bluuebunny/crossref_metadata_2025 huggingface dataset:
The metabolism of 3,5-di-tert.-butyl-4-hydroxytoluene in the rat and in man
Authors: Daniel J W , Gage J C , Jones D. I.
Published: August 2015

1. The major metabolites of 3,5-di-tert.-butyl-4-hydroxytoluene (BHT) in the rat are 3,5-di-tert.-butyl-4-hydroxybenzoic acid (BHT-acid), both free (9% of the dose) and as a glucuronide (15%), and S-(3,5-di-tert.-butyl-4-hydroxybenzyl)-N-acetylcysteine. 2. The mercapturic acid does not appear to derive from the usually accepted enzyme mechanism, and may involve a non-enzymic reaction between BHT free radical and cysteine. 3. The ester glucuronide and mercapturic acid found in rat urine are also the major metabolites in rat bile and must be responsible for the enterohepatic circulation. 4. Free BHT-acid is the main component in rat faeces. 5. In man, BHT-acid, free and conjugated, is a minor component in urine, and the mercapturic acid is virtually absent. The bulk of the radioactivity is excreted as the ether-insoluble glucuronide of a metabolite in which the ring methyl group and one tert.-butyl methyl group are oxidized to carboxyl groups, and a methyl group on the other tert.-butyl group is also oxidized, probably to an aldehyde group. 6. These differences in metabolism by the rat and by man are sufficient to account for the difference in excretion by the two species.
Rat small-intestinal galactosidases. Separation by ion-exchange chromatography and gel filtration
Authors: Asp Nils Georg , Dahlqvist Arne
Published: August 2015

1. The chromatography of rat small-intestinal β-galactosidase activities on gel-filtration and ion-exchange columns has been studied. Five different substrates were used to measure β-galactosidase activity (lactose, phenyl β-galactoside, o-nitrophenyl β-galactoside, p-nitrophenyl β-galactoside and 6-bromo-2-naphthyl β-galactoside) and the activity was measured at one acid and one more neutral pH value. 2. By gel filtration one acid β-galactosidase, hydrolysing lactose and the hetero-β-galactosides at about the same rate, and one more neutral β-galactosidase, hydrolysing lactose much more rapidly than the hetero-β-galactosides, were separated. 3. By ion-exchange chromatography the acid enzyme was fractionated into two components. These may be individual enzymes or different forms of the same enzyme.
A direct pathway for the metabolism of propionate in cell extracts from Moraxella Iwoffi
Authors: Hodgson B. , McGarry J. D.
Published: August 2015

1. Extracts from Moraxella lwoffi oxidize propionate, but at a low rate when compared with whole cells. 2. This oxidative activity requires the formation of propionyl-CoA. 3. Enzymes catalysing the formation of propionyl phosphate and propionyl-CoA are present. The presence of a propionyl-CoA hydrolase is considered to be an artifact, but partly responsible for the low rates of oxidation. 4. Enzymes catalysing the reduction of NAD+ and the formation of pyruvate with propionyl-CoA as substrate are also present. 5. That the only pathway for the metabolism of propionate in extracts is a direct one to acetate via pyruvate was confirmed by the use of 14C-labelled materials. 6. A possible sequence of enzyme-catalysed reactions that will account for the experimental observations is described.
Changes in the activities of enzymes of the biosynthetic pathway of the nicotinamide nucleotides in rat mammary gland during the lactation cycle
Authors: Greenbaum A. L. , Pinder S.
Published: August 2015

1. The activities of NMN pyrophosphorylase, NMN adenylyltransferase and NAD kinase in the mammary glands of rats at different stages of pregnancy, lactation and involution were measured. 2. NMN pyrophosphorylase has a low activity early in pregnancy, but its activity increases at parturition and in early lactation to reach a maximum at the tenth day of lactation, after which it remains constant until it declines abruptly in involution. 3. NMN adenylyltransferase is already quite active by the tenth day of pregnancy and its activity does not rise further in the second half of gestation. After a sharp rise in activity at parturition, the activity of the enzyme declines slowly throughout the period of lactation and, more sharply, in involution. 4. NAD kinase has a low activity for most of pregnancy, but its activity rises at parturition to a value at 2 days of lactation that is maintained until the tenth day. Between the tenth and fifteenth days of lactation the activity almost doubles, but falls sharply in mammary involution. 5. The relation of the activities of these enzymes to the rates of synthesis of NAD and NADP is discussed.
Role of neuraminic acid in the heterogeneity of alkaline phosphatase in sheep brain
Authors: Saraswathi S. , Bachhawat B. K.
Published: August 2015

1. Two alkaline phosphatase fractions from sheep brain obtained by DEAE-cellulose column chromatography were shown to be associated with different concentrations of NANA (N-acetylneuraminic acid). Enzyme II contains nearly three times as much NANA as does enzyme I. 2. Partial removal of NANA by neuraminidase digestion from these alkaline phosphatase fractions has different effects on their chromatographic properties. Though the enzymic release of NANA has no effect on the elution pattern of enzyme I from a DEAE-cellulose column, such a treatment shifts the elution pattern of enzyme II towards that of enzyme I. 3. However, this change in the elution pattern of enzyme II as a result of the removal of NANA does not produce any change in the kinetics of this fraction, and the differences between enzyme I and enzyme II with respect to their substrate affinities and Ki for phosphate inhibition are maintained even after the removal of NANA. 4. Results indicate that NANA is not the only factor responsible for the heterogeneity of alkaline phosphatase in sheep brain and enzyme I is not the result of the removal of NANA from enzyme II.
A method for the separation of peptides and α-amino acids
Authors: Tommel D. K. J. , Vliegenthart J. F. G. , Penders T. J. , Arens J F
Published: August 2015

1. Peptides and α-amino acids, occurring in mixtures from various sources, can be separated into one fraction containing the amino acids and several peptide fractions. This is achieved by chelation of the mixture with Cu2+ ions and subsequent chromatography of these chelates over the acetate form of diethylaminoethylcellulose or triethylaminoethylcellulose. 2. The amino acid fraction is obtained by elution with 0·01m-collidine–acetate buffer, pH8·0. 3. Peptide fractions are eluted with 0·01m-collidine–acetate buffer, pH4·5, 0·17n-acetic acid and 0·1n-hydrochloric acid respectively. 4. With the exception of aspartic acid and glutamic acid, which are partly found in the acidic peptide fraction, the amino acids are completely separated from the peptides. 5. Contamination of the acidic peptide fraction with glutamic acid and aspartic acid can be largely avoided by previous addition of an excess of arginine. 6. Copper is removed from the eluates by extraction with 8-hydroxyquinoline in chloroform.
A study of the heterogeneous 37s ribonucleic acid induced by foot-and-mouth-disease virus
Authors: Wild T. F. , Martin S J , Brown F.
Published: August 2015

1. The 37s RNA induced in baby-hamster kidney cells by infection with foot-and-mouth-disease virus was examined on sucrose gradients and by filtration through Sepharose 4B. 2. The RNA sedimented faster (37s) and as a broader band than the 35s RNA from purified virus. 3. Treatment with deoxyribonuclease, Pronase or amylase did not alter the sedimentation profile of the 37s RNA. 4. Treatment of individual fractions of the RNA with phenol, dimethyl sulphoxide or methylCellosolve did not decrease the sedimentation rate of the faster-sedimenting molecules. 5. Sedimentation in sucrose gradients of different ionic strengths or containing EDTA had no effect on the heterogeneous nature of the profile. 6. On filtration through Sepharose 4B columns, the 37s virus-induced RNA was eluted before viral RNA. 7. Only 20% of the rapidly sedimenting RNA was incorporated into complete virus particles.
The effect of ribonuclease on polysomes and ribosomes of bacteria and animal cells
Authors: Fenwick M. L.
Published: August 2015

The mildest treatment with ribonuclease that causes any disaggregation of the polysomes of Escherichia coli or HeLa cells simultaneously attacks the RNA of the constituent ribosomes. It is concluded that the susceptibility to ribonuclease of polysomes does not suggest that they are held together by a strand of messenger RNA. The RNA of the larger sub-unit of bacterial ribosomes has particularly sensitive regions resulting in a non-random degradation. The RNA of the smaller sub-unit of E. coli ribosomes is relatively resistant to ribonuclease attack. The same may be true of the respective sub-units of the intact HeLa-cell ribosome, but both sub-units become very sensitive to ribonuclease on dissociation from each other.
Restricted permeability of rat liver for glutamate and succinate
Authors: Hems R. , Stubbs Marion , Krebs H. A.
Published: August 2015

1. When rat liver slices were incubated aerobically with [U−14C]glutamate the concentration of 14C within the slices remained lower (about 50%) than in the medium. The maximal concentration of 14C in the liver was reached within minutes. In rat kidney-cortex slices by contrast, 14C reached concentrations more than six times those of the medium. 2. In both liver and kidney 14C appeared in the respiratory CO2, indicating penetration of glutamate carbon into the mitochondria. In kidney slices the rate of glutamate oxidation per unit weight was about five times that in liver slices. 3. Taking into account the conversion of glutamate into glucose that occurs in the kidney but not in the liver, the flux rates of glutamate through the kidney were calculated to be about 15 times those through the liver when the external glutamate concentration was 5mm. 4. Anaerobically the glutamate concentrations in medium and tissue rapidly became equal in both liver and kidney. Thus the maintenance of concentration gradients depended on the expenditure of energy. 5. [U−14C]Succinate behaved similarly to glutamate. [U−14C]Serine was taken up more rapidly by the kidney than by the liver slices, but the concentrations reached in the liver did not remain below those of the medium. [14C]Urea was distributed evenly between medium and tissue water. 6. Incubation of liver slices with [3H]inulin indicated an extracellular space of liver slices of 26%. 7. When glutamate was generated within liver slices or the perfused liver on addition of oxaloacetate, pyruvate and a source of nitrogen, the concentration of glutamate in the tissue after 1hr. was 70–97 times that in the medium. Thus the exit of glutamate from the liver cell, like its entry, is restricted. This is borne out by measurements of the specific activity of extra- and intra-cellular glutamate on addition of [U−14C]glutamate medium. 8. Liver homogenates removed added glutamate and dicarboxylic acids 20–30 times as fast as did the perfused liver. 9. It is concluded that a major permeability barrier restricts the entry and exit through the outer liver cell membrane.
Non-protein components of the cell surface of Staphylococcus aureus
Authors: James A. M. , Brewer J. E.
Published: August 2015

1. pH–mobility curves of various laboratory strains of Staphylococcus aureus are non-sigmoid in shape, and all pass through a maximum value in the range pH4–5. 2. The maxima in the curves are not due to incomplete washing of the cells, adsorption of buffer components or irreversible surface damage. 3. Mild oxidation of the cell-surface teichoic acid with sodium metaperiodate gives cells that have typical sigmoid pH–mobility curves, characteristic of either a simple carboxyl surface or a mixed carboxyl–amino surface. 4. The results are discussed in terms of a pH-dependent change in the configuration of the teichoic acid molecules at the surface.
Glucose metabolism in the mucosa of the small intestine. The effect of glucose on hexokinase activity
Authors: Shakespeare P. , Srivastava L M , Hübscher G.
Published: August 2015

1. The effect of three dietary components on hexokinase activity in the mucosa of rat small intestine was studied in vivo. Glucose, amino acids or an emulsion of monoglyceride with long-chain fatty acids were given by stomach tube to previously starved rats, and hexokinase activity was determined in the particle-free supernatant of mucosal homogenates. The formation of lactate from glucose and glucose 6-phosphate respectively was also measured. 2. When the three dietary components were given in isocaloric amounts, only glucose brought about an increase in hexokinase activity. 3. Intravenous injection of a similar amount of glucose to that given orally did not alter hexokinase activity. 4. An increase in the hexokinase activity of the particle-free supernatant prepared from mucosal homogenates was also observed after sacs of the small intestine of starved rats had been incubated in vitro in a medium containing glucose. Hexokinase activity increased to the values observed in corresponding preparations from fed rats, and this increase was strictly glucose-dependent.
Physicochemical characteristics of insulin secretion granules
Authors: Coore H. G. , Hellman B. , Pihl E. , Täljedal I.-B.
Published: August 2015

β-Granules were prepared from micro-dissected pancreatic islets of obese–hyperglycaemic mice. This fraction contained 60% of the insulin, 30% of the cytochrome oxidase, 16% of the acid phosphatase activity and 20% of the protein present in whole islets. The isolated granules retained a heavy metal during fractionation. Optimum conditions for granule stability were low ionic strength and pH6, the granules being unexpectedly fragile at pH7·4. The stability of the granules was unaffected by sucrose in the concentration range 50–320mm, but 1% (w/v) sodium deoxycholate released all insulin. A solubilizing effect was also noted with ATP and citrate. Spinning through 1·6m-sucrose yielded a further purification in relation to mitochondria and acid-phosphatase-carrying particles but virtually no purification in relation to protein. Electron microscopy revealed that the major contaminants were rough-surfaced vesicles and membranes. A separation of granules from acid phosphatase was achieved by phase distribution in polyethylene glycol and dextran. The location of the enzyme to the interphase was so pronounced in systems buffered with lithium phosphate that the technique may be used for future purification of acid-phosphatase-carrying particles from the β-cells.
Synthesis of 10,11-dihydrofarnesyl pyrophosphate from 6,7-dihydrogeranyl pyrophosphate by prenyltransferase
Authors: Popják G. , Holloway P. W. , Baron J. M.
Published: August 2015

The syntheses of 6,7-dihydrogeraniol and of its pyrophosphate are described. It is shown that this analogue of geranyl pyrophosphate is a substrate for liver prenyltransferase and that the product synthesized by this enzyme from it and isopentenyl pyrophosphate is 10,11-dihydrofarnesyl pyrophosphate. The Km value for 6,7-dihydrogeranyl pyrophosphate was determined to be 1·11±0·19μm as compared with 4·34±1·71μm for geranyl pyrophosphate. The maximum reaction velocity with the artifical substrate was, however, only about one-fourth of that observed with geranyl pyrophosphate. The binding of isopentenyl pyrophosphate to the enzyme was not affected by the artificial substrate.
Effect of polymerized orosomucoid on some strains of influenza virus
Authors: Barclay Grizel R. , Flewett T. H. , Keller Evelyn , Halsall H. B. , Spragg S. P.
Published: August 2015

1. Polymers of orosomucoid were produced in two molecular shapes, filamentous (‘chain’) and spherical (‘ball’), by heating the sodium salt of the monomer in either water or high concentrations of sodium chloride. An ‘intermediate’ state containing both shapes in various proportions was found in preparations obtained by polymerizing orosomucoid in intermediate concentrations of sodium chloride. 2. The filamentous form of polymer was found to inhibit strongly the haemagglutination of some (‘sensitive’) strains but not of other (‘insensitive’) strains of influenza virus; the ‘intermediate’ form feebly inhibited haemagglutination by ‘sensitive’ strains. 3. The filamentous form agglutinated both ‘sensitive’ and ‘insensitive’ strains of virus; the other forms of polymer did not. It also inhibited multiplication of both ‘sensitive’ and ‘insensitive’ strains when inoculated into embryonated and de-embryonated eggs. 4. The ‘intermediate’ and spherical forms of the polymer had no effect on the virus multiplication. 5. Polymers of orosomucoid from which neuraminic acid had been split off had no detectable effect on influenza viruses.
Some effects of chlorambucil on enzymes of glutathione metabolism in drug-sensitive and -resistant strains of the Yoshida ascites sarcoma
Authors: Harrap K R , Jackson R C , Hill Bridget T.
Published: August 2015

1. Yoshida ascites cells from a strain sensitive to chemotherapy with alkylating agents contained elevated activities of the two enzymes directly responsible for glutathione synthesis, in comparison with a resistant cell strain. The activities of the glutathione-degrading enzyme γ-glutamyltranspeptidase were comparable in both cell strains. 2. After parenteral administration of chlorambucil to rats carrying either strain of tumour, the activities of the glutathione-synthetic enzymes increased in the sensitive cells, but remained unchanged in the resistant cells. Drug treatment was without effect on the γ-glutamyltranspeptidase activity of either cell strain. 3. The activities of a number of enzymes concerned in the oxidoreduction of glutathione remained unchanged after the administration of chlorambucil to rats carrying either strain of cells.
The active chemical state of d-glyceraldehyde 3-phosphate in its reactions with d-glyceraldehyde 3-phosphate dehydrogenase, aldolase and triose phosphate isomerase
Authors: Trentham D. R. , McMurray C. H. , Pogson C I
Published: August 2015

Glyceraldehyde 3-phosphate exists as the geminal diol and the free aldehyde in the molar ratio 29:1 in aqueous solution. The rate constant of the conversion of diol into aldehyde is 8·7×10−2sec.−1 in the pH range 7·3–8·6 at 20°. The free aldehyde is the substrate for d-glyceraldehyde 3-phosphate dehydrogenase. Over a wide concentration range of enzyme the rate of conversion of diol into aldehyde is the rate-limiting process in the catalytic oxidation of d-glyceraldehyde 3-phosphate by NAD+. Aldolase and triose phosphate isomerase both liberate d-glyceraldehyde 3-phosphate as the aldehyde. This suggests that the relatively slow diol–aldehyde interconversion does not restrict the rate of glycolysis.
Activity of selected gluconeogenic and lipogenic enzymes in bovine rumen mucosa, liver and adipose tissue
Authors: Young Jerry W. , Thorp Sylvia L. , De Lumen Helen Z.
Published: August 2015

The activities of phosphoenolpyruvate carboxykinase, ‘malic enzyme’, citrate-cleavage enzyme and glucose 6-phosphate dehydrogenase were assayed in homogenates of rumen mucosa, liver and adipose tissue of cattle. Rumen mucosa cytoplasm contained activities of ‘malic enzyme’ approximately sevenfold those of phosphoenolpyruvate carboxykinase, suggesting that the conversion of propionate into lactate by rumen mucosa involves ‘malic enzyme’. Neither starvation for 8 days nor feeding with a concentrate diet for at least 3 months before slaughter produced enzyme patterns in the tissues different from those in cattle given only hay, except that the all-concentrate diet caused increased activities of glucose 6-phosphate dehydrogenase and ‘malic enzyme’ in adipose tissues. Rumen mucosa, liver and adipose tissue contained phosphoenolpyruvate carboxykinase activity. ‘Malic enzyme’ was absent in liver. Citrate-cleavage enzyme activity was present in liver and adipose tissue but was quite low in rumen mucosa. Liver contained much less glucose 6-phosphate dehydrogenase activity than rumen mucosa or adipose tissue.
Pyruvate kinase variants of the Alaskan king-crab. Evidence for a temperature-dependent interconversion between two forms having distinct and adaptive kinetic properties
Authors: Somero George N.
Published: August 2015

1. Pyruvate kinase of Alaskan king-crab leg muscle exists in two kinetically distinct forms, each of which displays a different temperature-dependence in the Km for phosphoenolpyruvate. 2. A ‘cold’ variant of the enzyme has hyperbolic kinetics and exhibits a minimal Km for substrate at 5°. At physiological concentrations of phosphoenolpyruvate the ‘cold’ enzyme is active only below 10°. A ‘warm’ pyruvate kinase has a minimal Km for substrate at about 12°. This enzyme displays sigmoidal kinetics and is likely to be inactive, at physiological substrate concentrations, at temperatures below 9°. 3. The combined activities of these two pyruvate kinases yield highly temperature-independent rates of catalysis, at physiological substrate concentrations, over the range of habitat temperatures encountered by the organism, namely 4–12°. 4. The two variants of pyruvate kinase do not appear to be isoenzymes in the conventional sense. Electrophoretic and electrofocus analyses revealed only single peaks of activity. 5. The results suggest that the ‘warm’ pyruvate kinase and the ‘cold’ pyruvate kinase are formed by a temperature-dependent interconversion of one protein species. This interconversion has major adaptive significance: as the temperature is lowered the ‘warm’ enzyme is converted into the ‘cold’ enzyme; the opposite situation obtains when the temperature is raised. Temperature changes thus mimic the effects noted for fructose 1,6-diphosphate on certain mammalian pyruvate kinases.
Control of the steady-state concentrations of the nicotinamide nucleotides in rat liver
Authors: Clark J. B. , Pinder S.
Published: August 2015

1. The effects of injecting nicotinamide, 5-methylnicotinamide, ethionine, nicotinamide+5-methylnicotinamide and nicotinamide+ethionine on concentrations in rat liver of NAD, NADP and ATP were investigated up to 5hr. after injection. 2. Nicotinamide induced three- to four-fold increases in hepatic NAD concentration even in the presence of 5-methylnicotinamide or ethionine, whereas 5-methylnicotinamide or ethionine alone did not cause marked changes in hepatic NAD concentration. 3. Nicotinamide alone also induced a twofold increase in hepatic NADP concentration. However, in the presence of 5-methylnicotinamide+nicotinamide, the NADP concentration decreased by 25% after 5hr., and in the presence of nicotinamide+ethionine by 30% in the same time. In the presence of 5-methylnicotinamide or ethionine alone hepatic NADP concentrations fell by 50% after 5hr. 4. 5-Methylnicotinamide inhibited the microsomal NAD+ glycohydrolase (EC 3.2.2.6) by 60% at a concentration of 1mm and the NADP+ glycohydrolase by 40% at the same concentration. 5. The rat liver NAD+ kinase (EC 2.7.1.23) was found to have Vmax. 4·83μmoles/g. wet wt./hr. and Km (NAD+) 5·8mm. This enzyme was also inhibited by 5-methylnicotinamide in a ‘mixed’ fashion. 6. The results are discussed with respect to the control of NAD synthesis. It is suggested that in vivo the NAD(P)+ glycohydrolases are effectively inactive and that the increased NAD concentrations induced by nicotinamide are due to increased substrate concentration available to both the nicotinamide and nicotinic acid pathways of NAD formation.
Intramitochondrial localization of δ-aminolaevulate synthetase and ferrochelatase in rat liver
Authors: McKay Roxane , Druyan R. , Getz G. S. , Rabinowitz M.
Published: August 2015

Intramitochondrial loci for δ-aminolaevulate synthetase and ferrochelatase, the initial and final enzymes in haem synthesis, have been found in rat liver. Two different methods of fractionation were applied to mitochondria: (a) sonication and density-gradient centrifugation; (b) treatment with digitonin and differential centrifugation. Similar results were obtained with each technique. δ-Aminolaevulate synthetase is distributed similarly to two known matrix enzymes, malate dehydrogenase and glutamate dehydrogenase. Ferrochelatase is firmly bound to the the inner mitochondrial membrane. These results are considered in terms of the regulation of haem synthesis and in relation to mitochondrial biogenesis.
The kinetic properties of citrate synthase from rat liver mitochondria
Authors: Shepherd D. , Garland P. B.
Published: August 2015

1. Citrate synthase (EC 4.1.3.7) was purified 750-fold from rat liver. 2. Measurements of the Michaelis constants for the substrates of citrate synthase gave values of 16μm for acetyl-CoA and 2μm for oxaloacetate. Each value is independent of the concentration of the other substrate. 3. The inhibition of citrate synthase by ATP, ADP and AMP is competitive with respect to acetyl-CoA. With respect to oxaloacetate the inhibition by AMP is competitive, but the inhibition by ADP and ATP is mixed, being partially competitive. 4. At low concentrations of both substrates the inhibition by ATP is sigmoidal and a Hill plot exhibits a slope of 2·5. 5. The pH optimum of the enzyme is 8·7, and is not significantly affected by ATP. 6. Mg2+ inhibits citrate synthase slightly, but relieves the inhibition caused by ATP in a complex manner. 7. At constant total adenine nucleotide concentration made up of various proportions of ATP, ADP and AMP, the activity of citrate synthase is governed by the concentration of the sum of the energy-rich phosphate bonds of ADP and ATP. 8. The sedimentation coefficient of the enzyme, as measured by activity sedimentation, is 6·3s, equivalent to molecular weight 95000.
Cathepsin B, the lysosomal thiol proteinase of calf liver
Authors: Snellman O.
Published: August 2015

Cathepsin B from calf liver was obtained by a method involving preparation of a lysosomal–mitochondrial pellet and treatment of this pellet with acetone. The material was extracted with an acid buffer, pH4·0, and then precipitated from the extract with acetone. The precipitate was dissolved in phosphate buffer, pH7·4, and subjected to gel filtration on Sephadex G-200 and G-100. The cathepsin B emerged in a range of molecular weight much lower than 50000 as a well-defined component. The purity of this material was checked by electrophoresis. To obtain maximum activity the enzyme had to be activated with a chelating agent and a reducing agent (i.e. EDTA and cysteine). A number of different substrates were used. The enzyme was active for the hydrolysis of both peptide bonds and ester bonds and had approximately equal reactivity in the two cases. The pH-dependence of the hydrolysis was the same with both substrates. The binding of the substrates was half-maximal at pH4·5 and at pH6·8. A thiol group occurred in the active centre but this group ought to have a much higher pK than that found in this enzyme.
Reversible activation of succinate dehydrogenase
Authors: McDonald-Gibson R. G. , Thorn M. B.
Published: August 2015

1. Treatment of particulate respiratory chain preparations in ways expected to raise or lower the concentration of endogenous soluble low-molecular-weight compounds respectively increased and diminished the capacity of succinate dehydrogenase to become activated reversibly and ‘spontaneously’ when preparations were diluted in tris acetate buffer and incubated at 37°. 2. Addition of critically low concentrations of recognized activators to preparations that failed to undergo reversible ‘spontaneous’ activation when incubated at 1mg. of protein/ml. conferred on them the capacity to do so. 3. Preparations with a diminished tendency to undergo reversible ‘spontaneous’ activation had an increased tendency to become irreversibly inactivated on prolonged incubation at 1mg. of protein/ml. in tris acetate. 4. Extraction procedures designed to demonstrate the presence of possible endogenous activators in enzyme preparations failed to reveal a single substance to which such a role could be conclusively attributed. A mixture of compounds was found, however, including certain amino acids that have been shown to act as activators. It is questionable whether these compounds would be present at sufficiently high concentrations to act as activators when enzyme preparations are diluted to 1mg. of protein/ml. 5. Despite the failure to demonstrate conclusively the presence of endogenous activators, the balance of evidence appears to favour the hypothesis that reversible ‘spontaneous’ activation of these preparations can best be explained by the presence of such substances, and a scheme describing the mechanism of activation and deactivation of succinate dehydrogenase is discussed in relation to these and other observations.
Solution properties of polygalacturonic acid
Authors: Stoddart R W , Spires I. P. C. , Tipton K F
Published: August 2015

1. The specimen of polygalacturonic acid used in these studies was shown to contain very little neutral sugar, methyl ester groups or ash, and only residues of galacturonic acid. Its electrophoretic homogeneity was examined in pyridine–acetic acid buffer at pH6·5 and in borate buffer at pH9·2. The distribution of effective particle weights was shown to be fairly narrow. 2. The pH-titration curve of the polymer gave a pK value of 3·7. 3. The interaction of the polymer with Ruthenium Red was studied and titration curves were obtained for the spectral shifts associated with the formation of a complex. 4. Optical-rotatory-dispersion studies showed that the Drude constant, λc, was dependent on pH. 5. Polygalacturonic acid was shown to display non-Newtonian properties in solution and to have an anomalously high relative specific viscosity at low concentrations. 6. Studies were made of the pH-dependence of the sedimentation coefficient of the polymer. 7. These results are discussed in terms of the structure of the molecule and their relevance to the properties of pectic substances.
Inhibition of bacterial growth by metal salts. A survey of effects on the synthesis of ribonucleic acid and protein
Authors: Blundell M. R. , Wild D. G.
Published: August 2015

Inhibition of the growth of Escherichia coli M.R.E. 600 by six different metal salts was accompanied by a greater decrease in the synthesis of RNA than in that of protein. The action of cobalt chloride was exceptional; inhibited cells made an excess of RNA to an extent depending on the concentration of Co2+, the time of incubation and the concentration of Mg2+ in the medium. Preferential synthesis of RNA in the presence of cobalt chloride was not confined to E. coli but occurred to various extents in some, but not all, of the other micro-organisms that were tested. Possible reasons for the special effect of Co2+ are discussed.
Periodate oxidation studies of human pituitary follicle-stimulating hormone
Authors: Kennedy J. F. , Butt W. R.
Published: August 2015

A preparation of human pituitary follicle-stimulating hormone was subjected to periodate oxidation, borohydride reduction and acid hydrolysis. Comparison of the analysis of the remaining intact carbohydrate and amino acid units with the analyses of the original material and identification of the carbohydrate fragments permit some structural assignments to the molecule of follicle-stimulating hormone. The results of radioimmunological assay of fragments of the molecule of follicle-stimulating hormone suggest that, although the carbohydrate component is essential for biological activity, it is not a requirement for immunological activity, which appears to be a function of the protein moiety.
Amino acid sequences of peptides from a chymotryptic digest of a urea-soluble protein fraction (U.S.3) from oxidized wool
Authors: Corfield M. C. , Fletcher J. C.
Published: August 2015

1. A chymotryptic digest of the protein fraction U.S.3. from oxidized wool was separated into 51 peptide fractions by chromatography on a column of cation-exchange resin. 2. The less acidic fractions were separated into their component peptides by a combination of cation-exchange-resin chromatography, paper chromatography and paper electrophoresis. 3. The amino acid sequences of 34 of these peptides were elucidated, and those of 14 others partially determined. 4. Overlaps between the tryptic and chymotryptic peptides from fraction U.S.3 have enabled ten extended amino acid sequences to be deduced, the longest containing 20 amino acid residues. 5. The relevance of the results to the structures of the helical and non-helical regions of wool is discussed.
A new approach to the analysis of hybridization of bacterial nucleic acids. Analysis of ribosomal ribonucleic acids of Bacillus subtilis
Authors: Avery R. J. , Midgley J. E. M.
Published: August 2015

A new graphical analytical technique is described for the hybridization of bacterial RNA with denatured homologous DNA immobilized on cellulose nitrate membrane filters. To a constant amount of DNA, various amounts of bacterial RNA were added and the percentage of input RNA bound was plotted against the DNA/RNA weight ratio in a given experiment. When RNA samples were used that hybridize to denatured DNA as a single species, the resulting curves (RNA-hybridization-efficiency curves) could be analysed to show the percentage of the DNA capable of specifically binding the RNA and could also be used to detect the presence of minor RNA contaminants in a purified specimen. The method could also estimate the relative amounts of two species of RNA in a mixture when these were hybridized independently to different DNA cistrons or cistron groups. As an example of RNA that can be studied in this way, the 16s and 23s ribosomal RNA species of Bacillus subtilis were chosen. These each behave in DNA–RNA hybridization as a single species and bind independently to different groups of DNA cistrons. The results obtained from hybridization-efficiency curves were compared with those obtained by the more usual method of saturating the specific DNA regions with excess of ribosomal RNA (hybridization-saturation curves). It was confirmed by both approaches that 0·15 (±0·02)% of B. subtilis DNA would hybridize with 16s ribosomal RNA, 0·30 (±0·02)% would hybridize with 23s ribosomal RNA, and 0·46 (±0·02)% would hybridize with (16s+23s) ribosomal RNA. This agreement suggested that mass-action equilibria between hybridized and free RNA had a negligible effect on the hybridization curves over the range of DNA and RNA concentrations employed.
The nature of the sex-linked differences in glutathione peroxidase activity and aerobic oxidation of glutathione in male and female rat liver
Authors: Pinto R E , Bartley W
Published: August 2015

1. Glutathione peroxidase activity in the livers of sham-operated female rats was about 60% higher than in similarly treated male rats. The value in the ovariectomized female was about the same as that in the castrated or sham-operated male. 2. Glutathione peroxidase activity changed during the oestrous cycle. The highest value was in oestrus, and was about 50% higher than the lowest activity, which was found in dioestrus. The activity in proestrus and in metoestrus was respectively about 20 and 30% higher than in dioestrus. 3. In the pregnant female 1 or 2 days before term, glutathione peroxidase activity was about 20% higher than that in the female in oestrus. 4. Subcutaneous implants of both oestra-diol and progesterone in the gonadectomized rats increased the glutathione peroxidase activity approximately to the values found in the female at oestrus. 5. The rate of aerobic oxidation of GSH in the female rat liver was about 80% higher than in the male and about 110% higher than in the gonadectomized rats. Treatment of gonadectomized rats with subcutaneous implants of oestradiol and of progesterone increased the rate of oxidation of GSH by about 100%. 6. In the presence of azide the rate of GSH oxidation in the male and in the female was respectively about 3·5- and 2·1-fold that in the absence of azide. In castrated or ovariectomized rats the increase due to the presence of azide was about 2·4-fold. In the gonadectomized rats treated with oestradiol or progesterone the rate of GSH oxidation in the presence of azide was about 2·2-fold that in its absence. 7. The rate of lipid peroxidation in female was 15–30-fold that in male or in gonadectomized rats. Treatment of the gonadectomized rats with oestradiol or with progesterone increased the rate of lipid peroxidation up to values that were even higher than in the female. In the presence of GSH the formation of malonaldehyde from peroxides was virtually eliminated. 8. The results suggest that the sex-linked differences in glutathione peroxidase activity, in the rate of GSH oxidation and in the rate of lipid peroxidation are due to the female sex hormones. 9. It is suggested that both the catalase activity and the rate of hydrogen peroxide formation are higher in the male than in the female. 10. Sex-linked changes in glutathione peroxidase, in the rate of GSH oxidation and in the rate of lipid peroxide formation are discussed in relation to the metabolism of oestrogens in the liver and also to the possible nature of those sex-linked changes.
Two fractions required for cell-free protein synthesis with components from rabbit reticulocytes
Authors: Cohen Brian B.
Published: August 2015

An extract was prepared from rabbit reticulocyte ribosomes after treatment with potassium chloride as described by Miller, Hamada, Yang, Cohen & Schweet (1967). This extract has been shown to convert monoribosomes into polyribosomes during protein synthesis in vitro (Cohen, 1968). The nature of this extract was studied in greater detail. Centrifugation of the extract through a sucrose density gradient separated the activity into a fast-sedimenting fraction. The two fractions were shown to have different functions in stimulating cell-free protein synthesis and their active components were shown to be protein or partly protein in nature. Each fraction was analysed by electrophoresis and in the analytical ultracentrifuge. It was concluded that the active component in the fast-sedimenting fraction had a sedimentation coefficient of 15·5s and that of the slow-sedimenting fraction 10·5s.
Inhibitory effect of pH5 enzyme from non-lactating bovine mammary gland on various stages of protein synthesis in the rat liver amino acid-incorporating system
Authors: Herrington M. D. , Hawtrey A. O.
Published: August 2015

1. pH5 enzyme from non-lactating bovine mammary gland was found to contain potent inhibitors of protein synthesis in the rat liver cell-free system. These inhibitors affect (a) formation of aminoacyl-tRNA where tRNA represents transfer RNA, (b) transfer of labelled amino acids from rat liver amino[14C]acyl-tRNA to protein in rat liver polyribosomes, and (c) incorporation of 14C-labelled amino acids into peptide by rat liver polyribosomes supplemented with rat liver pH5 enzyme. 2. Increasing amounts of pH5 enzyme from bovine mammary gland progressively inhibited the incorporation of labelled amino acids into protein by a complete incorporating system from rat liver. Approx. 80% inhibition was observed at a concentration of 2mg. of protein of pH5 enzyme from bovine mammary gland. The inhibitory effect of the bovine pH5 enzyme fraction could not be overcome by the addition of increasing amounts of rat liver pH5 enzyme. 3. Fractionation of bovine pH5 enzyme with ammonium sulphate into four fractions showed that all the fractions inhibited the incorporation of 14C-labelled amino acids in the rat liver system, but to varying extents. The highest inhibition observed (90%) was exhibited by the 60%-saturated-ammonium sulphate fraction. 4. Heat treatment of bovine pH5 enzyme at various temperatures caused only a partial loss of its inhibitory effect on labelled amino acid incorporation by the rat liver system. Treatment at 105° for 5min. resulted in the bovine pH5 enzyme fraction losing 30% of its inhibitory activity. 5. pH5 enzyme from bovine mammary gland strongly inhibited the charging of rat liver tRNA in the presence of its own pH5 enzymes. 6. The transfer of labelled amino acids from rat liver amino[14C]acyl-tRNA to protein in a system containing rat liver polyribosomes and pH5 enzyme was almost completely inhibited by bovine pH5 enzyme at a concentration of 2mg. of protein of the enzyme fraction. 7. One of the inhibitors of various stages of protein synthesis in rat liver present in bovine pH5 enzyme was identified as an active ribonuclease, and the second inhibitor present was shown to be tRNA.
The metabolism of [14C]nicotine in the cat
Authors: Turner D. M.
Published: August 2015

The metabolism of [2′−14C]nicotine given as an intravenous injection in small doses to anaesthetized and unanaesthetized cats has been studied. A method is described for the quantitative determination of [14C]nicotine and [14C]cotinine in tissues and body fluids. Nanogram amounts of these compounds have been detected. After a single dose of 40μg. of [14C]nicotine/kg., 55% of the injected radioactivity was excreted in the urine within 24hr., but only 1% of this radioactivity was unchanged nicotine. [14C]Nicotine is metabolized extremely rapidly, [14C]cotinine appearing in the blood within 2·5min. of intravenous injection. [14C]Nicotine accumulates rapidly in the brain and 15min. after injection 90% of the radioactivity still represents [14C]nicotine. Metabolites of [14C]nicotine have been identified in liver and urine extracts. [14C]Nicotine-1′-oxide has been detected in both liver and urine.
Chemical and biological properties of mycobactins isolated from various mycobacteria
Authors: Snow G. A. , White A. J.
Published: August 2015

Nine different strains of mycobacteria grown on media deficient in iron all produced mycobactins. Most strains produced one mycobactin in great preponderance. Mycobacteria from clearly distinct taxonomic groups gave mycobactins differing in the structure of their nuclei. One group of taxonomically related mycobacteria produced mycobactins having the same nucleus but with different distributions of side chains within the homologous mixtures. Simple methods are described for identifying mycobactins on a small scale; these may be of value in classifying mycobacteria. Structures are proposed for mycobactin A from Mycobacterium aurum, mycobactin R from M. terrae, mycobactin F, produced together with mycobactin H by M. fortuitum, and mycobactins M and N from M. marinum. The first three of these differ from known mycobactins in details of substitution and configuration of asymmetric centres in the nucleus. Mycobactins M and N are substantially different, having only small acyl groups (acetyl and propionyl respectively) at the hydroxamic acid centre of the mycobactic acid moiety. Both are homologous mixtures having long-chain saturated 3-hydroxy-2-methyl acid fragments in the cobactin moiety. All mycobactins so far isolated promote almost maximal growth of M. johnei at 30ng./ml. in liquid medium. The activity of some mycobactins extends to much lower concentrations, mycobactin S showing significant growth promotion at 0·3ng./ml. Mycobactin M or N in combination with mycobactins having a long side chain in the mycobactic acid moiety exerts a mutually antagonistic effect on the growth of M. johnei, the mixture giving less growth than either mycobactin separately. Mycobactin M also decreases the growth of M. kansasii and M. tuberculosis on liquid media. These antagonistic effects are probably caused by a lengthening of the lag phase.
Effects of thyroxine on the synthesis of folate coenzymes in rat liver
Authors: Pasquali P. , Landi L. , Bovina C. , Marchetti M.
Published: August 2015

1. The effects of thyroidectomy and of ‘acute’ and ‘chronic’ administration of thyroxine on the synthesis of folate coenzymes were studied by determining the liver contents of folate active derivatives and the enzymic activities involved in their biosynthesis. The effect of thyroxine on the same enzymes in vitro was also studied. 2. In thyroidectomized rats the liver contents of folate coenzymes did not change except for a slight decrease in the contents of 5-formyltetrahydrofolate and tetrahydrofolate compared with those in control rats. 3. In the same animals serine hydroxymethyltransferase and formyltetrahydrofolate synthetase activities decreased markedly. 4. The ‘chronic’ administration of thyroxine to thyroidectomized rats caused more evident variations in the liver contents of folate coenzymes and in particular a decrease in the contents of 5-formyltetrahydrofolate, tetrahydrofolate, 5(or 10)-formyl derivatives of tetrahydropteroylpolyglutamate and of 5(or 10)-formyl derivatives of pteroylpolyglutamate. 5. The enzymic activities did not show significant variations. 6. The ‘acute’ administration of thyroxine caused changes in the liver contents of some folate derivatives such as 10-formyldihydrofolate, 10-formylfolate, tetrahydrofolate and the 10-formyl derivative of dihydropteroylpolyglutamate. In these animals also the enzymic activities were unchanged. 7. No effect of thyroxine on enzymic activities in vitro was observed.
Depolarization of the intrinsic and extrinsic fluorescence of pepsinogen and pepsin
Authors: Teale F. W. J. , Badley R. A.
Published: August 2015

1. The effects on the intrinsic tryptophan emission anisotropy of pepsin and pepsinogen solutions produced by (a) changes in temperature, (b) increases in viscosity with added glycerol at constant temperature and (c) decreases in lifetime through collisional quenching by potassium iodide were measured at several excitation wavelengths. The rotational-relaxation times calculated from results provided by method (b) approximate to the theoretical values for the two proteins, on taking hydration and shape factors into account, on the basis of random orientation of the tryptophan groups within the macromolecules. Differences between the results provided by methods (b) and (c) are attributable to inter-tryptophan resonance-energy-transfer depolarization, and the anomalous values recorded in method (a) can be attributed to the temperature-dependence of the limiting anisotropies. 2. Two different monomeric conjugates of pepsin, each containing one extrinsic fluorescent group per macromolecule, gave widely different relaxation times. This difference may arise from a specific orientation of the emission dipole in the enzyme. In active-site-labelled pepsin (1-dimethylaminonaphthalene-5-sulphonylphenylalanine–pepsin) this orientation would be approximately parallel to the symmetry axis of the equivalent ellipsoid, whereas in the other conjugate (1-dimethylaminonaphthalene-5-sulphonyl-pepsin) the orientation may be roughly normal to this direction, or some independent rotation of parts of the protein molecule is possible.
Distribution of prostaglandins in rabbit kidney
Authors: Crowshaw Keith , Szlyk J. Z.
Published: August 2015

Three prostaglandins (PGE2, PGF2α and PGA2) are present in rabbit kidney medulla. An acidic lipid extract (0.165g) obtained from 2kg of frozen rabbit kidney cortex was separated by silicic acid chromatography to yield eluates containing fatty acids, possible non-polar prostaglandin metabolites, PGA, PGE and PGF compounds. Ultraviolet spectra of the eluates before and after treatment with sodium hydroxide did not yield chromophores typical of any known prostaglandins or related metabolites. By using more sensitive bioassay procedures (contraction of rabbit duodenum) weak activity equivalent to 60μg of PGE2 and 10μg of PGF2α was detected in the PGE and PGF eluates respectively. Extraction and bioassay of fresh kidney cortex revealed no prostaglandin-like activity. Attempts to biosynthesize prostaglandins in fresh homogenates of rabbit kidney cortex from endogenous precursors and from added arachidonic acid were unsuccessful. When freshly prepared homogenates of rabbit kidney cortex were incubated with added PGE1 no evidence of enzymic breakdown was obtained. It is concluded that rabbit kidney prostaglandins are present predominantly in the medulla and there are no cortical mechanisms for their biosynthesis or inactivation under normal conditions.