bluuebunny/crossref_metadata_2025 huggingface dataset:
A study of the thermal stability of ribosomes and biologically active subribosomal particles
Authors: Cox R. A. , Pratt Hester , Huvos Piroska , Higginson Betty , Hirst W.
Published: August 2015

1. The ability of Escherichia coli ribosomes to function in poly(U)-directed protein synthesis was measured at elevated temperatures by using thermostable supernatant factors from Bacillus stearothermophilus. The amount of polyphenylalanine synthesized at 55°C was about the same as at 37°C, but the rate of synthesis was increased approximately fivefold. At 60°C the activity of the ribosomes was halved. 2. E. coli ribosomes can sustain the loss of approx. 10% of the double-helical secondary structure of RNA without losing activity. 3. Within the active ribosome the double-helical secondary structure of the rRNA moiety is stabilized compared with isolated rRNA, as judged by enzymic hydrolysis and by measurements of E260. 4. The main products, over the range 0–55°C, of ribonuclease T1 digestion of the smaller subribosomal particle of E. coli were two fragments (s020,w 15S and 25.3S) of approximately one-quarter and three-quarters of the size of the intact molecule, revealing the presence of a ‘weak spot’ where intramolecular bonds appear insufficient to hold the fragments together. 5. Subribosomal particles of B. stearothermophilus were more stable to heating, by approx. 10°C, than those of E. coli, and the stabilization of double-helical secondary structure of the RNA moiety was more striking. 6. Rabbit reticulocyte ribosomes were active in poly(U)-directed protein synthesis at 45°C, and half the activity was lost after heating to 53°C. Active subribosomal particles of rabbit reticulocytes and of oocytes of Xenopus laevis, like the bacterial subribosomal particles, underwent a conformational change to a slower-sedimenting form on heating. The temperature range of the transition depended on the species. 7. Slower-sedimenting particles, whether produced by EDTA treatment or by heating, had different ‘melting’ profiles compared with active subribosomal particles, providing another indication of conformational differences. 8. Comparison of the properties of the various subribosomal particles revealed greater variation in the secondary structure of the protein moieties (judged by measurement of circular dichroism) than in the secondary structure of the RNA moieties, which appeared to have features in common.
Protein synthesis in vitro in a system from plant mitochondria (Short Communication)
Authors: Goswami Biswendu B. , Chakrabarti Symamalima , Dube Dipak K. , Roy S. C.
Published: August 2015

A mitochondrial system from 48h-germinating seeds of Vigna sinensis (Linn.) Savi is capable of incorporating l-[U-14C]valine into proteins and is practically insensitve to cycloheximide, but highly sensitive to chloramphenicol and fusidic acid, a potent inhibitor of peptide-chain elongation factor. A system consisting of mitochondrial S-100 fraction and ribosomes from the same source and other cofactors is capable of polyphenylalanine synthesis and behaves similarly with respect to these inhibitors.
Biosynthesis of mercapturic acids from allyl alcohol, allyl esters and acrolein
Authors: Kaye Clive M.
Published: August 2015

1. 3-Hydroxypropylmercapturic acid, i.e. N-acetyl-S-(3-hydroxypropyl)-l-cysteine, was isolated, as its dicyclohexylammonium salt, from the urine of rats after the subcutaneous injection of each of the following compounds: allyl alcohol, allyl formate, allyl propionate, allyl nitrate, acrolein and S-(3-hydroxypropyl)-l-cysteine. 2. Allylmercapturic acid, i.e. N-acetyl-S-allyl-l-cysteine, was isolated from the urine of rats after the subcutaneous injection of each of the following compounds: triallyl phosphate, sodium allyl sulphate and allyl nitrate. The sulphoxide of allylmercapturic acid was detected in the urine excreted by these rats. 3. 3-Hydroxypropylmercapturic acid was identified by g.l.c. as a metabolite of allyl acetate, allyl stearate, allyl benzoate, diallyl phthalate, allyl nitrite, triallyl phosphate and sodium allyl sulphate. 4. S-(3-Hydroxypropyl)-l-cysteine was detected in the bile of a rat dosed with allyl acetate.
Biosynthesis of growth hormone in the rat anterior pituitary gland. Stimulation of biosynthesis in vitro by insulin
Authors: Betteridge A. , Wallis M.
Published: August 2015

The effect of insulin on the incorporation of radioactive leucine into growth hormone was investigated by using rat anterior pituitary glands incubated in vitro. A 50% stimulation over control values was observed at insulin concentrations above 2μm (280munits/ml). The effect was specific for growth hormone biosynthesis, over the range 1–5μm-insulin (140–700munits/ml). Lower more physiological concentrations had no significant effect in this system. Above 10μm (1.4 units/ml) total protein synthesis was also increased. The stimulation of growth hormone synthesis could be partially blocked by the addition of actinomycin D, suggesting that RNA synthesis was involved. Insulin was found to stimulate the rate of glucose utilization in a similar way to growth hormone synthesis. 2-Deoxyglucose and phloridzin, which both prevented insulin from stimulating glucose utilization, also prevented the effect of insulin on growth hormone synthesis. If glucose was replaced by fructose in the medium, the effect of insulin on growth hormone synthesis was decreased. We conclude that the rate of utilization of glucose may be an important step in mediating the effect of insulin on growth hormone synthesis.
Studies on the sulphation of 3,4-dihydroxyphenylethylamine (dopamine) and related compounds by rat tissues
Authors: Jenner William N. , Rose Frederick A.
Published: August 2015

The formation of sulpho-conjugates of 3,4-dihydroxyphenylethylamine (dopamine) and related compounds was examined in preparations of rat tissues. Liver high-speed-supernatant preparations readily transferred sulphate from adenosine 3′-phosphate 5′-sulphato-phosphate to dopamine under standard conditions. The main product was identified as the 3-O-sulphate. The preparation also sulphated the 3- and 4-methoxy derivatives but to a lesser extent (44% and 95% respectively) relative to dopamine. Brain preparations possessed only half the activity of liver but formed both the 3- and 4-O-sulphates in the molar ratio of 1.7:1. l-3,4-Dihydroxyphenylalanine (l-dopa) in both tissue preparations did not yield any significant amount of sulpho-conjugate when the dopa decarboxylase present was inhibited. The sulphotransferase activity of preparations was doubled in the presence of dithiothreitol and it was concluded that l-tyrosine methyl ester sulphotransferase was the enzyme involved. A method for the preparation of authentic dopamine 3-O-sulphate and 4-O-sulphate was developed.
A phosphorylated light-chain component of myosin from skeletal muscle
Authors: Perrie W. T. , Smillie L. B. , Perry S. V.
Published: August 2015

1. The low-molecular-weight components of myosin from rabbit skeletal muscle migrated as four bands on polyacrylamide-gel electrophoresis in 8m-urea but only as three in systems containing sodium dodecyl sulphate. The two bands of intermediate mobility in 8m-urea (Ml2 and Ml3) had identical mobilities in sodium dodecyl sulphate. 2. The isolation of pure samples of all four low-molecular-weight components by DEAE-Sephadex chromatography is described. 3. The amino acid compositions of components Ml2 and Ml3 were identical. Further analyses showed the presence of 1 mol of phosphate/18500g of component Ml2 and less than 10% of this amount in component Ml3. Neither light component contained ribose. 4. Alkaline phosphatase from Escherichia coli converted component Ml2 into Ml3. Incubation with crude preparations of phosphorylase b kinase or protein kinase in the presence of ATP converted component Ml3 into Ml2. 5. Phosphorylation of component Ml3 with the kinases isolated from skeletal muscle and [γ-32P]ATP gave incorporation of 32P only into component Ml2 whether whole myosin or separated low-molecular-weight components were used. 6. High-voltage electrophoresis at pH6.5 and pH1.8 of a chymotryptic digest of 32P-labelled component Ml2 yielded one major radioactive peptide containing serine phosphate. 7. The amino acid sequence of this peptide was shown to be: Arg-Ala-Ala-Ala-Glu-Gly-Gly-(Ser,Ser(P))-Asn-Val-Phe. This sequence shows no obvious similarity to the site phosphorylated in the conversion of phosphorylase b into phosphorylase a by phosphorylase b kinase. 8. Evidence suggests that in vivo all the 18500-molecular-weight light chain is in the phosphorylated form. The extent of dephosphorylation that occurred during myosin extraction depended on the conditions employed.
Amino acid activation in mammalian brain. Purification and characterization of tryptophan-activating enzyme from buffalo brain
Authors: Liu C.-C. , Chung C.-H. , Lee M.-L.
Published: August 2015

l-Tryptophan-activating enzyme [l-tryptophan–tRNA ligase (AMP), EC 6.1.1.2] of water-buffalo brain was purified to near homogeneity by heat and pH treatments, ammonium sulphate fractionation, column chromatography on DEAE-cellulose, hydroxyapatite and Amberlite CG-50, and gel filtration on Sephadex G-200. The purified enzyme catalyses tryptophanyl-tRNA formation with yeast tRNA, but not with Escherichia coli tRNA. The enzyme exhibits multiple peaks of activity in Sephadex gel filtration with molecular weights corresponding to 155000, 105000 and 50000. However, only one peak of activity with molecular weight of 155000 can be detected when the enzyme is subjected to gel filtration at high concentration. Disc gel electrophoresis in the presence of sodium dodecyl sulphate reveals a single band with molecular weight of 55000. The activity of the enzyme is concentration dependent. Different Km and Vmax. values are obtained at different enzyme concentrations. These data suggest that this enzyme may exist in different quaternary structures, each with its own kinetic constants. The enzyme activity is inhibited by p-chloromercuribenzoate, and is not protected by the presence of the substrates, l-tryptophan, Mg2+, ATP, in any combination.
Cross-specificity in some vertebrate and insect glutathione-transferases with methyl parathion (dimethyl p-nitrophenyl phosphorothionate), 1-chloro-2,4-dinitrobenzene and S-crotonyl-N-acetylcysteamine as substrates
Authors: Clark A. G. , Smith J. N. , Speir T. W.
Published: August 2015

1. Enzymes catalysing the reaction between GSH and methylparathion (dimethyl p-nitrophenyl phosphorothionate), 1-chloro-2,4-dinitrobenzene and S-crotonyl-N-acetylcysteamine were separated by (NH4)2SO4 precipitation from homogenates of sheep, rat and mouse livers and from homogenates of cockroaches, houseflies and grass grubs. 2. Electrofocusing of the preparations from each of these species separated a number of zones, each of which catalysed the reaction of GSH with all three substrates. 3. Ion-exchange chromatography on CM-cellulose also separated a number of fractions in which activity towards the three substrates coincided. 4. In both separation methods patterns of the activities were consistent with the presence in all species of several GSH transferases each having a degree of cross specificity towards the three substrates.
The effects of chronic phenobarbitone administration and subsequent withdrawal on the activity of rat liver tryptophan pyrrolase and their resemblance to those of ethanol
Authors: Badawy Abdulla A.-B. , Evans Myrddin
Published: August 2015

Chronic phenobarbitone administration inhibits the apo-(tryptophan pyrrolase) activity in homogenates of rat liver and subsequent withdrawal enhances the enzyme activity by 2.5-fold. Similar effects have been previously produced by chronic ethanol administration and withdrawal, but, whereas NADH may cause the ethanol inhibition, that by phenobarbitone may be mediated by NADPH.
The nature and mode of action of the cellulolytic component C1 of Trichoderma koningii on native cellulose
Authors: Halliwell G. , Griffin M.
Published: August 2015

1. A purified cellulolytic component C1 was isolated free from associated activities of the cellulase complex and shown to act as a β-1,4-glucan cellobiohydrolase on both simple and complex forms of native cellulose. 2. The enzyme releases terminal cellobiose units from cellulose, its extent of action being determined principally by the product and by the nature of the substrate. 3. Component Cx of the cellulase system is not required for the action of component C1 (cellobiohydrolase). The enzyme synergizes extensively with cellobiase in extending the hydrolysis of native and of less-complex forms of cellulose to at least 70% with the liberation of glucose. 4. The cellobiohydrolase is relatively unstable, with an optimum at pH5 and a Km of 0.05mg/ml. The enzyme is inhibited by its product, from which it is released by cellobiase. 5. Of other compounds tested against the cellobiohydrolase the metal ions Cu2+, Zn2+, phenylmercuric and Fe3+are increasingly effective inhibitors. Glucose has no action at concentrations found inhibitory with cellobiose. 6. The relationship of the enzyme to the entire cellulase complex is discussed.
Glycyl-l-leucine hydrolase, a versatile ‘master’ dipeptidase from monkey small intestine
Authors: Das Manjusri , Radhakrishnan A. N.
Published: August 2015

1. A highly active and electrophoretically homogeneous dipeptidase was purified from the soluble extracts of monkey small-intestinal mucosa. 2. By gel-filtration studies the molecular weight of the enzyme was found to be 107000. It is composed of two identical, subunits of molecular weight 54000. 3. A paper-chromatographic method of dipeptidase assay was developed to overcome some of the difficulties encountered in the generally used spectrophotometric procedure. By using this method, the Km and k0 values of a few substrates were determined. 4. The substrate specificity of the enzyme was investigated in great detail with substrates of a wide range of possible structural types. The enzyme hydrolyses a very large proportion of the range of dipeptides tested. This enzyme, which exhibits such a wide range of action, has been termed the ‘master’ dipeptidase of the intestine. Glycylglycine, glycyl-l-proline, glycyl-l-histidine, l-prolylglycine and some of the arginine- and aspartic acid-containing dipeptides were not substrates and are possibly hydrolysed by other peptidases. These results therefore suggest that in the intestine the number of dipeptidases is rather limited. 5. In the light of these findings, the implications on the role of dipeptidases in intestinal peptide transport are discussed.
The direction of glycan synthesis in a bacterial peptidoglycan
Authors: Ward J. Barrie , Perkins Harold R.
Published: August 2015

A cell-free membrane preparation from a poorly lytic mutant of Bacillus licheniformis was used to synthesize radioactive peptidoglycan. The product was apparently un-cross-linked. When UDP-N-acetyl[14C]glucosamine was used and the final peptidoglycan subjected to Smith degradation, no radioactive glycerol was found. On the other hand, when peptidoglycan labelled with meso-diamino[14C]pimelic acid was first hydrolysed in 0.1m-HCl at 60°C for 2h and then subjected to alkaline conditions, radioactive lactyl-peptides were eliminated. The proportion of radioactive lactyl-peptide decreased with increasing time of incorporation. It is concluded that the glycan chains grow by extension at their reducing ends while remaining attached by some linkage labile to mild acid, such as a glycosyl link to undecaprenol pyrophosphate.
Assay for glutamine synthetase activity (Short Communication)
Authors: Vorhaben Jean E. , Wong Lyle , Campbell James W.
Published: August 2015

The use of phosphoenolpyruvate plus pyruvate kinase as an ATP-generating system in the assay for glutamine synthetase activity via the formation of γ-glutamylhydroxamate from glutamate and hydroxylamine with crude tissue preparations is shown to give values far in excess of the true glutamine synthetase activity of the tissue. This is due to the generation of pyruvate, which reacts with hydroxylamine to give a compound that is chromogenic with the ferric chloride reagent used for measuring γ-glutamylhydroxamate.
Folic acid metabolism in vitamin B12-deficient sheep. Depletion of liver folates
Authors: Smith Richard M. , Osborne-White William S.
Published: August 2015

1. Metabolism of folate was studied in six ewes in an advanced state of vitamin B12 deficiency as judged by voluntary food intake and in their pair-fed controls receiving vitamin B12. A group of four animals that were maintained throughout the experiment at pasture was also studied. 2. After 34–40 weeks on the cobalt-deficient diet urinary excretion of formiminoglutamate by four deficient animals was about 3.2mmol/day and this was not significantly decreased by injection of three of them with about 4.5μg of [2-14C]folate/kg body weight per day for 5 days. Three days after the last injection retention of [2-14C]folate by the livers of the deficient animals (5.5% of the dose) was lower than that of their pair-fed controls (26% of the dose) but there was no evidence of net retention of injected folate in the livers of either group. Urinary excretion of 14C indicated that renal clearance of folate may have been impaired in very severe vitamin B12 deficiency. 3. As estimated by microbiological assays total folates in the livers of animals at pasture (12.9μg/g) included about 24% of 5-methyltetrahydrofolate as compared with about 72% of a total of 12.5μg/g in three further ewes fed on a stock diet of wheaten hay-chaff and lucerne-chaff. Liver folates of vitamin B12-deficient animals (0.5μg/g) included about 88% of 5-methyltetrahydrofolate as compared with about 51% of a total of 5.2μg/g in pair-fed animals treated with vitamin B12. 4. Chromatography of liver folates of the pair-fed animals permitted quantitative estimates of the pteroylglutamates present. The results showed that the vitamin B12-deficient livers were more severely depleted of tetrahydrofolates and formyltetrahydrofolates than of methyltetrahydrofolates and that as the deficiency developed they were more severely depleted of the higher polyglutamates than of the monoglutamate within each of these classes. Results from animals injected with [2-14C]folate indicated an impairment of the exchange between pteroylmonoglutamates and pteroylpolyglutamates in the livers of deficient animals. 5. In vitamin B12-deficient animals with food intakes below 200g/day some of the liver folates were not completely reduced and some degradation of pteroylpolyglutamates was detected. The latter condition may have been associated with fatty liver. 6. The results are discussed in relation to current theories of vitamin B12–folate interactions.
Hydrolysis of GM1-ganglioside by human liver β-galactosidase isoenzymes
Authors: Ho Mae Wan , Cheetham Peter , Robinson Donald
Published: August 2015

1. GM1-ganglioside, specifically tritiated in the terminal galactose, was hydrolysed by two forms of ‘acid’ methylumbelliferyl β-galactosidase isolated on gel filtration. 2. Identification of GM1-ganglioside β-galactosidase activity with the ‘acid’ methyl-umbelliferyl β-galactosidases was based on the following: coincident elution profiles on gel filtration; simultaneous inactivation by heat and other treatments; stabilization of both activities by chloride ions; mutual inhibition of hydrolysis by the two substrates. 3. The two isoenzymes (I) and (II) showed general requirements for a mixture of anionic and nonionic detergents in the hydrolysis of the natural substrate. 4. Isoenzyme (I) differed from (II) in molecular size, pH–activity profile, relative resistance to dilution and in sensitivity to various inhibitors. 5. The most significant difference between the isoenzymes is in substrate saturation kinetics: (I) was hyperbolic whereas (II) was sigmoid. The apparent Michaelis constants were 28μm for (I) and 77μm for (II). Isoenzyme (I) was insensitive to GM2-ganglioside whereas (II) was inhibited, consistent with the hypothesis that GM1-ganglioside (and its analogue) acts as modifier in isoenzyme (II) but not in (I). 6. Isoenzyme (I) was membrane-bound whereas (II) was soluble; the former probably represents isoenzyme (II) bound to membrane components, thereby becoming activated. 7. Membranes may serve a dual role in enzyme catalysis involving lipids: as a medium where both enzyme and substrate are effectively concentrated, and as actual activator of enzymes through binding of the latter to specific membrane components.
Microbiological transformation of steroids. 1-Dehydrogenation of 17,21-dihydroxypregn-4-ene-3,20-dione with Glomerella cingulata
Authors: Pla Luis Costa
Published: August 2015

The micro-organism Glomerella cingulata dehydrogenates 17,21-dihydroxypregn-4-ene-3,20-dione to 17,21-dihydroxypregna-1,4-diene-3,20-dione in high yield practically without by-products.
Ribosome precursors accumulated by Escherichia coli during incubation with cobalt chloride
Authors: Blundell M. R. , Wild D. G.
Published: August 2015

The ribonucleoprotein particles that accumulate during inhibition of Escherichia coli by CoCl2 are ribosome precursors. The RNA that they contain can be transferred intact to completed ribosomes. The particles contain less protein than do ribosomes, but this protein appears identical with proteins from the appropriate ribosomal subunit. At least one of the particles is heterogeneous.
Phosphatidylinositol kinase. A component of the chromaffin-granule membrane
Authors: Phillips John H.
Published: August 2015

Phosphorylation of bovine chromaffin granules by ATP leads to the formation of diphosphoinositide in the granule membrane. Both phosphatidylinositol kinase and its substrate are components of this membrane, and triphosphoinositide is not formed under the conditions of the assay. The reaction is Mg2+-dependent and is stimulated by Mn2+and F−ions. The initial reaction is rapid, with a broad pH profile and a ‘transition’ temperature for its activation energy at 27°C. The apparent Km for ATP is 5μm. ATP, N-ethylmaleimide, Cu2+ions and NaIO4 are inhibitory. The phospholipids of chromaffin-granule membranes have been analysed: 6.8% of the lipid P is found in phosphatidylinositol, and only 2–3% in phosphatidylserine. Comparison of the rate of phosphorylation of intact and lysed granules suggests that the sites for phosphorylation are on the outer (cytoplasmic) surface of the granules, and diphosphoinositide may therefore make an important contribution to the charge of the chromaffin granule in vivo.
Identity of ‘acid’ β-glucosidase and glucocerebrosidase in human spleen
Authors: Ho Mae Wan
Published: August 2015

1. Glucocerebrosidase, in association with a membrane-bound ‘acid’ β-glucosidase, was separated from a soluble ‘neutral’ β-glucosidase that had no activity towards glucocerebroside as substrate. 2. Glucocerebrosidase, as well as ‘acid’ β-glucosidase activity depended upon the association of factor P (a heat-stable, soluble, acidic glycoprotein) with factor C (a heat-labile membrane-bound protein). 3. Factor C was solubilized under certain conditions. 4. Solubilized factor C, as well as membrane-bound factor C, could be alternatively stimulated by sodium taurocholate to give both glucocerebrosidase and ‘acid’ β-glucosidase activities. 5. Membrane-bound factor C reacted optimally with factor P whereas solubilized factor C was preferentially stimulated by taurocholate. 6. Factor P-dependent glucocerebrosidase activity differed in kinetic properties from the taurocholate-stimulated enzyme activity. 7. The results are discussed in the light of (a) identity of glucocerebrosidase and ‘acid’ β-glucosidase, (b) application in clinical diagnosis, (c) physiological significance of the enzyme system, and (d) polygenic inheritance in adult Gaucher's disease.
Biosynthesis of respiratory-tract mucins. A comparison of canine epithelial goblet-cell and submucosal-gland secretions
Authors: Stahl Glenn H. , Ellis Daniel B.
Published: August 2015

1. A modified canine tracheal organ culture system was used to investigate differences between mucous secretions of epithelial goblet cells and the submucosal glands. 2. Denuded explants were prepared by removing goblet, ciliated and basal cells from the surface epithelium leaving an intact basement membrane and viable submucosa. 3. Denuded explants actively incorporated radioactive precursors into secreted macromolecules when cultured in medium 199 containing label. 4. Chromatography on Bio-Gel A-150m and electrophoresis on 1% agarose gels indicated that epithelial goblet cell secretions were relatively more sulphated than submucosal glandular secretions. 5. The glandular structures were shown to respond to a parasympathomimetic agent.
Output of [14C]adenine nucleotides and their derivatives from cerebral tissues. Tetrodotoxine-resistant and calcium ion-requiring components
Authors: Pull Ian , McIlwain Henry
Published: August 2015

1. Neocortical tissues, exposed briefly to [14C]adenine and containing over 98% of their14C as adenine nucleotides, when superfused with glucose–bicarbonate salines released about 0.1% of their14C content/min to the superfusate. 2. Addition of unlabelled adenosine to the superfusing fluid increased the14C output three- to four-fold; half-maximal increase was given by about 40μm-adenosine, and reasons are adduced for considering the activity of adenosine kinase to be a major factor in conditioning the14C output. Adenosine similarly increased the enhanced14C output caused by electrical excitation of the superfused tissue; it brought about only a small increase in tissue glycolysis. 3. Output of14C from the [14C]adenine-labelled tissues was increased when Ca2+was omitted from the superfusing fluids, but electrical stimulation did not then liberate more14C. Nevertheless, such tissues still responded to electrical stimulation by increased glycolysis, and their14C output again became susceptible to increase by electrical stimulation when Ca2+was restored. 4. The six-fold increase in tissue glycolysis caused by electrical excitation was almost completely inhibited by tetrodotoxin at 0.1μm and above, but this was associated with about 50% inhibition only in the output of14C from tissues preincubated with [14C]adenine. The14C-labelled compounds of which output was most inhibited by tetrodotoxin were adenosine, inosine and hypoxanthine whereas output in a nucleotide fraction was little affected.
A rapid method for collecting and assaying gradient fractions for radioactive macromolecules (Short Communication)
Authors: Rubinstein Leonard , Rein Alan
Published: August 2015

A rapid method is described for collecting gradients after centrifugation, and assaying them for acid-precipitable radioactivity. An entire gradient is collected and processed on a single glass-fibre filter disc. This method allows one easily to assay six gradients for acid-precipitable radioactivity in less than 2h.
The molecular basis for biological inactivation of nucleic acids. The action of methylating agents on the ribonucleic acid-containing bacteriophage R17
Authors: Shooter Kenneth V. , Howse Ruth , Shah Sudhikumar A. , Lawley Philip D.
Published: August 2015

1. The inactivation of an RNA-containing bacteriophage after reaction with four methylating agents was studied. Measurements of the extent of methylation of the RNA and of the nature and amounts of the various reaction products were made. In experiments with dimethyl sulphate and methyl methanesulphonate inactivation can be quantitatively accounted for by methylation at two of the positions involved in hydrogen bonding: N-1 of adenine and N-3 of cytosine. In experiments with N-methyl-N-nitrosourea and N-methyl-N′-nitro-N-nitrosoguanidine methylation at N-1 of adenine and N-3 of cytosine accounts for only about one-half of the observed inactivation. Scission of the RNA chain during reaction accounts for a further 20% of the inactivation. To account for the remainder it seems necessary to postulate that formation of O6-methylguanine constitutes a lethal lesion. 2. Breaks in the RNA chain formed on reaction with the nitroso derivatives presumably result from methylation of the phosphate diester group followed by hydrolysis of the unstable triester thus formed.
Electron-paramagnetic-resonance studies on cobalt(II) carbonic anhydrase. Low-spin cyanide complexes
Authors: Cockle Stephen A.
Published: August 2015

The low-spin cyanide complexes of three Co(II) carbonic anhydrases were investigated by electron paramagnetic resonance (e.p.r.) at 9 and 35GHz. Well-defined and closely axial spectra were obtained only in the absence of oxygen. Several mole equivalents of cyanide were required for complete formation of the complexes in frozen solution, although large excesses caused abstraction of the cobalt. Experiments with [13C]cyanide showed that the low-spin complexes contained two CN- groups in an environment similar to that of the in-plane ligands in [Co(CN)5]3-. A combined e.p.r. and spectrophotometric titration confirmed the presence of two CN- ligands. A 5-co-ordinate square pyramidal structure involving three protein ligands was proposed. The dicyanide complex could be oxygenated reversibly, producing a characteristic new e.p.r. spectrum. The O2 molecule was thought to occupy the remaining octahedral metal site in a formally Co(III) species. The optical spectrum of the dicyanide lacked the prominent d–d bands of the high-spin monocyanide. Both e.p.r. and optical data indicated that the low-spin complex was formed much more fully in frozen solution than at room temperature. Differences in behaviour between the high- and low-activity enzymes suggested some variation in conformational flexibility at the metal binding site.
Characterization of plasma lipoproteins separated and purified by agarose-column chromatography
Authors: Rudel Lawrence L. , Lee Jason A. , Morris Manford D. , Felts James M.
Published: August 2015

1. A simple method for isolation of individual human plasma lipoprotein classes is presented. In this technique, lipoproteins are removed from plasma at d1.225 by ultracentrifugation, after which they are separated and purified by agarose-column chromatography. 2. Three major classes are obtained after agarose-column chromatography. Separation between classes is excellent; more than 95% of the lipoproteins eluted from the column are recovered in the form of a purified lipoprotein class. 3. Each lipoprotein class was characterized immunologically, chemically, electrophoretically and by electron microscopy. A comparison of the properties of the column-isolated lipoproteins was made with very-low-density lipoproteins, low-density lipoproteins, and high-density lipoproteins separated by sequential ultracentrifugation at densities of 1.006, 1.063 and 1.21 respectively. 4. By each criterion, peak-I lipoproteins from the agarose column are the same as very-low-density lipoproteins, peak-II lipoproteins are the same as low-density lipoproteins, and peak-III lipoproteins are the same as high-density lipoproteins. Thus the lipoprotein classes isolated by both methods are similar if not identical. 5. The agarose-column separation technique offers the advantage of a two- to three-fold saving in time. In addition, the column-elution pattern serves as a recording of the size distribution of lipoproteins in plasma. 6. The most complete characterization is reported for human plasma lipoproteins. The results with rhesus-monkey and rabbit lipoproteins were identical.
The kinetic mechanism and properties of the cytoplasmic acetoacetyl-coenzyme A thiolase from rat liver
Authors: Middleton B.
Published: August 2015

1. Cytoplasmic acetoacetyl-CoA thiolase was highly purified in good yield from rat liver extracts. 2. Mg2+ inhibits the rate of acetoacetyl-CoA thiolysis but not the rate of synthesis of acetoacetyl-CoA. Measurement of the velocity of thiolysis at varying Mg2+ but fixed acetoacetyl-CoA concentrations gave evidence that the keto form of acetoacetyl-CoA is the true substrate. 3. Linear reciprocal plots of velocity of acetoacetyl-CoA synthesis against acetyl-CoA concentration in the presence or absence of desulpho-CoA (a competitive inhibitor) indicate that the kinetic mechanism is of the Ping Pong (Cleland, 1963) type involving an acetyl-enzyme covalent intermediate. In the presence of CoA the reciprocal plots are non-linear, becoming second order in acetyl-CoA (the Hill plot shows a slope of 1.7), but here this does not imply co-operative phenomena. 4. In the direction of acetoacetyl-CoA thiolysis CoA is a substrate inhibitor, competing with acetoacetyl-CoA, with a Ki of 67μm. Linear reciprocal plots of initial velocity against concentration of mixtures of acetoacetyl-CoA plus CoA confirmed the Ping Pong mechanism for acetoacetyl-CoA thiolysis. This method of investigation also enabled the determination of all the kinetic constants without complication by substrate inhibition. When saturated with substrate the rate of acetoacetyl-CoA synthesis is 0.055 times the rate of acetoacetyl-CoA thiolysis. 5. Acetoacetyl-CoA thiolase was extremely susceptible to inhibition by an excess of iodoacetamide, but this inhibition was completely abolished after preincubation of the enzyme with a molar excess of acetoacetyl-CoA. This result was in keeping with the existence of an acetyl-enzyme. Acetyl-CoA, in whose presence the overall reaction could proceed, gave poor protection, presumably because of the continuous turnover of acetyl-enzyme in this case. 6. The kinetic mechanism of cytoplasmic thiolase is discussed in terms of its proposed role in steroid biosynthesis.
The effects of two disulphides on cholinesterase activity in the spectrophotometric assay
Authors: Augustinsson Klas-Bertil , Eriksson Håkan
Published: August 2015

1. 5,5′-Dithiobis-(2-nitrobenzoate) did not influence serum cholinesterase activity, whereas 2,2′-dithiodipyridine had an inhibitory effect. 2. The lowering of the molar extinction coefficients observed in the presence of physostigmine may be a result of a reaction between thiolate ions with carbamate moieties. 3. The use of 5,5′-dithiobis-(2-nitrobenzoate) is still recommended in investigations, especially where the quantitative aspects are significant.
Some properties and a suggested reclassification of mevaldate reductase
Authors: Beedle Alan S. , Rees Huw H. , Goodwin Trevor W.
Published: August 2015

Mevaldate reductase was purified 70-fold from rat liver. The partly purified enzyme had a molecular weight of 27000–30000, reduced certain aromatic aldehydes and was inhibited by barbiturates. These properties are similar to those of other animal tissue aldehyde reductases (EC 1.1.1.2) and it is suggested that mevaldate reductase be reclassified as one of this group.
A convenient spectrophotometric assay for the determination of l-ergothioneine in blood
Authors: Carlsson Jan , Kierstan Marek P. J. , Brocklehurst Keith
Published: August 2015

1. A convenient spectrophotometric assay for the determination of l-ergothioneine in solution including deproteinized blood haemolysate was developed. 2. The method consists of deproteinization by heat precipitation and Cu2+-catalysed oxidation of thiols such as glutathione and of l-ascorbic acid, both in alkaline media, and titration of l-ergothioneine (which is not oxidized under these conditions) by its virtually instantaneous reaction with 2,2′-dipyridyl disulphide at pH1. 3. This method and the results obtained with it for the analysis of human, horse, sheep and pig blood are compared with existing methods of l-ergothioneine analysis and the results obtained thereby.
The lactate dehydrogenase–reduced nicotinamide–adenine dinucleotide–pyruvate complex. Kinetics of pyruvate binding and quenching of coenzyme fluorescence
Authors: Südi J.
Published: August 2015

The stopped-flow kinetic studies described in this and the following paper (Südi, 1974) demonstrate that a Haldane-type description of the reversible lactate dehydrogenase reaction presents an experimentally feasible task. Combined results of these two papers yield numerical values for the six rate constants defined by the following equilibrium scheme, where E represents lactate dehydrogenase: [Formula: see text] The experiments were carried out at pH8.4 at a relatively low temperature (6.3°C) with the pig heart enzyme. Identification of the above two intermediates and determination of the corresponding rate constants actually involve four series of independent observations in these studies, since (a) the reaction can be followed in both directions, and (b) both the u.v. absorption and the fluorescence of the coenzymes are altered in the reaction, and it is shown that these two spectral changes do not occur simultaneously. Kinetic observations made in the reverse direction are reported in this paper. It is demonstrated that the fluorescence of NADH can no longer be observed in the ternary complex ENADHPyr. Even though the oxidation–reduction reaction rapidly follows the formation of this complex, the numerical values of k−4 (8.33×105m−1·s−1) and k+4 (222s−1) are easily obtained from a directly observed second-order reaction step in which fluorescent but not u.v.-absorbing material is disappearing. U.v.-absorption measurements do not clearly resolve the subsequent oxidation–reduction step from the dissociation of lactate. It is shown that this must be due partly to the instrumental dead time, and partly to a low transient concentration of ENAD+Lac in the two-step sequential reaction in which the detectable disappearance of u.v.-absorbing material takes place. It is estimated that about one-tenth of the total change in u.v. absorption is due to a `burst reaction' in which ENAD+Lac is produced, and this estimation yields, from kobs.=120s−1, k−2=1200s−1.
Pig liver pyruvate carboxylase. The reaction pathway for the carboxylation of pyruvate
Authors: Warren Graham B. , Tipton Keith F.
Published: August 2015

1. The reaction pathway for the carboxylation of pyruvate, catalysed by pig liver pyruvate carboxylase, was studied in the presence of saturating concentrations of K+ and acetyl-CoA. 2. Free Mg2+ binds to the enzyme in an equilibrium fashion and remains bound during all further catalytic cycles. MgATP2− binds next, followed by HCO3− and then pyruvate. Oxaloacetate is released before the random release, at equilibrium, of Pi and MgADP−. 3. This reaction pathway is compared with the double displacement (Ping Pong) mechanisms that have previously been described for pyruvate carboxylases from other sources. The reaction pathway proposed for the pig liver enzyme is superior in that it shows no kinetic inconsistencies and satisfactorily explains the low rate of the ATP[unk][32P]Pi equilibrium exchange reaction. 4. Values are presented for the stability constants of the magnesium complexes of ATP, ADP, acetyl-CoA, Pi, pyruvate and oxaloacetate.
Purification and regulatory properties of phosphoribulokinase from Hydrogenomonas eutropha H 16
Authors: Abdelal Ahmed T. H. , Schlegel Hans G.
Published: August 2015

1. Phosphoribulokinase was purified 286-fold from extracts of autotrophically grown cells. 2. The enzyme had a molecular weight of 237000 and showed a pH optimum of 9.0 in both crude extracts and purified preparation. MgCl2 was required for activity; full activation was obtained at 5mm-MgCl2 and the Km was approx. 0.5mm. 3. The ATP-saturation curve was sigmoidal and the degree of positive co-operativity increased at higher MgCl2 concentrations. The ATP-binding sites appeared to be non-interacting at low ribulose 5-phosphate concentrations. 4. Lineweaver–Burk plots for ribulose 5-phosphate showed abrupt transitions between apparently linear sections. The apparent Km and Vmax. values increased with increasing concentrations of ribulose phosphate. The transitions may be explained by a sequence of negative and positive co-operativity in the catalytic rate constants. 5. Phosphoribulokinase activity was inhibited by AMP and phosphoenolpyruvate and was activated by NADH. The presence of AMP or phosphoenolpyruvate increased s0.5 (substrate concentration required for half-maximal velocity) for both ribulose 5-phosphate and ATP but Vmax. was not changed. The sigmoidicity of the ATP-saturation curve increased in the presence of AMP but was not affected by phosphoenolpyruvate. The transitions in the ribulose 5-phosphate-saturation curves were more abrupt in the presence of either inhibitor. NADH lowered the s0.5 for both ribulose 5-phosphate and ATP. The activator did not affect the degree of positive co-operativity between ATP-binding sites, but the ribulose 5-phosphate-binding sites appeared to be non-interacting in its presence. 6. A sequence of positive and negative co-operativity in the interactions of AMP-binding sites was suggested by the Hill plots. In the presence of NADH (and phosphoenolpyruvate) the sensitivity to inhibition by AMP was less below a certain AMP concentration and increased above that concentration. 7. Examination of the interactions between ligands indicated that phosphoribulokinase can be regulated effectively by changes in effector concentrations similar to those reported to occur in vivo.
Regulation of hepatic lipogenesis by plasma free fatty acids: simultaneous studies on lipoprotein secretion, cholesterol synthesis, ketogenesis and gluconeogenesis (Short Communication)
Authors: Mayes Peter A. , Topping David L.
Published: August 2015

1. An inverse logarithmic relationship was found between lipogenesis and the concentration of serum free fatty acids perfusing the rat liver. 2. Increased concentrations of serum free fatty acids did not alter cholesterol synthesis, triglyceride secretion or increase gluconeogenesis. 3. No direct relationship was found between ketogenesis and either lipogenesis or gluconeogenesis.
An improved micro-method for avidin assay (Short Communication)
Authors: Elo Heikki , Tuohimaa Pentti
Published: August 2015

A sensitive method for avidin assay was devised. Tritiated biotin is bound to avidin and this complex is then bound to bentonite. Radioactivity is converted into a gas form by combustion and counted in an automatic proportional counter with 55% efficiency and background of 3.7c.p.m. The sensitivity is 1–2ng of avidin.
Lipogenesis in rat and guinea-pig isolated epididymal fat-cells
Authors: Saggerson E. David
Published: August 2015

Fat-cells were prepared from rat and guinea-pig epididymal adipose tissue and compared on the basis of the intracellular distributions and activities of enzymes and with respect to their utilization of various U-14C-labelled substrates for lipogenesis. 1. Compared with the rat, guinea-pig extramitochondrial enzyme activities differed in that aconitate hydratase, alanine aminotransferase, ATP–citrate lyase, lactate dehydrogenase, NAD–malate dehydrogenase, NADP–malate dehydrogenase and phosphoenolpyruvate carboxykinase activities were appreciably lower, whereas aspartate aminotransferase, glucose 6-phosphate dehydrogenase, NADP–isocitrate dehydrogenase and 6-phosphogluconate dehydrogenase activities were appreciably higher. Mitochondrial activities of citrate synthase, NADP–isocitrate dehydrogenase and pyruvate carboxylase were appreciably lower, whereas mitochondrial activities of aspartate aminotransferase, glutamate dehydrogenase, NAD–malate dehydrogenase and phosphoenolpyruvate carboxykinase were higher in the guinea pig compared with the rat. 2. In general guinea-pig fat-cells incorporated acetate and lactate into fatty acids more readily than rat fat-cells, whereas rat fat-cells incorporated glucose and pyruvate more readily than guinea-pig fat-cells. 3. Acetate stimulated the incorporation of glucose into fatty acids in rat fat-cells, but had no appreciable effect upon this process in guinea-pig fat-cells. Acetate greatly decreased the incorporation of lactate into fatty acids in cells from both species. 4. Lactate/pyruvate ratios produced by incubation of guinea-pig cells with glucose+insulin were very low compared with those found with rat cells under the same conditions. 5. With glucose (+insulin) or with glucose+acetate (+insulin) as substrates guinea-pig cells produced enough NADPH by the hexose monophosphate pathway to satisfy the NADPH requirements of lipogenesis. In rat fat-cells under the same conditions, hexose monophosphate-pathway NADPH provision was not sufficient to meet the requirements of lipogenesis. 6. These results are discussed, particularly in relationship to the disposition of cytosolic reducing equivalents in the cells.
Long-term effects of glucose on insulin release and glucose oxidation by mouse pancreatic islets maintained in tissue culture
Authors: Andersson Arne
Published: August 2015

Rates of glucose oxidation and insulin release in response to a wide range of glucose concentrations were studied in short-term experiments in isolated mouse pancreatic islets maintained in tissue culture for 6 days at either a physiological glucose concentration (6.7mm) or at a high glucose concentration (28mm). The curves relating glucose oxidation or insulin release to the extracellular glucose concentration obtained with islets cultured in 6.7mm-glucose displayed a sigmoid shape similar to that observed for freshly isolated non-cultured islets. By contrast islets that had been cultured in 28mm-glucose showed a linear relationship between the rate of glucose oxidation and the extracellular glucose concentration up to about 8mm-glucose. The maximal oxidative rate was twice that of the non-cultured islets and the glucose concentration associated with the half-maximal rate considerably decreased. In islets cultured at 28mm-glucose there was only a small increase in the insulin release in response to glucose, probably due to a depletion of stored insulin in those B cells that had been cultured in a high-glucose medium. It is concluded that exposure of B cells for 6 days to a glucose concentration comparable with that found in diabetic individuals causes adaptive metabolic alterations rather than degeneration of these cells.
Effects of fructose, sucrose and glucose feeding on plasma insulin concentrations and on adipose-tissue clearing-factor lipase activity in rat
Authors: Cryer Anthony , Riley Susan E. , Williams Edric R. , Robinson Donald S.
Published: August 2015

The rise in adipose-tissue clearing-factor lipase activity that results from feeding glucose to starved rats cannot be duplicated by giving equicaloric amounts of fructose or sucrose. An inability of the administered fructose and sucrose to raise the plasma insulin concentration probably accounts for this failure in enzyme response.
Calcium and the assays of human plasma clotting Factor XIII
Authors: Cooke Rodney D. , Holbrook J. John
Published: August 2015

1. A continuous fluorimetric assay for blood-clotting Factor XIII based on the incorporation of dansylcadaverine into casein was investigated. 2. Hammarsten casein was fractionated to yield the β-casein, which was dephosphorylated and acetylated to give a substrate which itself did not bind calcium and which was not cross-linked by the activated Factor. 3. The modified β-casein was used as substrate for a continuous fluorimetric assay and as substrate for the incorporation of radioactive glycine ethylester. 4. A linked fluorimetric assay for the zymogen is described. 5. The Km for calcium was redetermined and at 0.2mm was in the physiological range and much lower than the values reported by others using substrates which interact with calcium. 6. The Km for casein is about 14μm.