bluuebunny/crossref_metadata_2025 huggingface dataset:
Stepwise cleavage of rabbit immunoglobin G by papain and isolation of four types of biologically active Fc fragments
Authors: Utsumi Sayaka
Published: August 2015

Four types of Fc fragments of different sizes were isolated by papain treatment of rabbit immunoglobulin G under various conditions and by subsequent chromatographic procedures. 1. Brief digestion at neutral pH without reduction produced a molecule in which the Fab and Fc fragments were still linked by a pair of labile disulphide bridges, and the Fc fragment released by cleaving these bonds, called 1Fc fragment, contained a portion of the ‘hinge’ region including an interchain disulphide bridge. Both complement-binding and guinea-pig skin-binding activities were retained by this fragment, which had mol. wt. 48000. 2. Prolonged digestion at neutral pH of immunoglobulin G whose labile inter-heavy-chain disulphide bridges had been reduced removed the ‘hinge’ region, giving mFc fragments (mol. wt. 46000), which lacked the capacity to bind guinea-pig skin but retained the antigenic as well as the complement-binding activities of 1Fc fragment completely. 3. Digestion at pH5·0 yielded a smaller fragment, sFc (mol. wt. 40000), which was no longer able to bind complement. Though the antigenic structure was intact, sFc fragment was curiously unable to precipitate with antibodies to the N-terminal determinants. 4. Fragment stFc (mol. wt. 25000), representing the C-terminal portion of Fc fragment, was formed from all the larger fragments by digestion at pH4·5. Only the C-terminal antigenic determinants were retained by stFc fragment.
Chemical changes associated with aging of collagen in vivo and in vitro
Authors: Deshmukh Kalindi , Nimni Marcel E.
Published: August 2015

Collagen extracted from rat skin by neutral-salt solutions contains less aldehydes than the more insoluble collagen fractions. The concentration of aldehydes in collagen is directly related to its capacity to form stable cross-linked gels, which do not redissolve on cooling and become more insoluble in a variety of reagents. Whereas the absorption spectrum of neutral-salt-soluble collagen treated with N-methylbenzothiazolone hydrazone resembles that of acetaldehyde, the more insoluble collagen fractions show increasing amounts of a component that behaves like an αβ-unsaturated aldehyde. The ratio between α- and β-sub-units present in a particular fraction of soluble collagen seems to be constant and independent of the age of the animal. Neutral-salt-soluble collagen, which has a low concentration of β-components, will generate intramolecular bonds if gelled at 37°. These intramolecular bonds seem to precede the formation of stable intermolecular cross-links, since these gels can redissolve when cooled to yield a soluble collagen with a higher content of β-components of intramolecular origin.
Studies on the cathepsins in elastic cartilage
Authors: Ali S. Y. , Evans Lois
Published: August 2015

1. The presence of several enzymes in rabbit ear cartilage was examined by a quantitative method that permits the incubation of a fixed weight of cartilage sections (18μm.) with an appropriate exogeneous substrate. 2. As the presence of cathepsins B and D in cartilage has already been established, evidence is now provided to show that cathepsins A and C are also present and are maximally active at pH5. 3. Cathepsin A was recognized by its hydrolysis of benzyloxycarbonyl-glutamyl-tyrosine and cathepsin C by its hydrolysis of glycyl-tyrosine amide; the cartilage also hydrolysed benzyloxycarbonyl-glutamyl-phenylalanine and benzoyl-dl-phenylalanine 2-naphthyl ester at pH5. 4. The acid phosphatase activity and the DNA content of cartilage have also been measured to provide a basis for comparison with the cathepsin activity of cartilage obtained from other sites and species.
Immune reactions in polysaccharide media. The effect of hyaluronate, chondroitin sulphate and chondroitin sulphate–protein complex on the precipitin reaction
Authors: Hellsing Krister
Published: August 2015

The influence of the connective-tissue polysaccharides hyaluronate, chondroitin 4-sulphate and a chondroitin 4-sulphate–protein complex (PP-L) from cartilage on the precipitin reaction was investigated. In a system consisting of 125I-labelled human serum albumin and the immunoglobulin G fraction from rabbit anti-albumin sera, the precipitation is greatly increased in the region of antigen excess. This effect depends on the concentration, molecular weight and configuration of the polysaccharide. The increase parallels a decrease in the amount of soluble immune complexes in the supernatant. It is suggested that the effect is due to steric exclusion of the complexes from the domains of the polysaccharides. The possibility that such a mechanism might enhance precipitation of antigen–antibody complexes in certain pathological conditions is discussed.
Immune reactions in polysaccharide media. Investigation on complex-formation between some polysaccharides, albumin and immunoglobulin G
Authors: Hellsing Krister
Published: August 2015

Serum albumin and immunoglobulin G were chromatographed on columns of dextran, hyaluronate and chondroitin 4-sulphate. The partition of the two proteins between hyaluronate and buffer was also measured by equilibrium dialysis. The results accord with the view that there is no complex-formation between the polysaccharides and the proteins in 0·05m-phosphate buffer, pH7·4, containing sodium chloride (0·1m). The observations support the hypothesis that the previously described polysaccharide enhancement of the precipitin reaction is due to exclusion and not to non-specific complex-formation.
The preferential loss of the polylysine- or polyornithine-stimulated activity of Clostridium perfringens polynucleotide phosphorylase during proteolysis
Authors: Fitt P. S. , Wille Helga
Published: August 2015

1. An improved method for the purification of Clostridium perfringens polynucleotide phosphorylase (nucleoside diphosphate–polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is described. The product was stable and was highly stimulated by polylysine or polyornithine. 2. It migrated as a single enzyme during sucrose-density-gradient centrifugation, and no separation of polymerization and phosphorolytic activities was observed. 3. Trypsin digestion caused a rapid, preferential loss of the polylysine- or polyornithine-stimulated activity, which was prevented by low concentrations of polyornithine. 4. The protection by polyornithine was not specific. 5. It is concluded that charge effects on the clostridial polynucleotide phosphorylase itself are primarily responsible for the stimulation of this enzyme by polylysine or polyornithine.
Composition of Pseudomonas aeruginosa slime
Authors: Brown M. R. W. , Foster J. H. Scott , Clamp J. R.
Published: August 2015

1. The slime produced by eight strains of Pseudomonas aeruginosa on a number of different media was demonstrated to be qualitatively the same. Small quantitative differences may be occasioned by differences in the extraction procedure, the growth medium or the strain of organism used. 2. The slime was shown to be predominantly polysaccharide with some nucleic acid material and a small amount of protein. 3. The hydrolysed polysaccharide fraction consists mainly of glucose with smaller amounts of mannose. This accounts for some 50–60% of the total slime. In addition, there is some 5% of hyaluronic acid. The nucleic acid material represents approx. 20% of the total weight, and is composed of both RNA and DNA. 4. Minor components are protein, rhamnose and glucosamine, the protein being less than 5% of the total. 5. Hyaluronic acid is produced in greater quantities from nutrient broth than from chemically defined media, and is more firmly attached to the cells than the other components.
Studies on the mechanism of activation and inactivation of pyruvate, phosphate dikinase. A possible regulatory role for the enzyme in the C4 dicarboxylic acid pathway of photosynthesis
Authors: Hatch M. D. , Slack C. R.
Published: August 2015

1. The activity of pyruvate,Pi dikinase in leaves of maize and Amaranthus palmeri rapidly falls on transferring illuminated plants to darkness. Illumination of dark-treated plants results in an immediate rapid increase in activity of the enzyme, the final activity reached being dependent on the intensity of the incident light. 2. Activation of the enzyme in extracts of dark-treated maize leaves after gel filtration on Sephadex G-25 requires a thiol and Pi. The Pi requirement for activation can be replaced by arsenate. Activation of the enzyme is inhibited by AMP and GMP and possibly also by ADP and ATP. Activation of the enzyme after gel filtration on Sephadex G-200 also requires a heat-labile component that is excluded by Sephadex G-25. 3. The active enzyme isolated from illuminated leaves is inactivated by ADP in the presence of a thiol, the rate of inactivation being very much faster in air than in an oxygen-free atmosphere. Reactivation of the ADP-inactivated enzyme requires a thiol, Pi and a component excluded by Sephadex G-25 but considerably retarded by Sephadex G-200. 4. The active enzyme is rapidly and irreversibly inactivated in the absence of a thiol. Inactivation is accelerated by both sodium diethyldithiocarbamate and tetraethylthiuram disulphide, and the enzyme inactivated by these reagents is completely reactivated by incubation with dithiothreitol. This reactivation does not require Pi. The inactive enzyme from dark-treated leaves is stabilized by diethyldithiocarbamate and can be partially activated by dithiothreitol alone; complete reactivation requires both dithiothreitol and Pi. 5. The enzyme activity is markedly inhibited by the thiol reagents p-chloromercuribenzoate, γ-(p-arsenophenyl)-n-butyrate and an equimolar mixture of arsenite and 2,3-dimercaptopropan-1-ol. 6. The processes of activation and inactivation observed in vitro are discussed in relation to the regulation of pyruvate,Pi dikinase activity in the leaf.
Changes in the patterns of synthesis of ribonucleic acid species in immature rat uterus in response to oestradiol-17β
Authors: Billing R J , Barbiroli B , Smellie R. M. S.
Published: August 2015

1. After treatment of immature rats with diethylstilboestrol, the wet weight and RNA content of uterine tissue increased rapidly, reaching a peak at 40hr. After an initial lag of a few hours, the acid-soluble ribose and protein contents also rose to maxima at 40hr. No increase in DNA content occurred until at least 24hr. after treatment. 2. The RNA from immature rat uterus isolated at various times up to 6hr. after administration of oestradiol-17β was labelled by injecting [3H]uridine and [3H]guanosine intraperitoneally 30min. before the animals were killed. It was fractionated on columns of kieselguhr coated with methylated serum albumin and the radioactivity in fractions corresponding to transfer RNA, 7s RNA, ribosomal RNA, Q1-RNA, Q2-RNA and DNA-like RNA was determined. 3. The radioactivity of the whole RNA increased steadily for 6hr. after hormone treatment. The earliest changes occurred in the Q1-RNA (ribosomal RNA precursor), whereas at longer time-intervals the radioactivity of the ribosomal RNA, 7s RNA and transfer RNA increased by four- to five-fold. The radioactivity of the DNA-like RNA increased by about 50%, but only at the longer time-intervals. 4. It is concluded that one of the earliest changes in response to oestradiol treatment is a major increase in synthesis of ribosomal RNA followed later by a similar increase in synthesis of transfer RNA and by a much smaller increase in synthesis of DNA-like RNA. The change in synthesis of ribosomal RNA in immature rat uterus may represent one of the most important responses to oestradiol treatment.
Adenine nucleotides and magnesium ions in relation to control of mammalian cerebral-cortex hexokinase
Authors: Bachelard H S , Goldfarb P. S. G.
Published: August 2015

1. The kinetics of inhibition of brain soluble cytoplasmic hexokinase by ADP were examined in relation to variations in the concentrations of Mg2+ and ATP. The type of inhibition observed was dependent on the Mg2+/ATP ratio. 2. ADP at Mg2+/ATP ratios 2:1 exhibited inhibition of the ‘mixed’ type; at Mg2+/ATP ratios 1:1 the inhibition appeared to be competitive with regard to ATP. 3. Inhibition by free ATP was observed when the Mg2+/ATP ratio was less than 1:1. The inhibition was also of the ‘mixed’ type with respect to MgATP2−. 4. The inhibitions due to ADP and to free ATP were not additive. The results suggested that there may be up to four sites in the soluble enzyme: for glucose, glucose 6-phosphate, ADP and MgATP2−. 5. The ‘free’ non-particulate intracellular Mg2+ concentration was measured and concluded to be about 1·5mm. 6. The concentrations in vivo of Mg2+ and ATP likely to be accessible to a cytoplasmic enzyme are suggested to be below those that yield maximum hexokinase rates in vitro. The enzymic rates were measured at relevant suboptimum concentrations of Mg2+ and ATP in the presence of ADP. Calculations that included non-competitive inhibition due to glucose 6-phosphate (56–65% at 0·25mm) resulted in net rates very similar to the measured rates for overall glycolysis. This system may therefore provide a basis for effective control of cerebral hexokinase.
Electrophoretic differentiation of myofibrillar proteins in the pig
Authors: Parsons A. L. , Parsons J. L. , Blanshard J. M. V. , Lawrie R. A.
Published: August 2015

1. Starch-gel electrophoretograms of myosin and tropomyosin preparations in 8m-urea, from longissimus dorsi and psoas muscles of the pig, were characterized by laser densitometry. 2. The typical pattern for freshly prepared myosin from both muscles was similar, there being five electrophoretically distinct components. 3. The number of electrophoretically distinct components in both muscles increased after freeze-drying, but the effect of freeze-drying was more marked in longissimus dorsi. 4. Extraction with 8m-urea containing 2% β-mercaptoethanol decreased the number of major electrophoretically distinct components of the fresh myosin of both muscles to four. 5. Although there was also some simplification of the patterns after freeze-drying the greater susceptibility of the myosin from longissimus dorsi was still evident. 6. The typical pattern for freshly prepared tropomyosin in 8m-urea differed in the two muscles: in each case it was more complex than that of the corresponding myosins. 7. The pattern of tropomyosin from neither longissimus dorsi nor psoas was altered significantly after freeze-drying. 8. Electrophoretograms of pig longissimus dorsi tropomyosin in 8m-urea differed from those of longissimus dorsi tropomyosin from sheep, ox and rabbit. 9. Extraction of the tropomyosins in 8m-urea and 2% β-mercaptoethanol simplified the electrophoretic pattern to two major components with samples from pig, sheep and ox, and to one major component with samples from rabbit. 10. It was concluded that classification of skeletal muscles as ‘red’ or ‘white’ is insufficient to account for the degree of functional specialization which the electrophoretograms suggest.
The use of maleic anhydride for the reversible blocking of amino groups on polypeptide chains
Authors: Butler P. J. G. , Harris J. I. , Hartley B S , Leberman R.
Published: August 2015

1. Maleic anhydride was shown to react rapidly and specifically with amino groups of proteins and peptides. Complete substitution of chymotrypsinogen was achieved under mild conditions and the extent of reaction could be readily determined from the spectrum of the maleyl-protein. 2. Maleyl-proteins are generally soluble and disaggregated at neutral pH. Trypsin splits the blocked proteins only at arginine residues and there is frequently selectivity in this cleavage, e.g. in yeast alcohol dehydrogenase and pig glyceraldehyde 3-phosphate dehydrogenase. 3. The group is removed by intramolecular catalysis at acid pH. The half-time was 11–12hr. at 37° at pH3·5 in ∈-maleyl-lysine or in maleyl-chymotrypsinogen. 4. The unblocking reaction can be used as the basis for a ‘diagonal’-electrophoretic separation of lysine peptides and N-terminal peptides, as shown by studies with β-melanocyte-stimulating hormone.
Experimental conditions affecting ribonucleic acid polymerase in isolated rat liver nuclei. Effect of nucleoside triphosphate concentration, temperature, ammonium sulphate and heparin
Authors: Novello F. , Stirpe F.
Published: August 2015

1. The conditions affecting the activity of RNA polymerase in isolated rat liver nuclei were studied with Mg2+ or Mn2+ as activating ions. 2. The enzyme assayed with Mg2+ and at low ionic strength is saturated by a lower concentration of nucleotide substrates than if assayed with Mn2+ at low ionic strength or with either ion at high ionic strength. 3. At low and at high ionic strength the incorporation of AMP is affected in a similar way by variations in the temperature of incubation. Preincubation at 37° impairs the AMP incorporation. 4. Heparin stimulates the RNA polymerase activity in the presence of Mn2+. 5. Both ammonium sulphate and heparin ‘restart’ the reaction if added after 15min., the effect being more marked with ammonium sulphate than with heparin, and also more marked in the presence of Mn2+ than of Mg2+. 6. α-Amanitin abolishes the effect of ammonium sulphate and of heparin.
Effects of magnesium, manganese and adenosine triphosphate ions on pyruvate carboxylase from baker's yeast
Authors: Cazzulo J. J. , Stoppani A. O. M.
Published: August 2015

1. Pyruvate carboxylase from baker's yeast acts with either MgATP2− or MnATP2− as substrate. The optimum pH for the enzyme reaction is 8·0 with MgATP2− and 7·0 with MnATP2−. 2. When the reaction velocity is plotted against MgATP2− (or MnATP2−) concentration slightly sigmoid curves are obtained, either in the presence or in the absence of acetyl-CoA (an allosteric activator). In the presence of excess of free Mg2+ (or Mn2+) the curves turn into hyperbolae, whereas in the presence of excess of free ATP4− the apparent sigmoidicity of the curves increases. 3. The sigmoidicity of the plots of v against MgATP2− (or MnATP2−) concentration can be explained by the inhibitory effect of free ATP4−, the concentration of which, in the experimental conditions employed, is significant and varies according to the total concentration of the ATP–magnesium chloride (or ATP–manganese chloride) mixture. Free ATP4− behaves as a negative modifier of yeast pyruvate carboxylase. 4. The effect of high concentrations of Mg2+ (or Mn2+) on the kinetics of yeast pyruvate carboxylase can be explained as a deinhibition with respect to ATP4−, instead of a direct enzyme activation. 5. At pH6·5 manganese chloride is more effective than magnesium chloride as enzyme activator even in the presence of a great excess (16-fold) of the latter. This is consistent with a significant contribution of the MnATP2− complex to the activity of yeast pyruvate carboxylase, in medium conditions resembling those existing inside the yeast cell (pH6·25–6·75; 12mm-magnesium chloride and 0·75mm-manganese chloride). 6. The physiological significance of the enzyme inhibition by free ATP4− is doubtful since the Mg2+ and Mn2+ concentrations reported to exist inside the yeast cell are sufficient to decrease ATP4− concentrations to ineffective values.
Kinetic complexity of chloroplastal deoxyribonucleic acid and mitochondrial deoxyribonucleic acid from higher plants
Authors: Wells Richard , Birnstiel Max
Published: August 2015

1. Chloroplasts and mitochondria were isolated by aqueous and non-aqueous cell-fractionation techniques. In a variety of higher plants the mitochondrial DNA bands in a caesium chloride gradient at 1·706g.cm.−3, whereas chloroplastal DNA has a buoyant density of 1·697g.cm.−3. 2. In total cellular DNA of moderate molecular weight, the chloroplastal DNA is found within the Gaussian distribution of the nuclear DNA and is not resolved as a satellite. 3. Both chloroplastal DNA and mitochondrial DNA from lettuce renature rapidly. 4. The kinetic complexity of mitochondrial DNA is > 108 daltons. 5. Chloroplastal DNA is made up from fast and slow renaturing sequences with kinetic complexities of 3×106 and 1·2×108 daltons respectively. 6. From the discrepancy between analytical and kinetic complexity it is concluded that chloroplastal DNA is extensively reiterated.
Purification and properties of α-galactosidases from Vicia faba seeds
Authors: Dey P. M. , Pridham J. B.
Published: August 2015

Two forms of α-galactosidase, I and II, exist in Vicia faba seeds and these have been purified 3660- and 337-fold respectively. They behaved as homogeneous preparations when examined by ultracentrifugation, disc electrophoresis and gel filtration. The apparent molecular weights of enzymes I and II, as determined by gel filtration, were 209000 and 38000 respectively. The carbohydrate contents of enzymes I and II were 25% and 2·8% respectively, and the enzymes differed in their aromatic amino acid compositions. Enzyme I was split into six inactive subunits in the presence of 6m-urea. α-Galactosidases I and II showed different pH optima and Km and Vmax. values with p-nitrophenyl α-d-galactoside and raffinose as substrates, and also differed in their thermal stabilities.
Studies on the positional integrity of glyceride fatty acids during digestion and absorption in rats
Authors: Raghavan S. S. , Ganguly J.
Published: August 2015

1. Rats previously starved for 24hr. were separately given by intraduodenal injections 0·5ml. of a dispersion containing 10mg. of sodium taurocholate, with 50mg. of glycerol 1,3-dioleate 2[1−14C]-palmitate, glycerol 1,2-dioleate 3[1−14C]-palmitate, a mixture of [1−14C]palmitic acid and triolein, or a mixture of [1−14C]-palmitic acid and oleic acid. 2. At the end of 30min., the net amounts, and the radioactivity, of the neutral-lipid components recovered from the intestinal lumen and mucosa, and the position of the labelled palmitic acid in the mucosal triglycerides, were determined. 3. When glycerol 1,3-dioleate 2[1−14C]-palmitate was administered, most of the labelled acid was retained in the di- and monoglycerides of the lumen; the triglycerides were the major components containing the radioactivity in the mucosa and 75–80% of the labelled acid was located at the β-position of these triglycerides. 4. When glycerol 1,2-dioleate 3[1−14C]-palmitate was administered, the labelled acid was readily split off in the lumen and virtually no radioactivity could be traced in the monoglyceride fraction; in the intestinal mucosa, triglycerides were again the chief components containing most of the radioactivity, and 80–85% of the labelled acid was esterified at the outer positions of the glycerol. 5. When [1−14C]palmitic acid mixed with triolein was administered, the concentrations of free fatty acids increased markedly in the intestinal lumen and mucosa, and 80–88% of the radioactivity of the mucosal triglycerides was located at the outer positions of the glycerol. 6. When [1−14C]palmitic acid mixed with oleic acid was administered, the labelled acid accumulated in the lumen as well as in the cell, and it was randomly incorporated into all three positions of the mucosal triglycerides.
The subcellular distribution of [N-Me−3H]-acetylcholine synthesized by brain in vivo
Authors: Chakrin L. W. , Whittaker V P
Published: August 2015

1. Three forms of acetylcholine occur in subcellular fractions of brain tissue: free acetylcholine, present in the high-speed supernatant from eserinized sucrose homogenates; stable bound acetylcholine, present in synaptic vesicles; and labile bound acetylcholine, present in the cytoplasm of synaptosomes (detached presynaptic nerve terminals). 2. The relationship between these forms has been investigated by isolating the subcellular fractions from the cortical tissue of cats and guinea pigs excised 1hr. after infiltration of [N-Me−3H]choline into the cortex in vivo. 3. Since choline is a ubiquitous metabolite, means were devised for isolating the radioactive acetylcholine on columns of the weak acid ion-exchange resin IRF-97; control experiments with samples of extracts treated with acetylcholinesterase showed that the radioactivity attributed to acetylcholine migrated to the choline peak after cholinesterase treatment. 4. The specific radioactivities of the various forms of acetylcholine were different: labile bound (synaptosomal cytoplasmic) acetylcholine had the highest, stable bound (vesicular) acetylcholine the next highest, and the high-speed-supernatant form the lowest. 5. It is concluded that the various forms of acetylcholine could not have arisen during fractionation from a single pre-existing pool of acetylcholine.
The hydroxylation of p-coumaric acid by an enzyme from leaves of spinach beet (Beta vulgaris L.)
Authors: Vaughan P. F. T. , Butt V S
Published: August 2015

1. An enzyme from the leaves of spinach beet (Beta vulgaris L.) that catalyses the hydroxylation of p-coumaric acid to caffeic acid in the presence of ascorbate has been purified about 1000-fold on a protein basis. 2. It is activated by high concentrations of ammonium sulphate and sodium chloride. 3. The preparation shows both hydroxylase and catechol oxidase activities, in a constant ratio throughout the purification procedure; they are similarly activated by salts. 4. Ascorbate acts as a reductant in quantities equivalent to the caffeic acid produced by hydroxylation. 5. Ascorbate can be replaced by tetrahydrofolic acid, NADH, NADPH or 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine, but not by caffeic acid. Among these, the pteridine is the most effective, but the reaction is not inhibited by aminopterin. In experiments with saturating concentrations of NADH and the pteridine, these reductants compete in the reaction and are equivalent on a molar basis. 6. No cofactor has been separated from the enzyme by prolonged dialysis. 7. The relation of the enzyme to other hydroxylases and phenolases is discussed.
Regulation of glycolysis and l-glycerol 3-phosphate concentration in rat epididymal adipose tissue in vitro. Role of phosphofructokinase
Authors: Halperin M L , Denton R M
Published: August 2015

1. Attempts were made to define the role of phosphofructokinase in glycolytic control and the factors regulating the concentration of l-glycerol 3-phosphate in rat epididymal fat pads incubated in vitro. 2. Glycolysis rates were altered by anoxia or by additions of insulin, adrenaline or both to the incubation medium, and the changes in rate were related to changes in the steady-state concentrations of hexose phosphates, adenine nucleotides, l-glycerol 3-phosphate and citrate in the whole tissue. Measurements were also made of the lactate/pyruvate concentration ratio in the medium after incubation. 3. The mass-action ratios of phosphofructokinase, calculated from the whole-tissue concentrations of products and substrates, were less than 0·1% of the value of the ratio at pH7·4 at equilibrium. 4. Only in the presence of adrenaline could the observed stimulation of glycolytic flux be related to a possible activation of phosphofructokinase since, in this situation, the concentration of one substrate, fructose 6-phosphate, was not altered and the concentration of the other, ATP, was decreased. Increased glycolytic flux in the presence of insulin may be explained by an observed increase in the concentration of the substrate, fructose 6-phosphate. Under anaerobic conditions, glycolytic flux was decreased but this did not appear to be the result of inhibition of phosphofructokinase, since the concentrations of both substrates, fructose 6-phosphate and ATP, were decreased. The changes in glycolytic flux with insulin and anoxia may be secondary to changes in the rate of glucose uptake. 5. Changes in l-glycerol 3-phosphate concentration appear to be related both to changes in the concentration of dihydroxyacetone phosphate and to changes in the NADH/NAD+ concentration ratio in the cytoplasm. They do not seem to be related directly to alterations in glycolytic rate.
Partial purification and properties of carbamoyl phosphate synthetase of Alaska pea (Pisum sativum L. cultivar Alaska). Purification and general properties
Authors: O'neal T. Denny , Naylor Aubrey W.
Published: August 2015

1. Carbamoyl phosphate synthetase was purified up to 45-fold from Alaska pea seedling (Pisum sativum L. cultivar Alaska). 2. The enzyme was most active with and had the lowest Km for l-glutamine as compared with NH4+. 3. The purest preparations utilized very poorly or not at all l-asparagine and urea as nitrogen donors. 4. At saturating concentrations of components of the reaction, the Km for l-glutamine was 1·2×10−4m, and the Km for ATP was approx. 3·9×10−4m. 5. Although the enzyme was very labile, stability was improved by glutamine, asparagine, ammonium sulphate, dithiothreitol and especially l-ornithine. 6. Free ATP was markedly inhibitory, and MgATP2− and Mg2+ appeared to be the actual substrates utilized. 7. Fe2+ and Mn2+ were also utilized, but not as readily as Mg2+ except at low concentrations. K+ increased activity significantly. 8. Of the four nucleotides tested (ITP, ATP, GTP and UTP) only ATP served as an effective phosphate donor.
Lipid peroxide formation in microsomes. General considerations
Authors: Wills E. D.
Published: August 2015

1. Liver microsomes form lipid peroxide when incubated with ascorbate or NADPH, but not with NADH. Increasing the concentration of ascorbate beyond the optimum (0·5mm) decreases the rate of lipid peroxide formation, but this effect does not occur with NADPH. Other reducing agents such as p-phenylenediamine or ferricyanide were not able to replace ascorbate and induce lipid peroxide formation. 2. The rate of ascorbate-induced peroxidation is optimum at pH6·0 whereas the rate of the NADPH system is optimum at pH7·0. Both systems require phosphate for maximum activity. 3. Lipid peroxide formation occurs at the maximum specific rate in very dilute microsome suspensions (0·15mg. of protein/ml.). 4. Treatment of microsomes with deoxycholate and other detergents causes membrane disintegration and inhibits lipid peroxide formation. 5. Lipid peroxide formation is accompanied by a rapid uptake of oxygen and there is a large excess of oxygen utilized for each molecule of malonaldehyde measured in the peroxide method. 6. Boiled microsomes form lipid peroxide in the presence of ascorbate, but not if NADPH is added. 7. Lipid peroxide formation induced by NADPH is strongly inhibited by p-chloromercuribenzoate, weakly inhibited by N-ethylmaleimide and unaffected by iodoacetamide. Ascorbate-induced peroxidation in untreated microsomes is unaffected by p-chloromercuribenzoate, but inhibited if boiled microsomes are used. These experiments may be interpreted on the basis that a ferredoxin-type protein forms part of the system in which NADPH induces lipid peroxide formation. 8. Most heavy-metal ions, with the exception of inorganic iron (Fe2+ or Fe3+), which activates, inhibit both ascorbate-induced and NADPH-induced peroxidation. Mg2+ increases the rate of peroxidation whereas Ca2+ inhibits it. 9. Lipid peroxide formation is inhibited strongly by GSH and weakly by cysteine. Ascorbate-induced peroxidation is much more sensitive than NADPH-induced peroxidation. 10. Peroxidation is strongly inhibited by addition of low concentrations (0·01–0·1mm) of cytochrome c or of haemoglobin. 11. It is considered that lipid peroxide formation occurs as a result of the operation of the microsomal electron-transport chain switching from hydroxylation to oxidize unsaturated lipids of the endoplasmic reticulum.
The rate-determining step in pepsin-catalysed reactions, and evidence against an acyl-enzyme intermediate
Authors: Cornish-Bowden A. J. , Greenwell P. , Knowles J. R.
Published: August 2015

To delineate further the pathway of pepsin-catalysed reactions, three types of experiments were performed: (a) the enzyme-catalysed hydrolysis of a number of di- and tri-peptide substrates was studied with a view to observing the rate-determining breakdown of a common intermediate; (b) the interaction of pepsin with several possible substrates for which ‘burst’ kinetics might be expected was investigated; (c) attempts were made to trap a possible acyl-enzyme intermediate with [14C]methanol in both a hydrolytic reaction (with N-acetyl-l-phenylalanyl-l-phenylalanylglycine) and in a ‘virtual’ reaction (with N-acetyl-l-phenylalanine) under conditions where extensive hydrolysis or 18O exchange is known to occur. It is concluded that (i) intermediates in pepsin-catalysed reactions (aside from the Michaelis complex) occur subsequently to the rate-determining transition state, and (ii) an acyl-enzyme intermediate, if such is formed, cannot be trapped with [14C]methanol in these systems.
An aspartic acid residue at the active site of pepsin. The isolation and sequence of the heptapeptide
Authors: Bayliss R. S. , Knowles J. R. , Wybrandt Grith B.
Published: August 2015

Pepsin reacts stoicheiometrically with the active-site-directed irreversible inhibitor N-diazoacetyl-l-phenylalanine methyl ester, with concomitant loss of all proteolytic and peptidolytic activity. The reagent esterifies a unique aspartic acid residue in pepsin, which is in the sequence:Ile-Val-Asp-Thr-Gly-Thr-Ser
Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs
Authors: Barnes M. J. , Constable B. J. , Kodicek E.
Published: August 2015

1. After the administration of labelled proline to guinea pigs deprived of ascorbic acid for 15 days, the dorsal skin was examined 5 days later in an attempt to detect the presence of hydroxyproline-deficient collagen (protocollagen). The extent of incorporation of proline into skin collagens indicated a severe impairment of collagen synthesis. 2. A comparison of proline and hydroxyproline specific radioactivities in diffusible peptides obtained by treatment with collagenase of either purified skin collagens or direct hot-trichloroacetic acid extracts of skin failed to indicate the presence of protocollagen. Possible reasons for this are discussed. 3. The incorporation results did not indicate an inability of normal collagen, i.e. collagen hydroxylated to the normal degree, to cross-link in scurvy. 4. Incorporation of labelled proline into aortic elastin isolated from the same animals did not indicate a decrease in elastin biosynthesis in ascorbic acid deficiency, beyond that attributable to the inanition accompanying the vitamin deficiency. The proline/hydroxyproline specific-radioactivity ratio in elastin from scorbutic guinea pigs was about 6:1 in contrast with the 1:1 ratio in control groups. It is concluded that the formation of elastin hydroxyproline was ascorbate-dependent and that a hydroxyproline-deficient elastin is formed and retained in scurvy. The formation of desmosines was unimpaired in scorbutic animals. 5. Studies with chick embryos confirmed the formation of elastin hydroxyproline from free proline. Incorporation of free hydroxyproline into elastin hydroxyproline was negligible. 6. Digestion of solubilized samples with collagenase indicated that the hydroxyproline in guinea-pig aortic elastin preparations was not derived from contamination by collagen. It is suggested that most if not all of the hydroxyproline in the guinea pig elastin preparations investigated can be considered an integral part of the elastin molecule.
α1 acute-phase globulins of rats. Isolation and some properties
Authors: Gordon A. H. , Louis L. N.
Published: August 2015

1. A method is described for the isolation of certain of the α1-globulins of rat plasma that are known to increase in concentration after tissue damage (acute-phase globulins). 2. Although apparently homogeneous when examined by disc electrophoresis at pH9, these proteins could be subdivided further by isoelectric fractionation. 3. Treatment with neuraminidase removed approx. 60% of the sialic acid originally present in these proteins and gave almost completely homogeneous material of decreased mobility when examined by disc electrophoresis in polyacrylamide gel. When subjected to immunoelectrophoresis this material gave a single arc. 4. The homogeneity of the isolated materials was examined by ultracentrifugation. The single peak thus found is consistent with molecular weights of 45000–46000. 5. The isolated materials were shown to be glycoproteins containing approx. 15% of carbohydrate, and to have isoelectric points in the range pH4·4–4·8.
The sulphation of chondroitin sulphate in embryonic chicken cartilage
Authors: Robinson H. C.
Published: August 2015

1. Whole tissue preparations and subcellular fractions from embryonic chicken cartilage were used to measure the rate of incorporation of inorganic sulphate into chondroitin sulphate in vitro. 2. In cartilage from 14-day-old embryos, [35S]sulphate is incorporated to an equal extent into chondroitin 4-sulphate and chondroitin 6-sulphate at a rate of 1·5nmoles of sulphate/hr./mg. dry wt. of cartilage. 3. Microsomal and soluble enzyme preparations from embryonic cartilage catalyse the transfer of sulphate from adenosine 3′-phosphate 5′-sulphatophosphate into both chondroitin 4-sulphate and chondroitin 6-sulphate. 4. The effects of pH, ionic strength, adenosine 3′-phosphate 5′-sulphatophosphate concentration and acceptor chondroitin sulphate concentration on the soluble sulphotransferase activity were examined. These factors all influence the activity of the sulphotransferase, and pH and incubation time also influence the percentage of chondroitin 4-sulphate formed.
Assay and properties of diaminopimelate epimerase from Bacillus megaterium
Authors: White P. J. , Lejeune B. , Work Elizabeth
Published: August 2015

1. Diaminopimelate epimerase from a soluble extract of Bacillus megaterium N.C.I.B. 7581 was purified about 25-fold by fractionation with ammonium sulphate and chromatography on calcium phosphate gel–cellulose. The product was impure but was unstable on further purification. 2. Quantitative assay methods for the enzyme were devised in which meso- or ll-diaminopimelic acid may be the substrate. 3. Between 25° and 45° at pH7·0 enzyme action leads to an equilibrium mixture containing 65% meso-isomer and 35% ll-isomer. 4. The initial rate of epimerization was 2–3 times as fast with ll-diaminopimelic acid as substrate as with the meso-isomer; a number of other amino acids were not racemized by the enzyme. The Michaelis constants at 37° were 6·7mm (ll-isomer) and 100mm (meso-isomer); with both substrates enzyme activity was maximal at pH7–8. The relative rates of epimerization of ll-diaminopimelic acid at 25°, 37° and 45° were 0·77:1·00:1·15. 5. A thiol compound (of which 2,3-dimercaptopropan-1-ol was the most effective) was needed as an activator of the purified enzyme. 6. Carbonylbinding reagents and several other compounds did not inhibit diaminopimelate epimerase. 7. Pyridoxal phosphate did not stimulate enzymic activity even in preparations that had been almost completely freed of derivatives of vitamin B6 (as shown by microbiological assay).
Studies on a nucleoprotein prepared from rat liver polysomes by digestion with T1 ribonuclease
Authors: Hawtrey A. O.
Published: August 2015

1. Treatment of rat liver polysomes in a buffer containing 2·5mm-magnesium chloride with T1 ribonuclease at a concentration of 330units/ml. of reaction medium at 37° for 2hr. leads to the production of an insoluble nucleoprotein. 2. On the bases of analysis for protein and RNA and of u.v.-absorption spectra the nucleoprotein appears to have lost approx. 60% of the structural RNA originally present in the ribosome. Degradation of 3H-labelled polysomes (structural RNA labelled with orotic acid) with T1 ribonuclease leads to nucleoprotein preparations retaining approx. 30% of the radioactivity originally present in the polysomes. By means of sucrose-density-gradient centrifugation it is shown that the nucleoprotein preparations are free of single 73s ribosomes and ribosomal subunits. No evidence for the presence of 28s and 18s structural RNA was obtained on examination of extracted nucleoprotein-particle RNA by means of sucrose-density-gradient centrifugation. 3. Digestion of washed polysomes carrying 14C-labelled nascent peptide chains with T1 ribonuclease gives a nucleoprotein particle that retains approx. 70% of the original labelled chains. Treatment of labelled nucleoprotein particles with 1mm-puromycin in the absence of transfer factors releases 20% of the labelled chains. Addition of GTP (0·48μmole) increases this release to 37%. 4. Treatment of nucleoprotein particles carrying 14C-labelled peptide chains with either EDTA (50mm) or ammonium chloride (0·5m) brings about a small release of labelled material (approx. 15%). 5. Disruption of nucleoprotein particles carrying 14C-labelled peptide chains with either sodium dodecyl sulphate or 2m-lithium chloride, followed by addition of transfer RNA as marker and chromatography on Sephadex G-200, show in both cases that considerable amounts of labelled peptide material move well ahead of the added transfer RNA marker. Further, if nucleoprotein particles carrying labelled peptide chains are treated with 0·3m-potassium hydroxide at 20° for 24 hr., neutralized to pH7·6, and then chromatographed on Sephadex G-200, the labelled peptide material moves much closer to the added transfer RNA marker. These results suggest that a proportion of the nascent 14C-labelled peptides on the nucleoprotein are attached to transfer RNA or large fragments of transfer RNA. 6. [3H]Polyuridylic acid binds to nucleoprotein particles in 1mm-magnesium chloride. The rate of binding is rapid when measured at 20°.
The development of gluconeogenesis in rat liver. Experiments in vivo
Authors: Philippidis Helen , Ballard F. J.
Published: August 2015

1. The injection of substrate amounts of lactate into newborn rats produced an increase in the concentration of phosphoenolpyruvate in liver. Similar experiments with foetal rats showed no increase in phosphoenolpyruvate concentration although pyruvate formation was observed. 2. The administration of pyruvate to foetal rats was also without effect on the hepatic phosphoenolpyruvate concentration, although a 20-fold increase in this was observed when pyruvate was injected into newborn animals. 3. Analogous experiments with aspartate produced qualitatively similar differences between foetal and newborn rats. 4. When [14C]-lactate, -pyruvate or -aspartate was injected into foetal or newborn rats incorporation of radioactivity into liver glucose was observed only in the newborn animals. 5. Lactate/pyruvate ratios of 213 in foetal liver and 13·5 in the livers of newborn rats indicated a relatively reduced environment in the cytosol of foetal liver. This difference in redox state was illustrated experimentally by a greater conversion of pyruvate into lactate and an increased formation of malate in foetal liver. 6. Although both the substrate-loading and tracer experiments indicated a block in gluconeogenesis in foetal liver at the stage of conversion of oxaloacetate into phosphoenolpyruvate, gluconeogenesis was also hindered by a highly reduced environment.
The pepsins of normal human gastric juice
Authors: Etherington D J , Taylor W H
Published: August 2015

1. The frequency of occurrence, under defined conditions, of the different human pepsins in the gastric juices of 50 normal subjects was investigated by agar-gel electrophoresis. 2. From a total of eight proteolytic zones located in the zymograms, no significant differences of occurrence existed between the sexes, or between subjects with or without gastric symptoms. 3. Two zones, numbered 3 and 5, occurred in all normal gastric juices. Zone 3 always exhibited the greatest proteolytic activity, then zone 5. The remaining enzymic zones were less well-marked and occurred less frequently. 4. A minor zone, 3a, was demonstrated within zone 3. The corresponding pepsin, 3a, has a mobility towards the anode 6–7% greater than has pepsin 3. 5. Of the eight zones, 1,2,3,3a and 5, at least, represent unique pepsins.
Binding sites of cholinesterases. Alkylation by an aziridinium derivative
Authors: O'Brien R. D.
Published: August 2015

1. Alkylation of acetylcholinesterase and butyrylcholinesterase by 2-chloro-N-(chloroethyl)-N-methyl-2-phenylethylamine was observed. This alkylating agent was more potent than related compounds previously described, and less stable (half-life 8·5min. at 23°). 2. Alkylation had effects on hydrolysis of substrates varying from activation for indophenyl acetate to inhibition for acetylcholine, and intermediate effects with five other substrates. The effects were on Vmax. and not Km. 3. Alkylation caused a variety of changes in sensitivity to inhibition by five carbamates, five organophosphates and four other inhibitors, varying from total protection against tetraethylammonium to mildly enhanced sensitivity to urea. 4. The findings suggested the existence of three binding sites, one of which was anionic and another hydrophobic, in addition to the esteratic site.
Studies on the Biosynthesis of the ergosterol side chain
Authors: Akhtar M , Parvez M. A. , Hunt P. F.
Published: August 2015

1. A convenient synthesis of 24-methylene[23,25−3H3]dihydrolanosterol is described. 2. A general anaerobic–aerobic method for the incorporation of sterols into whole yeast cells is also described and illustrated by experiments with 3H-labelled lanosterol. 3. The method was used to convert labelled 24-methylene-dihydrolanosterol into ergosterol, in good yield, by Saccharomyces cerevisiae. 4. Degradation of the biosynthetic ergosterol provided confirmation of the conversion, which supports the proposed mechanism for the biosynthesis of the ergosterol side chain. 5. Mechanisms for the further conversion of the 24-methylene side chain into the ergosterol side chain are discussed and it was shown that a compound, [3α−3H1]-ergost-7-en-3β-ol, with a fully saturated side chain, can also be efficiently incorporated into ergosterol. 6. This result was confirmed by a procedure involving formation of the 5,8-epidioxide and subsequently the 5,8-epidioxy-22,23-epoxide of the biosynthetic ergosterol.
The distribution of polyphenols in the tea plant (Camellia sinensis L.)
Authors: Forrest G. I. , Bendall D S
Published: August 2015

1. Methods for the separation and determination of the polyphenolic components of the tea plant by thin-layer chromatography and colorimetric reactions have been devised. 2. High concentrations of catechins, flavonols and depsides were found to be restricted to the young vegetative and floral shoots, whereas leucoanthocyanins or flavylogens were characteristic of the more bulky axial tissues of the plant. 3. In the young shoots cell growth was correlated with an increasing degree of flavonoid B-ring hydroxylation. 4. Maximal flavylogen concentrations occurred in the outer protective layers of stem and of seed coat. 5. Mature leaves were shown to contain derivatives of the flavones apigenin and luteolin. 6. Developing seedlings showed a steady increase in polyphenol complexity; flavylogens were concentrated at shoot and root apices and accumulated at the stem base. 7. It is postulated that the flavanols (leucoanthocyanins and catechins), because they can co-polymerize, are of use to the plant for protection of wood and bark against infection and decay.
Studies on the polyphenol metabolism of tissue cultures derived from the tea plant (Camellia sinensis L.)
Authors: Forrest G. I.
Published: August 2015

1. The growth characteristics on various media of solid and liquid suspension cultures derived from the stem of the tea plant are described; chlorophyll and anthocyanin synthesis occurred in the light. 2. Only the simplest catechins and leucoanthocyanins were present in callus tissue, although oligomeric and polymeric leucoanthocyanin fractions were also represented. Light caused an increase in all monomeric components analysed, but inhibited polymerization of the leucoanthocyanins. 3. The polyphenol oxidase activity of cultures was comparable with that of the apical regions of the intact plant, and was inversely correlated with growth rate. 4. Growth was stimulated by hormonal variation, and inhibited by high concentrations of sucrose and by high light-intensity; polyphenol concentrations were generally inversely correlated with growth rate. 5. From the inability of callus tissue and of cultured root apices to synthesize complex catechins, it is inferred that complex catechin formation in intact plants is associated with the process of cell vacuolation.