The antibiotic A23187 carries Ca2+ across Müller-Rudin membranes made from 1,2-dierucoyl-sn-glycero-3-phosphocholine and n-decane. The conductance of the membranes is not increased by the Ca2+-transport. The flux depends linearly on Ca2+ concentration and ionophore concentration (above pH 6). It increases with increasing pH, approximately by a factor of 4-5 between pH 6 and pH 8. Maximal Ca2+-fluxes of about 10(-10) mol-cm-2-s-1 were found. A counter transport of H+ could not be detected. The complex formation between A23187 and Ca2+ in egg phosphotidylcholine vesicles was studied spectroscopically. The results are consistent with the formation of a 2:1 complex. Optical absorption measurements on single phophatidylcholine membranes were used to calculate the concentration of membrane-bound ionophore A23187.


Permeability characteristics of erythrocyte ghosts prepared under isoionic conditions by a glycol-induced osmotic lysis. A detailed study has been made of the permeability characteristics of human erythrocyte ghosts prepared under isoionic conditions by a glycol-induced lysis (Billah, M.M., Finean, J.B., Coleman, R. and Michell, R.H. (1976) Biochim. Biophys. Acta 433, 45-54). Impermeability to large molecules such as dextran (average molecular weight 70 000) was restored immediately and spontaneously after each of the 5-7 lyses that were required to remove all of the haemoglobin. Permeabilities to smaller molecules such as MgATP2-, [3H]inositol and [14C]choline were initially high but could be greatly reduced by incubation at 37 degrees C for an hour. The extent of such resealing decreased as the number of lyses to which the ghosts had been subjected increased. Both removal of haemoglobin and permeabilities to small molecules were affected significantly by pH, CA3+ concentrations and divalent cation chelators. Maximum resealing was achieved in ghosts prepared in the basic ionic medium (130 mM KCl, 10 nM NaCl, 2 mM MgCl2, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES)) at pH 7.0 (0 degrees C) and with a calcium level around 10(-5) M. Acidic pH facilitated the removal of haemoglobin whilst the presence of divalent cation chelators showed down its release. Retention of K+ by ghosts leaded with K+ during the first lysis and subsequently incubated at 37 degrees C was substantial but lation chelators slowed down its released. Retention of K+ by ghosts loaded with K+ during the first lysis and subsequently incubated at 37 degrees C was substantial but little K+ could be retained within the haemoglobin-free ghosts. Permeability of the ghosts to K+ after one lysis was affected by temperature, pH, Ca2+ concentrations and by the presence of divalent cation chelators.


Studies on membrane fusion. III. The role of calcium-induced phase changes. The interaction of phosphatidylserine vesicles with Ca2+ and Mg2+ has been examined by several techniques to study the mechanism of membrane fusion. Data are presented on the effects of Ca2+ and Mg2+ on vesicle permeability, thermotropic phase transitions and morphology determined by differential scanning calorimetry, X-ray diffraction, and freeze-fracture electron microscopy. These data are discussed in relation to information concerning Ca2+ binding, charge neutralization, molecular packing, vesicle aggregation, phase transitions, phase separations and vesicle fusion. The results indicate that at Ca2+ concentrations of 1.0-2.0 mM, a highly cooperative phenomenon occurs which results in increased vesicle permeability, aggregation and fusion of the vesicles. Under these conditions the hydrocarbon chains of the lipid bilayers undergo a phase change from a fluid to a crystalline state. The aggregation of vesicles that is observed during fusion is not sufficient range of 2.0-5.0 mM induces aggregation of phosphatidylserine vesicles but no significant fusion nor a phase change. From the effect of variations in pH, temperature, Ca2+ and Mg2+ concentration on the fusion of vesicles, it is concluded that the key event leading to vesicle membrane fusion is the isothermic phase change induced by the bivalent metals. It is proposed that this phase change induces a transient destabilization of the bilayer membranes that become susceptible to fusion at domain boundaries.


Bis-(4-methylumbelliferyl) phosphate as a substrate for the surface membrane-associated phosphodiesterase activity of pig platelets. It was found that the newly-available compound, bis-(4-methylumbelliferyl) phosphate, could be used as a substrate for the pig platelet surface membrane-associated phosphodiesterase activity, usually assayed with bis-(p-nitrophenyl) phosphate. This enzyme activity is distinct from the phosphodiesterase activity towards 5'-dTMP-P-nitrophenyl ester, which is probably associated with intracellular membrane structures in platelets. Consequently, the use of the 4-methylumbelliferyl derivative as substrate for the phosphodiesterase activity provides a sensitive, fluorimetric assay for this marker enzyme of the platelet surface membrane.


A specific polyadenylase from Escherichia coli K12. A polyadenylase, degrading specifically poly(A) sequences was isolated from Escherichia coli K12. The enzyme was purified about 850 times to practically electrophoretic homogeneity. It was free of poly(A) polymerase activity, as well as of the well known E. coli RNAases I and II. It is stimulated by bivalent cations like Mg2+ and Mn2+ and splits poly(A) to 3'-AMP and therefore it can be considered as an exonuclease. The enzyme does not degrade any other ribohomopolymer or RNA.


Kinetic formulations for the oxidation and the reduction of glyoxylate by lactate dehydrogenase. Chicken liver lactate dehydrogenase (L-lactate : NAD+ oxidoreductase, EC 1.1.1.27) irreversibly catalyses the oxidation of glyoxylate (hydrated form) (I) to oxalate (pH = 9.6) and the reduction of (non-hydrated form) (II) to glycolate (pH = 7.4). (I) attaches to the enzyme in the pyruvate binding site and (II) attaches to the enzyme at the L-lactate binding site. The oxidation of (I) (pH = 9.6) is adapted to the following mechanism: (see book). The abortive complexes, E-NADH-I and E-NAD+-II, are responsible for the inhibition by excess substrate in the reduction and oxidation systems, respectively. When lactate dehydrogenase and NAD+ are preincubated, E-NAD+- NAD+ appears and causes inhibition by excess NAD+ in the glyoxylate-lactate dehydrogenase-NAD+ and L-lactate-lactate dehydrogenase-NAD+ systems; the second NAD+ molecule attaches to the enzyme at the L-lactate binding site.


Purification and properties of aldehyde dehydrogenase from Proteus vulgaris. NADP-linked aldehyde dehydrogenase (aldehyde : NADP+ oxidoreductase, EC 1.2.1.4) was purified from Proteus vulgaris to the stage of homogeneity as judged by ultracentrifugation and polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be 130000 by gel filtration. The enzyme which was crystallized from ammonium sulfate solution, lost its activity. The enzyme did not require coenzyme A, and the reaction was completely dependent on ammonium ions which could be partially replaced by Rb+ or K+. The optimum pH was about 9. Broad substrate specificity was observed and Km values for propionaldehyde, acetaldehyde and isovaleraldehyde were 1.7 - 10(-5), 4 - 10(-5) and 3 - 10(-5) M, respectively. The physiological role of the enzyme in living cells is obscure, but might account for another degradative pathway of L-leucine in P. vulgaris differing from the established pathway.


Glucokinase of pea seeds.

Glucokinase of pea seeds. 1. Glucokinase (ATP : D-glucose 6-phosphotransferase, EC 2.7.1.2) was extracted from pea seeds and purified by fractionation with (NH4)2SO4 and chromatography on DEAE-cellulose and Sephadex. 2. The relative rates of phosphorylation of glucose, mannose and fructose (final concentration 5 mM) were 100, 64 and 11. 3. The Km for glucose of pea-seed glucokinase was 70 muM and the Km for mannose was 0.5 mM. The Km for fructose was much higher (30 mM). 4. Mg2+ ions were essential for activity. Mn2+ could partially replace Mg2+. 5. Enzyme activity was not inhibited by glucose 6-phosphate. A number of other metabolites had no effect on glucokinase activity. 6. Pea-seed glucokinase was inhibited by relatively low concentrations of ADP.


Purification and properties of polyphosphoinositide phosphomonoesterase from rat brain. 1. On subcellular fractionation of rat brain homogenate, polyphosphoinositide phosphomonoesterase activity was greater in the cytosol than the membranous fractions. 2. The enzyme was purified from the cytosol by column chromatography on DEAE-cellulose, calcium phosphate gel and Sephadex G-100. 3. The final preparation of the enzyme showed a 430-fold purification over the whole homogenate and appeared to be homogeneous since it gave a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on isoelectric focusing. The enzyme has a relatively low molecular weight and an isoelectric point of 6.8. 4. The phosphatase showed a high affinity for triphosphoinositide. Without added Mg2+, the Km was 25 muM and V was 33 mumol Pi released/min/mg protein. 5. The enzyme hydrolysed diphosphoinositide at a slower rate than triphosphoinositide. In the presence of 10 mM Mg2+, the Km values for triphosphoinositide and diphosphoinositide were 5 muM and 25 muM respectively and V was the same for each substrate. 6. Both Mg2+ and Ca2+ activated the enzyme. While Ca2+ produced maximum activation at 100 muM, a much higher concentration of Mg2+ (10 mM) was required to elicit comparable activation. The enzyme did not show an absolute requirement for Mg2+ or Ca2+ as it exhibited low activity in the presence of 0.5 mM EDTA or EGTA. 7. The phosphatase showed maximum activity between 7.4 and 7.6. A drop in pH to 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity. 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity.


Calf thymus alkaline phosphatase. I. Properties of the membrane-bound enzyme. A membrane fraction from calf thymocytes was used to investigate molecular and catalytic properties of membrane-bound alkaline phosphatase (ortho-phosphoric-monoester phosphohydrolase EC 3.1.3.1). The principal findings were: 1. Solubilization of membranes with the non-ionic detergent Triton X-100 increases alkaline phosphatase activity by 30-40%. The enzyme activity elutes in a single peak (Stokes' radius = 7.7 nm) after chromatography in Sepharose 6B in the presence of Triton X-100. The activity also sediments as a single component of approx. 6.4 S during centrifugation in sucrose gradients containing Triton X-100. 2. Ion-exchange chromatography and isoelectric focusing in the presence of Triton X-100 indicate substantial charge heterogeneity. Two overlapping bands, a peak at pH 5.92 with a pronounced shoulder at pH 5.29, are apparent by isoelectric focusing. 3. The pH optimum for hydrolysis of p-nitrophenylphosphate (pNPhP) by the undissolved enzyme(s) is 9.57. Half-maximal activity occurs at pH 8.65 and ph 10.45. Triton X-100 has no effect on the pH profile. 4. Catalytic activity is affected by amines, especially analogues of ethanolamine. Diethanolamine exerts a unique stimulatory effect, but does not change the pH dependency. Increasing the concentration of diethanolamine from 0 to 1 M causes a 6-fold increase in Km and a 10-fold increase in the rate of hydrolysis of pNPhP. Glycine is inhibitory. 5. EDTA causes an irreversible loss of activity with t1/2 (1 mM EDTA, pH 8.2, 23 degrees C) = 3.5 h. Optimal activity is achieved in 0.1--1.0 mM Mg2+, although this does not cause the degree of activation reported to occur with the purified enzymes. Other divalent ions are inhibitory. Concentrations required to reduce activity to 50% of control are: Zn2+, 4.0 muM (no added Mg2+) and 30 muM (in the presence of 1 mM Mg2+); Mn2+, 0.25 mM (+/- Mg2+); Ca2+, 20 mM (+/- Mg2+). 6. Monovalent cations have little effect on activity. In the absence of added Mg2+, 50--150 mM Na+ is partially inhibitory, but markedly less so in the presence of 1 mM Mg2+. K+ has no significant effect. 7. Of the substrates tested, pNPhP (Km = 44 muM) was most rapidly hydrolyzed. Other substrates (rate relative to pNPhP) were alpha-naphthylphosphate (0.79), 2'-AMP (0.80), 5'-AMP (0.70), 3'-AMP (0.63), alpha-glycerophosphate (0.47) and glucose 6-phosphate (0.35). Phosphodiesterase activity was less than or equal to 10% of the phosphomonoesterase activity (for pNPhP) as evidenced by the lack of hydrolysis of bis(p-nitrophenyl)-phosphate and cyclic 3',5'-AMP. The ability of these substances to inhibit hydrolysis of pNPhP reflected their capacity as substrates, i.e. the most inhibitory were the most rapidly hydrolyzed.


Purification and properties of one component of acid phosphatase produced by Aspergillus niger. One component, the i form, of acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) produced by Aspergillus niger was purified from the mycelial extract. The purified enzyme was homogenous on Sephadex G-200 gel filtration, disc electrophoresis and heat inactivation. The purified enzyme was studied and the following results were obtained: 1. The enzyme catalyzed the hydrolysis of a wide variety of phosphomonoesters, but not that of bis(p-nitrophenyl)phosphate, adenosine 3',5'-cyclic monophosphate, fructose 1,6-diphosphate, adenosine 5'-diphosphate or adenosine 5'-triphosphate. 2. Fluoride, orthophosphate, arsenate, borate, molybdate and (+)-tartrate acted as inhibitors. This enzyme was inactivated by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide, and was not affected by p-chloromercuribenzoate, N-acetylimidazole, p-diazobenzenesulfonic acid and tetranitromethane. From these results, tryptophan was estimated to play an important role in the enzyme activity. 3. The apparent molecular weight was 310000 by Sephadex G-200 gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate suggested that the molecular weight of the subunit was approximately 89000. 4. The purified enzyme contained 29% carbohydrate consisting of glucosamine, mannose and galactose. The amino acid composition of this enzyme was not specific compared with other known acid phosphatases.


Cell-specific differences in membrane beta-glucosidase from normal and Gaucher cells. Two isozymes of membrane-bound beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) with activity towards 4-methylumbelliferyl-beta-D-glucopyranoside have been identified in human cells. One of these isozymes was found to have a pH optimum of 5.0, a Km of 0.4 mM and to be rapidly inactivated at pH 4.0 ("acid-labile"). The second isozyme had a pH optimum of 4.5, a Km of 0.8 mM and was stable at pH 4.0 ("acid-stable"). Cultured long-term lymphoid lines and peripheral blood leukocytes contained both isozymes while cultured skin fibroblasts contained only the "acid-stable" form in detectable amounts. The specific activity of the "acid-stable" isozyme was severely reduced in cultured skin fibroblasts, cultured long-term lines and peripheral leukocytes from patients with Gaucher's disease. The specific activity of the "acid-labile" enzyme in the latter two cell types was apparently unaffected. The beta-glucosidase activity in all three cell types examined was predominantly particulate but the enzyme could be solubilized with low concentrations of Triton X-100. The solubilized enzyme required sodium taurocholate (0.2%) for maximum activity. Solubilized beta-glucosidase did not exhibit the cell-specific differences in pH optimum and Km shown by the membrane-bound enzyme.


Benzamidine as an inhibitor of proacrosin activation in bull sperm. Epididymal and ejaculated sperm contain a zymogen form of acrosin (acrosomal proteinase, EC 3.4.21.10) which is converted to active enzyme prior to fertilization. Benzamidine at concentrations greater than 10 mM has been shown to inhibit the conversion of proacrosin to acrosin. Based on this inhibition, a procedure was developed for extracting and quantitating the proacrosin content of bull sperm. Sperm were isolated from semen and washed by centrifugation through 1.3 M sucrose and the outer acrosomal membrane removed by homogenization. When 25 mM benzamidine was added to the semen and wash solutions, 98% or more of the acrosin activity in the sperm homogenate was present as proacrosin. Proacrosin can be extracted from the sperm homogenate by dialysis at pH 3, which solubilized the proenzyme and removed benzamidine. Benzamidine has been useful in isolating proacrosin and provides a new method for studying the activation of proacrosin in intact sperm. Neutralization of sperm extracts, after removal of benzamidine, resulted in rapid activation of proacrosin with a pH optimum of 8.5, and activation was complete within 15 min over a pH range of 7.0 to 9.5. Rapid activation also occurred during the washing of sperm in the absence of benzamidine, and this activation correlated with a swelling of the acrosomal membrane. This rapid activation appears to result from a small amount of acrosin activity consistently present in the sperm extract. These results indicate an autocatalytic conversion of proacrosin to acrosin and suggest that disruption of the acrosomal membrane may trigger this activation.


Studies on aspartase. IV. Reversible denaturation of Escherichia coli aspartase. Aspartase (L-aspartate ammonia lyase, EC 4.3.1.1) of Escherichia coli, denatured in 4 M guanidine-HCl, was renatured in vitro by simple dilution with a concomitant restoration of the activity. While the native enzyme exhibited a marked negative Cotton effect centered at 233 +/- 1 nm in optical rotatory dispersion, the enzyme denatured in 4 M guanidine-HCl retained little optical activity. Upon dilution of the denatured enzyme, however, more than 90% of the ordered structure was recovered in 1 min, while the restoration of the activity proceeded much more slowly. Estimation of molecular weights by gel permeation chromatography indicated that the tetrameric enzyme is subject to reversible dissociation into monomeric subunits under the experimental conditions. Various environmental factors such as temperature, pH and protein concentration exhibited profound influence on the rate and extent of the reactivation. In order to examine the correlation between the restoration of the activity and the quaternary structure, electron microscopic inspection of the kinetic processes of reversible denaturation was attempted. Upon dilution of the denatured enzyme at 4 degrees C, neither the activity nor tetrameric images were detected over several min. Upon the temperature shift up to 25 degrees C, however, the activity regain was rapidly proceeded concomitant with the appearance of tetrameric molecules. These results are compatible with the possibility that the subunit assembly is an essential prerequisite, thought not sufficient, for enzyme activity.


Inhibition of sterol synthesis by citrinin in a cell-free system from rat liver and yeast. Citrinin, a fungal metabolite known as an antibiotic, strongly inhibited the labeled acetate incorporation into nonsaponifiable lipids by a cell-free system from rat liver but not the labeled mevalonate incorporation. Of the enzymes involved in cholesterol synthesis, two enzymes, acetoacetyl-CoA thiolase (EC 2.3.1.9) and 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34), were specifically inhibited by the antibiotic. The concentration required for 50% inhibition was 0.2 mM for the former enzyme and 0.5 mM for the latter. Essentially the same results were obtained with a cell-free system from yeast although higher concentrations of the antibiotic were required for inhibition.