bluuebunny/crossref_metadata_2025 huggingface dataset:
Ribitol dehydrogenase from Klebsiella aerogenes. Purification and subunit structure
Authors: Taylor Susan S. , Rigby Peter W. J. , Hartley Brian S.
Published: August 2015

Ribitol dehydrogenase has been purified to homogeneity from several strains of Klebsiella aerogenes. One strain yields 3–6g of pure enzyme from 1kg of cells. The enzyme is a tetramer of four subunits, mol.wt. 27000. Preliminary studies of the activity of the enzyme are reported. Peptide ‘maps’ together with the amino acid composition indicate that the subunits are identical.
Studies of triose phosphate isomerase by hydrogen exchange
Authors: Browne Christopher A. , Waley Stephen G.
Published: August 2015

The3H–H exchange of chicken muscle and rabbit muscle triose phosphate isomerases was studied. Their behaviour was mostly very similar. ‘Exchange-in’ (acquisition of radioactivity when protein was incubated in3H2O) was measured at 37°C and at pH7.5, and the rates of exchange of the native and liganded enzymes were compared. Inhibitors and substrates retarded exchange, substrates showing the most marked effect; structural rearrangements in the enzyme may thus play some part in catalysis. The inhibitor phosphoglycollate affected the rabbit enzyme, but had little or no effect on the chicken enzyme. ‘Exchange-out’ (loss of radioactivity from protein previously labelled by incubation in3H2O) was measured by hollow-fibre dialysis. When ligand was removed during the course of dialysis (by replacing buffer that contained ligand with buffer that lacked ligand) there was a prompt decrease in the number of labelled H atoms of the protein. Analysis of the curves provides some information about the number and half-lives of the responsive H atoms. Ligands decrease the motility of the protein and affect about one-fifth of the chain. Low concentrations of glycerol 3-phosphate have an effect that is greater than expected.
Analysis of the code relating sequence to conformation in globular proteins. An informational analysis of the role of the residue in determining the conformation of its neighbours in the primary sequence
Authors: Robson Barry , Pain Roger H.
Published: August 2015

1. The effect exerted by a residue on the conformation of neighbouring residues was analysed by using data from nine globular proteins of known sequence and conformation. 2. An information measure was used which estimated the role of a residue in influencing neighbouring conformations and also its tendency to influence the lengths of runs of residues in that conformation. This measure was estimated for each residue in all conformations defined by domains on the ϕ, ψ diagram. 3. Plots of the information measure yielded an intercept, which was a measure of intra-residue information for a residue. The slope was a measure of the statistical co-operativity or tendency of the residue to influence the occurrence of its neighbours in runs of a particular conformation. Both parameters are a function of the residue type. Statistical co-operativity is found in the α1-helical (H1) and β-pleated-sheet (P2) conformations and, to a lesser extent, in their distorted variants H2 and P1. 4. The directional nature of these influences for H1 and P2 conformations is illustrated by plots of the information measure against the distance m from the residue, for m=−10 to +10. 5. The results for statistical co-operativity are discussed in relation to theories of helix–coil and pleated-sheet–coil transitions. The value of the information-theory-derived parameters in obtaining s parameters for the Zimm & Bragg (1959) equations is illustrated. 6. Directional effects are discussed with particular relation to mechanisms of the termination of helices and the involvement of the αII conformation and also to discontinuities in pleated-sheet conformations.
A comparison of two methods for fitting the integrated Michaelis–Menten equation
Authors: Nimmo Ian A. , Atkins Gordon L.
Published: August 2015

The methods of Atkins & Nimmo (1973) and Fernley (1974) for fitting the integrated Michaelis–Menten equation were compared by using the same sets of simulated experimental data. The method of Fernley (1974) is to be preferred because it gives precise and unbiased estimates of the Michaelis–Menten parameters over a wide range of substrate concentrations. However, the estimates may not be symmetrically distributed, especially at low substrate concentrations.
X-ray fibre diffraction of cartilage proteoglycan aggregates containing hyaluronic acid (Short Communication)
Authors: Atkins E. D. T. , Hardingham T. E. , Isaac D. H. , Muir H.
Published: August 2015

Ordered conformations of proteoglycan–hyaluronic acid aggregates in the intercellular matrix in cartilage were observed by X-ray diffraction. The sodium salt form of three samples, (a) aggregated proteoglycan, (b) disaggregated proteoglycan and (c) reconstituted disaggregated proteoglycan, give essentially similar X-ray fibre-type diffraction photographs. The patterns correlate with the chondroitin 4-sulphate component and can be interpreted as twofold helical conformations, similar to that observed previously for the free acid form of chondroitin 4-sulphate (Isaac & Atkins, 1973). The information takes us one step nearer the situation found in cartilage in vivo.
A new method for the preparation of uniformly 14C-labelled compounds by using Anacystis nidulans
Authors: Tovey Keith C. , Spiller Gerald H. , Oldham Kenneth G. , Lucas Norman , Carr Noel G.
Published: August 2015

1. Growth conditions were devised which enabled Anacystis nidulans to be grown on high specific radioactivity 14CO2 (at over 98% isotopic abundance of 14C) in a closed system with greater than 85% substrate utilization. 2. The DNA and RNA content of A. nidulans was estimated by various methods and compared with that of Chlorella pyrenoidosa. 3. A procedure was developed for the quantitative extraction and separation of the DNA and RNA from A. nidulans. 4. The fractionation and analysis of the DNA and RNA includes methods for the preparation of the uniformly 14C-labelled deoxyribonucleoside monophosphates and the uniformly 14C-labelled ribonucleoside monophosphates. 5. The base 6-methylaminopurine was identified as a component of the DNA. 6. The preparation and total yield of uniformly 14C-labelled amino acids from A. nidulans are also briefly described. The results are discussed in relation to the use of other micro-organisms for the preparation of compounds labelled with 14C.
Studies with a reconstituted muscle glycolytic system. The rate and extent of glycolysis in simulated post-mortem conditions
Authors: Scopes Robert K.
Published: August 2015

The reconstituted glycolytic system described previously (Scopes, 1973) was used to simulate post-mortem glycolytic metabolism in muscle. The effects of the following factors have been investigated: ATPase (adenosine triphosphatase) amount, AMP deaminase amount, percentage of the phosphorylase in the a form and the effect of diluting the glycolytic enzyme complex as a whole. It was confirmed that the rate of metabolism was solely dependent on the amount of ATPase present and that various concentrations of the glycolytic enzymes had no effect over a wide range encompassing the variation found in anatomically different muscles. The extent of metabolism, represented by the value of the ‘ultimate’ pH, depended markedly on the amount of phosphorylase in the a form; as little as 1% of the a form resulted in a considerably lower pH than in its absence. To a lesser extent the amount of AMP deaminase also affected the ultimate pH, but this was probably only significant for comparisons of genetically distinct muscles with widely differing amounts of AMP deaminase. The reconstituted system behaved almost identically with regard to post-mortem glycolytic metabolism compared with intact muscle tissue. It is concluded that the controlling effectors found with the reconstituted system apply to intact muscle also.
The effects of potassium, 5-hydroxytryptamine, adrenocorticotrophin and angiotensin II on the concentration of adenosine 3′:5′-cyclic monophosphate in suspensions of dispersed rat adrenal zona glomerulosa and zona fasciculata cells
Authors: Albano Janet D. M. , Brown Barry L. , Ekins Roger P. , Tait Sylvia A. S. , Tait James F.
Published: August 2015

Dispersed rat adrenal cells prepared from both the capsule and the decapsulated gland were used to investigate the effects on cyclic AMP accumulation of known stimuli of steroidogenesis [ACTH (adrenocorticotrophin), angiotensin II, K+ions and 5-hydroxytryptamine]. Since glomerulosa-cell preparations from capsular strippings are normally contaminated with a proportion of fasciculata cells, cells purified by fractionation on a bovine serum albumin gradient were also used. The results showed that: (1) ACTH and angiotensin II stimulated cyclic AMP accumulation in both fractionated and unfractionated zona fasciculata cells; (2) 5-hydroxytryptamine and an increased extracellular K+concentration (from 3.6 to 8.4mm) had no effect on cyclic AMP concentrations in fasciculata cell preparations; (3) the addition of ACTH, angiotensin II, 5-hydroxytryptamine or K+to the incubation medium resulted in increased cyclic AMP concentrations in unpurified zona glomerulosa cell preparations; (4) fractionation and hence the virtual elimination of fasciculata contamination, did not affect the response to 5-hydroxytryptamine and increased K+concentration. However, the responses to ACTH and angiotensin II were markedly lowered but not abolished. These results strongly suggest a link between cyclic AMP production and steroidogenesis in the zone of the adrenal gland that specifically secretes aldosterone. All four agents used stimulated both steroid output and cyclic AMP accumulation. However, at certain doses of 5-hydroxytryptamine, K+and angiotensin II the significant increases in corticosterone output were not accompanied by measurable increases in cyclic AMP accumulation.
Short Communication. Effect of duration of the period after castration on the response of the rat ventral prostate to androgens
Authors: Lesser Barry , Bruchovsky Nicholas
Published: August 2015

If 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) is administered to castrated rats before prostatic atrophy has commenced, secretory activity of the prostate is stimulated, but not proliferation. Once the number of cells has fallen below normal, hormone induces proliferation until the normal cellular complement has been restored; then proliferation ceases, but secretory activity is stimulated.
Synthesis of polyprenyltoluquinols from homogentisate and polyprenyl pyrophosphates in particulate fractions of Euglena and sugar beet
Authors: Thomas Graham , Threlfall David R.
Published: August 2015

Chloroplast-rich particles of sugar beet and Euglena gracilis are able to carry out the light-, H2O2- and O2-independent syntheses of a nonaprenyltoluquinol, an octaprenyltoluquinol and a phytyltoluquinol from homogentisate and nonaprenyl, octaprenyl and phytyl pyrophosphate. The formation of these compounds probably takes place by the concomitant polyprenylation (or phytylation) and non-oxidative decarboxylation of homogentisate.
Turnover of myelin and other structural proteins in the developing rat brain
Authors: Sabri M. I. , Bone A. H. , Davison A. N.
Published: August 2015

1. Protein metabolism of myelin and other subcellular components from developing rat brain was studied for periods from 5h to 210 days after intraperitoneal injection of [3H]lysine and [14C]glucose. 2. Half-lives for total brain proteins (t0.5) were 27 days after [3H]lysine and 4 days after [14C]glucose injection. 3. Factors accounting for the difference in the turnover rates obtained with different precursors, and the problem of reutilization of the label were investigated. 4. The catabolism of purified myelin proteins was studied and the half-lives of individual myelin proteins were calculated. 5. Myelin basic proteins turned over at two different rates. Half-life of the fast component of myelin basic proteins was 19–22 days and the slow component exhibited a high degree of metabolic stability. 6. Proteolipid protein underwent slow turnover. High-molecular-weight Wolfgram (1966) proteins underwent (relatively) fast metabolism (t0.5 of 17–22 days).
Phosphodiesterase II in epithelial cells from guinea-pig and rat small intestine
Authors: Flanagan Peter R. , Zbarsky S. H.
Published: August 2015

Phosphodiesterase II activity was determined by using a synthetic substrate, the 2,4-dinitrophenyl ester of thymidine 3′-phosphate. The enzyme activity was determined in fractions obtained by differential centrifugation of homogenates of epithelial cells from the small intestinal mucosa of guinea pigs and rats. In guinea-pig preparations phosphodiesterase II occurred with highest specific activity in those fractions rich in succinate dehydrogenase and acid phosphatase. A lysosomal location for the guinea-pig enzyme was indicated by its structure-linked latency and by its association with particles that under-went a characteristic decrease in equilibrium density when Triton WR-1339 was injected into the animals. With rat preparations a much greater proportion of the phosphodiesterase II activity was found in the soluble fraction after ultracentrifugation. The rat enzyme exhibited a lower degree of latency and administration of Triton WR-1339 had no effect. The rat enzyme further differed from that of the guinea pig in other respects; it was more labile at 60°C, it exhibited a lower pH optimum and it had a higher molecular weight as determined by gel-filtration chromatography.
A rapid method for the preparation of microvilli from rabbit kidney
Authors: Booth Andrew G. , Kenny A. John
Published: August 2015

A simple method for the isolation of microvilli from kidney brush border is described. The method depends on the preferential aggregation of other subcellular structures by bivalent metal ions. MgCl2 is added to a homogenate of cortical tissue prepared from frozen rabbit kidneys. Aggregated material is removed by a low-speed centrifugation and the supernatant centrifuged at 15000g to yield a pellet enriched in microvilli. This is resuspended and given a second treatment with Mg2+. The purified preparation is obtained after four short differential centrifugations. The six brush-border enzymes that were monitored were enriched 11–17-fold compared with the original homogenate and were obtained in about 10% yield. Marker enzymes for other subcellular components showed the preparation to be essentially free of mitochondria and to be less contaminated with endoplasmic reticulum and baso-lateral plasma membranes than are conventional brush-border preparations. The main contamination was of lysosomal origin, about half of which was attributable to adsorbed acid hydrolases rather than to intact lysosomes. The aggregated components in the low-speed pellet bound less Mg2+than did the microvillus fraction. A possible mechanism for the role of Mg2+is discussed.
Phosphatidylinositol cleavage in lymphocytes. Requirement for calcium ions at a low concentration and effects of other cations
Authors: Allan David , Michell Robert H.
Published: August 2015

The soluble activity in lymphocytes which converts phosphatidylinositol into 1,2-diacylglycerol and inositol phosphates requires Ca2+ions. At pH7 maximum activity occurs at [Ca2+]free ≈ 0.7μm whereas at pH5.5 the equivalent value is approx. 50μm. At [Ca2+]free ≈ 1μm, a concentration similar to common intracellular values, essentially all activity is confined to the peak of activity at pH7.0. Previous reports of requirements for larger amounts of Ca2+may reflect the fact that the Ca2+-buffering capacity of phosphatidylinositol means that high substrate concentrations can effectively decrease [Ca2+]free. Cations which displace Ca2+from association with phosphatidylinositol can, at low [Ca2+]free, enhance enzyme activity. Phosphatidylinositol breakdown in intact cells might be controlled, at least in part, by changes in intracellular [Ca2+]free.
Effects of iodination on the distribution of peptide hormones in aqueous two-phase polymer systems
Authors: Desbuquois Bernard , Aurbach G. D.
Published: August 2015

1. The effect of iodination on the distribution of peptide hormones into the aqueous two-phase dextran–polyethylene glycol system and on the solubility of these hormones in aqueous polyethylene glycol and in water was assessed. Hormones that were studied included insulin, glucagon and parathyroid hormone. 2. The partition coefficient of native insulin in the dextran–polyethylene glycol system showed a minimum (about 1) near the isoelectric point of the hormone (pH 5). Partial iodination of insulin (one atom per molecule) caused little change in the distribution of the hormone. More extensive iodination markedly decreased the partition coefficient in the region of the isoelectric point and displaced the pH value at which the partition coefficient was a minimum towards lower values. 3. The solubility of native insulin in aqueous polyethylene glycol and in water showed a pH-dependence similar to that observed for the distribution in the dextran–polyethylene glycol system. Iodination of insulin decreased the solubility of the hormone in polyethylene glycol and in water in parallel, and decreased the pH value at which solubility was a minimum. The changes in solubility correlated with the degree of iodination and accounted for the changes in distribution observed at high concentrations of insulin. 4. Comparable effects of iodination on distribution and solubility were also observed with glucagon. 5. At concentrations of insulin below its maximum solubility, serum proteins caused a decrease in the partition coefficient of iodinated hormone, but not of native hormone. These effects correlated with the degree of iodination and resulted from a co-precipitation of iodinated insulin with serum proteins.
Association of xanthine oxidase with the bovine milk-fat-globule membrane. Catalytic properties of the free and membrane-bound enzyme
Authors: Briley Michael S. , Eisenthal Robert
Published: August 2015

1. The catalytic properties of xanthine oxidase in bovine milk (EC 1.2.3.2) are dependent on the state of the enzyme, i.e. whether free or bound to the fat-globule membrane. Oxidase activity of the membrane-bound enzyme towards NADH is enhanced relative to that towards xanthine. This reflects a change in the relative Km values and enables the ratio of xanthine to NADH oxidase activities (X/N) to be used as a parameter for the relative amounts of free and membrane-bound xanthine oxidase in milk fractions. 2. Chromatography of buttermilk on Sepharose 2B yielded an excluded fraction, BM1, with xanthine oxidase activity. The remaining xanthine oxidase activity was eluted as a single broad peak. This was further resolved on Sephadex G-200 into an excluded fraction, BM2, and free xanthine oxidase. Fractions BM1 and BM2 had X/N values in the range 45–65, which is characteristic of membrane-bound xanthine oxidase. Purified xanthine oxidase has a mean X/N value of 110.3. Addition of fraction BM1, heated to remove associated enzyme activities, to purified xanthine oxidase progressively enhanced its NADH oxidase activity to a value where its X/N value was characteristic of membrane-bound xanthine oxidase. This was shown to be due to binding of free enzyme to heated fraction BM1. The binding constant and stoicheiometry were determined. 4. Proteolytic digestion of fraction BM1 liberated free xanthine oxidase from the fat-globule membrane with a corresponding alteration in X/N value.
The macromolecular properties of blood-group-specific glycoproteins. Characterization of a series of fractions obtained by solvent fractionation
Authors: Creeth J. Michael , Bhaskar K. Ramakrishnan , Donald Alastair S. R. , Morgan Walter T. J.
Published: August 2015

1. The glycoprotein components of a human ovarian-cyst fluid were isolated by a solvent [95% (w/w) phenol]-extraction procedure; the phenol-insoluble water-soluble glycoprotein was further fractionated by (NH4)2SO4 and by ethanol to yield eight fractions. 2. The fractions were analysed in terms of amino acids, fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid. Variations occurred, particularly in the proportion of peptide; these were partly correlated with varying extent of serological activity. 3. The fractions were characterized physicochemically in terms of buoyant density and degree of spreading in a density gradient, sedimentation velocity and molecular weight; their partial specific volumes and specific refraction increments were also determined. 4. The fractions showed wide variations in their sedimentation-velocity and density-gradient patterns, and gave evidence of pauci-dispersity in density. The fraction regarded as the most typical blood-group-specific glycoprotein sedimented as a single rapidly spreading peak and was of high molecular weight. 5. Significant correlations were observed between the physical properties of the glycoprotein fractions and the amount of their peptide component. The buoyant densities and sedimentation coefficients varied in a manner that suggested the existence of two families of glycoproteins. 6. It is suggested that variability in the extent of glycosylation, or in the degree of cross-linking, might account for the two families of glycoproteins, and that the extent of cross-linkage might also be a factor determining the solubility of these glycoproteins in hot saturated (NH4)2SO4.
Adenosine triphosphatase activity in the neural lobe of the bovine pituitary gland
Authors: Vilhardt Hans , Hope Derek B.
Published: August 2015

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential and density-gradient ultracentrifugation and the distribution of adenosine triphosphatase (ATPase) activity was studied. It was shown that all the activity was membrane-bound. 2. On the basis of ionic requirements the ATPase activity was grouped into three categories: (a) Mg2+-dependent, (b) Ca2+-dependent and (c) Mg2++Na++K+-dependent (ouabain-sensitive) ATPases. The activity in the absence of bivalent cations was negligible. The ratio between the activities of the three ATPases varied between the different subcellular fractions. 3. Preincubation of the subcellular fractions with deoxycholate increased the activity of the Mg2++Na++K+-dependent enzyme, whereas the Mg2+- and Ca2+-activated ATPases were either unaffected or slightly inhibited. Triton X-100 solubilized the Mg2+- and Ca2+-ATPases; however, the activity of the Mg2++Na++K+-ATPase was abolished by the concentration of Triton X-100 used. 4. All the subfractions displayed unspecific nucleotide triphosphatase activity towards GTP, ITP and UTP. These substrates inhibited the hydrolysis of ATP by all three ATPases. ADP also inhibited the ATPases. 5. Polyacrylamide-gel electrophoresis of extracts containing the Mg2+- and Ca2+-dependent ATPase activity solubilized by Triton X-100 revealed the presence of two enzymes; one activated by either Mg2+or Ca2+and the other activated only by Ca2+. 6. In sucrose density gradients the distribution of vasopressin was different from that of all three types of ATPases. It is therefore suggested that the neurosecretory granules do not possess ATPase activity.
A solid-phase radioimmunoassay for plasma progesterone
Authors: Dighe Kailas K. , Hunter William M.
Published: August 2015

A detailed procedure is presented for the assay of plasma progesterone. The routine assay is based on the use of antiserum which is covalently linked to microcrystalline cellulose, the double-antibody method being used as a reference separation system. This procedure gives high precision accompanied by small and acceptable losses of antiserum titre but without loss of sensitivity when compared with the double-antibody method. Ethanol is first added to the plasma (10vol. of plasma+1vol. of ethanol) after which a single extraction with light petroleum yields a constant recovery [92.4±1.2 (s.d.)% of added [3H]progesterone] and obviates the need for tracer recoveries on each sample being assayed. Distortions of the response curve owing to solvent residues have been almost eliminated. The assay can measure progesterone at all stages of the menstrual cycle when volumes of 200μl of plasma are used and this permits the detection of the periovulatory rise at its inception. Detailed specificity studies are presented for the assay end point itself and these are related to the responses to be expected in extracts of plasma. Progesterone-like activity was found in urine and a fourfold increase in excretion rates was observed between the follicular and luteal phase of the normal menstrual cycle.
Different molecular forms of plasminogen and plasmin produced by urokinase in human plasma and their relation to protease inhibitors and lysis of fibrinogen and fibrin
Authors: Müllertz Sten
Published: August 2015

Urokinase-activated human plasma was studied by gel electrophoresis, gel filtration, crossed immunoelectrophoresis and electroimmunoassay with specific antibodies and by assay of esterase and protease activity of isolated fractions. Urokinase induced the formation of different components with plasminogen+plasmin antigenicity. At low concentrations of urokinase, a component with a KD value of 0.18 by gel filtration and post β1 mobility by gel electrophoresis was detected. The isolated component had no enzyme or plasminogen activity. In this plasma sample fibrinogen was not degraded for 10h, but when fibrin was formed, by addition of thrombin, fibrin was quickly lysed, and simultaneously a component with a KD value of 0 and α2 mobility appeared, which was probably plasmin in a complex with α2 macroglobulin. This complex showed both esterase and protease activity. After gel filtration with lysine buffer of the clotted and lysed plasma another two components were observed with about the same KD value by gel filtration as plasminogen (0.35), but β1 and γ mobilities by gel electrophoresis. They appeared to be modified plasminogen molecules, and possibly plasmin with γ mobility. Similar processes occurred without fibrin at higher urokinase concentrations. Here a relatively slow degradation of fibrinogen was correlated to the appearance of the plasmin–α2 macroglobulin complex. The fibrin surface appeared to catalyse the ultimate production of active plasmin with a subsequent preferential degradation of fibrin and the formation of a plasmin–α2 macroglobulin complex. The gel filtration and electrophoresis of the plasma protease inhibitors, α1 antitrypsin, inter-α-inhibitor, antithrombin III, and C1-esterase inhibitor indicated that any complex between plasmin and these inhibitors was completely dissociated. The β1 and post β1 components appear to lack correlates among components occurring in purified preparations of plasminogen and plasmin.
Studies on chemical modification of thionucleosides in the transfer ribonucleic acid of Escherichia coli
Authors: Rao Yarlagadda S. Prasada , Cherayil Joseph D.
Published: August 2015

35S-labelled tRNA from Escherichia coli was treated with chemical reagents such as CNBr, H2O2, NH2OH, I2, HNO2, KMnO4 and NaIO4, under mild conditions where the four major bases were not affected. Gel filtration of the treated tRNA showed desulphurization to various extents, depending on the nature of the reagent. The treated samples after conversion into nucleosides were chromatographed on a phosphocellulose column. NH2OH, I2 and NaIO4 reacted with all the four thionucleosides of E. coli tRNA, 4-thiouridine (s4U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 2-thiocytidine (s2C) and 2-methylthio-N6-isopentenyladenosine (ms2i6A), to various extents. CNBr, HNO2 and NaHSO3 reacted with s4U, mnm5s2U and s2C, but not with ms2i6A. KMnO4 and H2O2 were also found to react extensively with thionucleosides in tRNA. Iodine oxidation of 35S-labelled tRNA showed that only 6% of the sulphur was involved in disulphide formation. Desulphurization of E. coli tRNA with CNBr resulted in marked loss of acceptor activities for glutamic acid, glutamine and lysine. Acceptor activities for alanine, arginine, glycine, isoleucine, methionine, phenylalanine, serine, tyrosine and valine were also affected, but to a lesser extent. Five other amino acids tested were almost unaffected. These results indicate the fate of thionucleosides in tRNA when subjected to various chemical reactions and the involvement of sulphur in aminoacyl-tRNA synthetase recognition of some tRNA species of E. coli.
A comparison of wild-type and mutant ribitol dehydrogenases from Klebsiella aerogenes
Authors: Burleigh Bruce D. , Rigby Peter W. J. , Hartley Brian S.
Published: August 2015

A ribitol dehydrogenase (ribitol–NAD+ oxidoreductase, EC. 1.1.1.56) having increased specificity and catalytic efficiency toward xylitol was isolated from mutant strains of Klebsiella aerogenes, which were selected for increased growth rate on xylitol over the ribitol dehydrogenase constitutive wild-type organism. 2. The mutant enzyme was purified to homogeneity and its general characteristics were compared with those of the previously purified wild-type enzyme. 3. Initial-velocity steady-state kinetic parameters were determined for both wild-type and mutant enzymes and the results compared. 4. The results are interpreted in terms of a model in which the mutant enzyme results from a small change of amino acid sequence, which affects both the stability and conformational equilibria of the molecule.
The solubility of fibrinogen in solutions containing dextrans of various molecular weights (Short Communication)
Authors: Rampling Michael W.
Published: August 2015

The effect of dextrans of various molecular weights on the solubility of fibrinogen was investigated. The results indicate that the prime effect responsible for the observed changes of solubility was steric exclusion. This applies over the whole molecular-weight range of dextrans used, i.e. from 5700 to 76000.
The reconstitution of microsomal redox chains. A comparative analysis of the effectiveness of membrane self-assembly and template binding of electron carriers
Authors: Archakov Alexander I. , Bachmanova Galina I. , Devichensky Vyacheslav M. , Karuzina Irina I. , Zherebkova Natalya S. , Alimov Gennady A. , Kuznetsova Galina P. , Karyakin Alexander V.
Published: August 2015

A comparative analysis was made of the effectiveness of three methods for the reconstitution of microsomal electron-transfer chains, namely, self-assembly, incorporation of electron carriers into liposomes (non-specific template) and incorporation into ‘ghosts’ of microsomal vesicles (specific template). It was shown that when the ‘ghosts’ of the microsomal vesicles were used as a specific template extra cytochrome b5 and NADH-specific flavoprotein were incorporated into them, but cytochrome P-450 and NADPH-specific flavoprotein were not incorporated into the membrane. As a result of the self-assembly and incorporation into liposomes all the electron carriers were present in the reconstituted membrane. Cytochrome P-450 reactivation took place and the inactive form, cytochrome P-420, was converted into the active form, cytochrome P-450. Of the four enzyme hydroxylation systems studied, i.e. NADPH- and NADH-dependent p-hydroxylation of aniline, and NADPH- and NADH-dependent N-demethylation of dimethylaniline, only the NADH-dependent demethylation of dimethylaniline (60% of the initial value) and NADH-dependent p-hydroxylation of aniline (30% of the initial value) were reconstituted by self-assembly. NADPH oxidase and NADH oxidase activities were only properly reconstituted by self-assembly and incorporation into liposomes. In contrast, the NADPH-specific system of peroxidation of unsaturated fatty acids was reconstituted by specific template-binding.
The metabolic sequence by which some 4,4-dimethyl sterols are converted into cholesterol
Authors: Gibbons Geoffrey F.
Published: August 2015

Cholest-8(14)-enol is the major radioactive component of the 4-di-demethyl sterol fraction biosynthesized from 4,4-dimethyl[2-3H2]cholest-8(14)-enol by rat liver microsomal fractions, and therefore the first steps in the biosynthesis of cholesterol from the latter compound probably involve removal of the 4-methyl groups. 4,4-Dimethylcholesta-8,14-dienol therefore is not an intermediate in this process, although its presence in the incubation medium at a concentration of 0.146mm almost completely inhibits the demethylation of 4,4-dimethyl[2-3H2]cholest-8(14)-enol. Nor is cholesta-8,14-dienol an intermediate in the conversion of cholest-8(14)-enol into cholest-7-enol and cholesterol. With 4,4-dimethyl[2-3H2]cholesta-8,14-dienol as the cholesterol precursor, 4,4-dimethylcholest-8(9)-enol becomes heavily labelled and there is very little radioactivity associated with cholesta-8,14-dienol.In this case, the most heavily labelled 4-di-demethyl sterols are cholest-7-enol and cholesterol with the former predominating. There is little or no radio-activity associated with cholest-8(14)-enol. A similar labelling pattern amongst the 4-di-demethyl sterols was observed with dihydro[14C]lanosterol as the precursor. The first step therefore in the synthesis of cholesterol from the 4,4-dimethyl[2-3H2]dienol is reduction of the Δ14(15) bond and not removal of the 4α-methyl group. Depending on the nature of the precursor, addition of the soluble fraction of the cell to the microsomal fraction resulted in a two- to four-fold stimulation of 4-di-demethyl sterol biosynthesis from the 4,4-dimethyl sterols studied. Under these conditions, 4,4-dimethylcholesta-8,14-dienol is the most efficient precursor of cholesterol and cholest-7-enol, and dihydrolanosterol is better than 4,4-dimethylcholest-8(14)-enol.
Yeast protein synthesis. Preparation and analysis of a highly active cell-free system
Authors: Sissons Christopher H.
Published: August 2015

A detailed description is given of the techniques for preparing, handling and assaying a cell-free protein-synthesizing system from yeast, analogous to crude (S-30) Escherichia coli extracts. Its basic characteristics are described. The rate of poly(U)-directed polyphenylalanine synthesis was at least fivefold higher than in previously reported yeast cell-free systems, approaching that of crude mammalian cell-free systems. Fractionation of the S-30 extracts lowered activity. Organelles and their fragments present in the S-30 extract neither contributed to nor inhibited cytoplasmic protein synthesis. There was a component localized in the high-speed supernatant that caused an inhibition of polyphenylalanine synthesis. Poly(U) programmed the synthesis of long-chain polyphenylalanine, in contrast with the only other yeast system in which this has been examined (Bretthauer & Golichowski, 1968). Preincubation techniques inactivated the system and probably a small proportion only of the ribosomes was active.
Distribution and metabolism of phenylalanine and tyrosine during tularaemia in the rat (Short Communication)
Authors: Powanda Michael C. , Dinterman Richard E. , Wannemacher Robert W. , Herbrandson Gerald D.
Published: August 2015

Tularaemia in rats causes a doubling of the serum phenylalanine/tyrosine ratio and a more than twofold increase in muscle phenylalanine which cannot be accounted for by decreased hepatic phenylalanine hydroxylase (EC 1.14.16.1) activity, but appear to result from a generalized movement of amino acids from muscle to liver during infection.
Steady-state kinetics of catecholamine transport by chromaffin-granule ‘ghosts’
Authors: Phillips J H
Published: August 2015

Resealed chromaffin-granule ‘ghosts’ were used to study the steady-state kinetics of catecholamine transport. The pump has a high affinity for (-)-noradrenaline, (-)-adrenaline, tyramine and 5-hydroxytryptamine (serotonin), but a lower affinity for (+)-noradrenaline. The measured rates of incorporation do not conform to Michaelis–Menten kinetics, but affinity constants for the former substrates are in the range 8–18μm. Reserpine is a potent inhibitor. Incorporation as a function of ATP concentration also fails to show simple kinetics; the affinity constant for ATP is deduced to be about 3mm at 1mm-MgCl2. Adenylyl (βγ-methylene)diphosphonate is a competitive inhibitor at low concentrations, but inhibits more strongly at high concentrations. The pump has a transition temperature at 29°C and does not seem to be identical with the Mg2+-stimulated adenosine triphosphatase of chromaffin granules.
The biosynthesis of granulose by Clostridium pasteurianum
Authors: Robson R L , Robson R M , Morris J G
Published: August 2015

1. Mutant strains of Clostridium pasteurianum were obtained, which are unable to synthesize granulose (an intracellularly accumulated amylopectin-like α-polyglucan). 2. These mutants lacked either (a) ADP-glucose pyrophosphorylase (EC 2.7.7.27), or (b) granulose synthase (i.e. ADP-glucose–α-1,4-glucan glucosyltransferase, EC 2.4.1.21). 3. Although both of these enzymes were constitutively synthesized by the wild-type organism, massive deposition of granulose in a sporulating culture coincided with a threefold increase in the specific activity of ADP-glucose pyrophosphorylase. 4. The soluble ADP-glucose pyrophosphorylase was partially purified (33-fold). Its ATP-saturation curve was not sigmoidal and its activity was not enhanced by phosphorylated intermediates of glycolysis, pyruvate, NAD(P)H or pyridoxal 5′-phosphate. ADP at relatively high concentrations acted as a competitive inhibitor (Ki=19mm). 5. The dependence of granulose synthase on a suitable polyglucan primer was demonstrated by using enzyme obtained from a granulose-free mutant strain (lacking ADP-glucose pyrophosphorylase). 6. Partial purification of granulose synthase from wild-type strains was facilitated by its being bound to the native particles of granulose. No activator was discovered, but ADP, AMP and pyridoxal 5′-phosphate were competitive inhibitors, ADP being most effective (Ki about 0.2mm). 7. It would appear that the synthesis of granulose in Cl. pasteurianum is not subject to the positive, fine control that is a feature of glycogen biosynthesis in most bacteria.
Synthesis of cyclohexylpuromycin and its reaction with N-acetylphenylalanyl-transfer ribonucleic acid on rat liver ribosomes
Authors: Ariatti M , Hawtrey A O
Published: August 2015

1. Cyclohexylpuromycin, an anlogue of puromycin in which a cyclohexane ring replaces the aromatic benzene ring of the L-phenylalanyl moeity of the nucleoside., has been synthesized and examined for its ability to release N-acetylphenylalanine from tRNA attached to rat liver ribosomes. 2.dl-Cyclohexylpuromycin was active in reacting with N-[3H]acetylphenylalanyl-tRNA on rat liver ribosomes to form N-E13H]lacetylphenylalanycyclohexypuromycin. 3. The reaction product N-acetylphenylalanylcyclohexylpuromycin and the corresponding analogue N-acetylphenylalanylpuromycin were chemically synthesized for evaluation of the structure of the released N-acetylphenylalanyl-containing material. 4. The results obtained suggest that the model of Raacke (1971) for purmycin reactivity needs further examination with regard to the role played by the aromatic ring system of the Lphenylalanyl moiety of the nucleoside
A comparsion of glycopeptides from the transferrins of several species
Authors: Graham I , Williams J
Published: August 2015

1. The carbohydrate compositions of human, pig and cattle transferrins and duck ovotransferrin have been determined. 2. Glycopeptides have been prepared from these transferrins and their carbohydrate compositions and amino acid sequences determined. One of the glycopeptides from human transferrin carries the C-terminal residue of the protein. 3. Each tranferrrin yielded two glycopeptides that appeared to be identical in carbohydrate composition but different in amino acid sequence. The two glycopeptides have been distinguished as type A, in which the residue following Asn(CHO)(where CHO represents a carbohydrate moiety) is a basic amino acid and type B in which Asn(CHO) is followed by a neutral aliphatic amino acid. Cattle transferrin is exceptional in having two glycopeptides in which this position is occupied by serine. 4. It is suggested that each molecule of human and cattle transferrin and duck ovotransferrin carries an average of two carbohydrate prosthetic groups. Hen and pig transferrins appear to carry only one carbohydrate group per mol of protein. 5. The N-terminal sequences of hen and duck ovotransferrins and of cattle, human and pig transferrins were also determined.
The amino acid sequence of ribonuclease U2 from Ustilago sphaerogena
Authors: Sato S , Uchida T
Published: August 2015

1. RNAase (ribonuclease) U2, a purine-specific RNAase, was reduced, aminoethylated and hydrolysed with trypsin, chymotrypsin and thermolysin. On the basis of the analyses of the resulting peptides, the complete amino acid sequence of RNAase U2 was determined, 2. When the sequence was compared with the amino acid sequence of RNAase T1 (EC 3.1.4.8), the following regions were found to be similar in the two enzymes; Tyr-Pro-His-Gln-Tyr (38-42) in RNAase U2 and Tyr-Pro-His-Lys-Tyr (38-42) in RNAase T1, Glu-Phe-Pro-Leu-Val (61-65) in RNAase U2 and Glu-Trp-Pro-Ile-Leu (58-62) in RNAase T1, Asp-Arg-Val-Ile-Tyr-Gln (83-88) in RNAase U2 and Asp-Arg-Val-Phe-Asn (76-81) in RNAase T1 and Val-Thr-His-Thr-Gly-Ala (98-103) in RNAase U2 and Ile-Thr-His-Thr-Gly-Ala (90-95) in RNAase T1. All of the amino acid residues, histidine-40, glutamate-58, arginine-77 and histidine-92, which were found to play a crucial role in the biological activity of RNAase T1, were included in the regions cited here. 3. Detailed evidence for the amino acid sequence of the sequence of the proteins has been deposited as Supplementary Publication SUP 50041 (33 PAGES) AT THE British Library (Lending Division)(formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975), 145, 5.
Oxidation of polymines by diamine oxidase from human seminal plasma
Authors: Hölttä E , Pulkkinen P , Elfving K , Jänne J
Published: August 2015

1. Diamine oxidase [amine-oxygen oxidoreductase (deaminating)(pyridoxal-containing), EC 1.4.3.6] was purified from human seminal plasma more than 1,700-fold. The enzyme appeared to be homogeneous on polyacrylamide-gel electrophoresis at two different pH values. 2. The general properties of the enzyme were comparable with those described for other diamine oxidases from different mammalian sources. The molecular weight of the enzyme was calculated to be about 182,000. 3. The enzyme had highest affinity for diamines, but polyamines spermidine and spermine were also degraded at concentrations that can be considered physiological in human semen. 3. The possible degradation of spermine by diamine oxidase in human semen in vivo may give rise to the formation of cytotoxic aldehydes that conceivably can influence the motility and survival of the spermatozoa.
The co-polymeric structure of pig skin dermatan sulphate. Distribution of L-iduronic acid sulphate residues in co-polymeric chains
Authors: Cöster L , Malmström A , Sjöberg I , Fransson L
Published: August 2015

1. Pig skin dermatan sulphate was degraded by periodate oxidation followed by alkaline elimination or by chondroitinase-ABC to quantify irregular repeating units, i.e. those containing D-GlcUA (D-glucuronic acid) and L-IdUA-SO4 (sulphated iduronic acid). 2. Previous results of periodate oxidation (Fransson, 1974) indicated repeating sequences in pig skin dermatan sulphate containing, on average, 3D-GlcUA, 9 L-IdUA-SO4 or 28 L-IdUA units in addition to N-acetylgalactosamine sulphate. However, complete digestion with chondroitinase-ABC yielded, at the most, 3-4 disulphated disaccharides/chain. Consequently, more than one-half of the L-IdUA-SO4 residues were present in monosulphated periods, i.e. IdUA-(SO4)-GalNAc. 3. To determine the location of L-IdUA-SO4 residues along the copolymeric chain dermatan sulphate was digested with testicular hyaluronidase. (This enzyme cleaves GalNAc-GlcUA bonds within block regions containing D-GlcUA.) By NaB3H4 reduction GalNAc residues located in the reducing end of the fragments were converted into [3H]GalNAcOH (N-acetylgalactosaminitol). Finally, the radioactive product was fragmented by periodate oxidation followed by alkaline elimination. The bulk of the radioactivity was associated with periodate-resistant oligosaccharides indicating that clusters of GlcUA-GalNAc-SO4 periods are often adjacent to a varying number of (n = 1-4) of L-IdUA-SO4-containing periods. 4. To study the distribution of L-IdUA-SO4-containing periods in relation to blocks of IdUA-GalNAc-SO4 periods different fractions of hyaluronidase-degraded dermatan sulphate were degraded separately. In all types of fragments (mol. wts. 1,500-10,000) L-IdUA-SO4-containing periods were demonstrated. In short fragments reducing terminal GalNAc-6-SO4 (6-sulphated N-acetylgalactosamine) was found confirming that these sequences were joined to relatively long D-GlcUA-containing block sequences via GalNAc-6-SO4. Moreover, low-molecular-weight oligosaccharides composed of alternating sequences were encountered. An octasaccharide derived from the carbohydrate sequence -GalNAc-GlcUA-GalNAc-IdUA-GalNAc-GlcUA-GalNAc-IdUA-GalNAc-GlcUA-GalNAc (- indicates the position of cleavage by hyaluronidase) was identified.
Structural equivalents of latency for lysosome hydrolases
Authors: Baccino F M , Zuretti M F
Published: August 2015

1. Structure-linked latency, a trait for most lysosome hydrolase activities, is customarily ascribed to the permeability-barrier function performed by the particle-limiting membrane, which shields enzyme sites from externally added substrates. 2. The influence of various substrate concentrations on the reaction rate has been measured for both free (non-latent) and total (completely unmasked by Triton X-100) hydrolase activities in rat liver cell-free preparations. The substrates were: β-glycerophosphate, phenolphthalein mono-beta-glucuronide. p-nitrophenyl N-acetyl-beta-D-glucosaminide and p-nitrophenyl β-D-galactopyranoside. The ratio (free activity/total activity) X 100 is called fractional free activity at any given substrate concentration. 3. The fractional free activity of β-glucuronidase and β-N-acetylglucosaminidase were clearly independent of substrate concentration, over the range examined, in both homogenates and lysosome-rich fractions. The fractional free activity of acid phosphatase appeared to be either unaffected (homogenate) or even depressed (lysosome-rich fraction) by increasing the β-glycerophosphate concentration. The fractional free activity of β-galactosidase consistently showed a non-linear increase with increasing substrate concentration in both homogenates and lysosome-rich fractions. 4. Procedures such as treatment with digitonin, hypo-osmotic shock and acid autolysis, although effective in causing varying degrees of resolution of the latency of lysosome hydrolase activities, were unable to modify appreciably the pattern of dependence or independence of their fractional free activities on substrate concentration, as compared with that exhibited by control preparations. Ouabain did not affect the free β-N-acetylglucosaminidase activity of liver homogenates at all. 5. Preincubation of control preparations with β-glycerophosphate or p-nitrophenyl α-galactoside did not result in any significant stimulation of the free hydrolytic activity toward these substrates. 6. The results consistently support the view that the membrane of “intact” lysosomes is virtually impermeable to all the substrates tested, except for p-nitrophenyl α-galactoside, for which the evidence is contradictory. Moreover the progressive unmasking of the hydrolase activities produced by these procedures in vitro reflects the increasing proportion of enzyme sites that are fully accessible to their substrates rather than a graded increase in the permeability of the lysosomal membrane.
Aerobic lactate production by mammary tissue
Authors: Elkin A R , Kuhn N J
Published: August 2015

Glucose uptake and L-lactate production were measured in cell, slice and intact tissue preparations of mammary glands from late-pregnant and lactating rats. The tissues showed extensive conversion of glucose into lactate in vitro, but not in vivo. Therefore aerobic lactate formation is not a normal feature of mammary tissue, but occurs in vitro as the result of some metabolic derangement.