Showing papers in "Biochemical Journal in 1968"
TL;DR: The apparent micellar molecular weights of the protein-sodium dodecyl sulphate complexes were measured by the dye-solubilization method; they were all found to have approximately the same miceLLar molecular weight irrespective of the molecular weight of theprotein to which they were attached.
Abstract: 1. The binding of sodium dodecyl sulphate to proteins by equilibrium dialysis was investigated. 2. Most of the proteins studied bound 90–100% of their weight of sodium dodecyl sulphate. 3. The glycoproteins studied bound 70–100% of their weight of sodium dodecyl sulphate, calculated in terms of the polypeptide moiety of the molecule. 4. Proteins not containing S·S groups bound about 140% of their weight of sodium dodecyl sulphate. 5. Reduction of four proteins containing S·S groups caused a rise in sodium dodecyl sulphate binding to 140% of the weight of protein. 6. The apparent micellar molecular weights of the protein–sodium dodecyl sulphate complexes were measured by the dye-solubilization method; they were all found to have approximately the same micellar molecular weight (34000–41000) irrespective of the molecular weight of the protein to which they were attached.
472 citations
TL;DR: The N-acetyl-β-glucosaminidase of human spleen has been separated by gel electrophoresis into two components, an acidic form A and a basic form B, and evidence is presented to indicate that the A form contains a number of sialic acid residues.
Abstract: 1. The N-acetyl-beta-glucosaminidase of human spleen has been separated by gel electrophoresis into two components, an acidic form A and a basic form B. 2. The two forms are readily separated on DEAE-cellulose and have been concentrated 50-fold and sevenfold respectively. 3. They show similar K(m) values towards 4-methylumbelliferyl N-acetyl-beta-d-glucosaminide, and have the same pH optima when compared in citrate, phosphate or acetate buffers. They are inhibited to a similar extent by acetate, heparin, N-acetylgalactosaminolactone, N-acetyl-beta-d-galactosamine and N-acetyl-beta-d-glucosamine. Specificity for C-4 orientation is not absolute and p-nitrophenyl beta-galactosaminide is also hydrolysed but at a rate only 11.6% of that for the corresponding glucosaminide. 4. N-Acetyl-beta-glucosaminidase B is stable over a wider pH range than is N-acetyl-beta-glucosaminidase A, and is less easily denatured by heat. 5. Tissue fractionation indicates that both the A and B forms are present in the lysosomal fraction, whereas the supernatant contains the A form only. 6. Evidence is presented to indicate that the A form contains a number of sialic acid residues.
438 citations
TL;DR: Increments in invertase activity, microscopic homogeneity and low nucleic acid content indicate that the microvillus plasma membrane has been extensively purified.
Abstract: 1. A technique is described for the removal of subcellular contaminants from intact rat intestinal brush borders, and for the subsequent separation of a microvillus membrane fraction from a fibrillar residue. 2. Increments in invertase activity, microscopic homogeneity and low nucleic acid content indicate that the microvillus plasma membrane has been extensively purified. Multiple membrane preparations have been shown to be highly reproducible with respect to their invertase specific activity, cholesterol content and phospholipid content. Alkaline phosphatase, leucine aminopeptidase, Mg2+- and Ca2+-dependent adenosine triphosphatase and seven separate disaccharidases were shown to be predominantly confined to the membrane fraction. 3. The fibrillar fraction has been shown to contain approximately 30% of the total protein of purified brush borders, plus most of the residual nucleic acid contaminant. No evidence was found for the localization of any specific enzyme in this fraction.
418 citations
TL;DR: Investigation of whether the use of maleylation to introduce glyoxyloyl groups (Dixon, 1968) could be extended to the introduction of pyruvoyl groups reports the effects of introducing methyl groups into the molecule of maleic anhydride.
Abstract: Butler, Harris, Hartley & Leberman (1967) showed that maleic anhydride could be used for the reversible blocking of amino groups. The maleyl group could be removed because the protonated form of the free carboxyl group catalysed the hydrolysis of the amide bond, presumably by intramolecular general acid catalysis (cf. Bender, Chow & Chloupek, 1958). The present paper reports the effects of introducing methyl groups into the molecule of maleic anhydride. 2,3-Dimethylmaleic anhydride. We had wished to investigate whether the use of maleylation to introduce glyoxyloyl groups (Dixon, 1968) could be extended to the introduction of pyruvoyl groups. We therefore treated arginine with 2,3-dimethylmaleic anhydride (Fluka A.-G., Buchs, Switzerland) as follows. To a solution of arginine hydrochloride (5M) in water was added, with stirring, dimethylmaleic anhydride to a final concentration of 5-5m, and the pH was maintained at 8 by the addition of N-NaOH. Uptake ofbase ceased after about an hour and suitable samples were then applied to paper for high-voltage electrophoresis in the buffer systems used previously (Perham, 1967). Material was detected on the paper by means of the ninhydrincadmium reagent (Heilmann, Barrollier & Watzke, 1957) and also by the Sakaguchi reaction (Jepson & Smith, 1953). It was immediately apparent that arginine alone and no product could be detected after electrophoresis at pH 3 5, but that at pH 6 5
394 citations
TL;DR: The results are discussed in connexion with the organ distribution of tumours induced by the compounds under study and in relation to the possible importance of alkylation of cellular components for the induction of cancer.
Abstract: 1. N[(14)C]-Methyl-N-nitrosourea, [(14)C]dimethylnitrosamine, [(14)C]dimethyl sulphate and [(14)C]methyl methanesulphonate were injected into rats, and nucleic acids were isolated from several organs after various time-intervals. Radioactivity was detected in DNA and RNA, partly in major base components and partly as the methylated base, 7-methylguanine. 2. No 7-methylguanine was detected in liver DNA from normal untreated rats. 3. The specific radioactivity of 7-methylguanine isolated from DNA prepared from rats treated with [(14)C]dimethylnitrosamine was virtually the same as that of the dimethylnitrosamine injected. 4. The degree of methylation of RNA and DNA produced in various organs by each compound was determined, and expressed as a percentage of guanine residues converted into 7-methylguanine. With dimethylnitrosamine both nucleic acids were considerably more highly methylated in the liver (RNA, about 1% of guanine residues methylated; DNA, about 0.6% of guanine residues methylated) than in the other organs. Kidney nucleic acids were methylated to about one-tenth of the extent of those in the liver, lung showed slightly lower values and the other organs only very low values. N-Methyl-N-nitrosourea methylated nucleic acids to about the same extent in all the organs studied, the amount being about the same as that in the kidney after treatment with dimethylnitrosamine. In each case the RNA was more highly methylated than the DNA. Methyl methanesulphonate methylated the nucleic acids in several organs to about the same extent as N-methyl-N-nitrosourea, but the DNA was more highly methylated than the RNA. Dimethyl sulphate, even in toxic doses, gave considerably less methylation than N-methyl-N-nitrosourea in all the organs studied, the greatest methylation being in the brain. 5. The rate of removal of 7-methylguanine from DNA of kidneys from rats treated with dimethylnitrosamine was compared with the rate after treatment of rats with methyl methanesulphonate. No striking difference was found. 6. The results are discussed in connexion with the organ distribution of tumours induced by the compounds under study and in relation to the possible importance of alkylation of cellular components for the induction of cancer.
361 citations
TL;DR: In this paper, simple thermodynamic expressions are used to describe the properties of uncharged binary and ternary polymer solutions, in particular the sedimentation equilibrium of binary systems and the osmotic pressures and "incompatible" phase separations.
Abstract: 1. Simple thermodynamic expressions are used to describe the properties of uncharged binary and ternary polymer solutions, in particular the sedimentation equilibrium of binary systems and the osmotic pressures and ‘incompatible’ phase separations of ternary systems. 2. Sedimentation-equilibrium experiments were performed on four samples of dextran and two of polyethylene glycol. The critical points of the phase diagrams were determined for the mixed solutions of polyethylene glycol–dextran–water and of polyethylene glycol–bovine serum albumin–0·2m-sodium chloride solution. Osmotic pressures were measured on a single-phase mixed solution of a polyethylene glycol and a dextran. By use of the simple thermodynamic expressions consistent values of second virial and interaction coefficients for the materials used were obtained from these experiments. 3. The interpretation of the values of the second virial and interaction coefficients, on the basis of three models of molecular interaction, is discussed.
326 citations
324 citations
TL;DR: In the rat ventral prostate gland the biosynthesis of putrescine, a precursor of spermidine and spermine, is shown to occur by the direct decarboxylation of l-ornithine.
Abstract: In the rat ventral prostate gland the biosynthesis of putrescine, a precursor of spermidine and spermine, is shown to occur by the direct decarboxylation of l-ornithine. Some properties of a soluble pyridoxal phosphate-dependent l-ornithine decarboxylase are described. The findings are discussed in relation to other enzymic reactions involved in the biosynthesis of polyamines by the prostate gland.
317 citations
TL;DR: It is suggested that early myelin is extruded glial plasma membrane, which only later becomes mature myelin.
Abstract: 1. The lipids of whole brain and subcellular fractions of the rat were analysed during development. 2. The deposition of cholesterol occurred in two phases, one related to increasing wet weight of the brain and the second to myelination. Cerebroside accumulation was related only to myelination. 3. The composition of myelin isolated from 12-day-old rat brain was different in some respects from that of the adult. In the former there was an increase of phospholipid in relation to cholesterol and a marked deficiency in cerebroside. 4. It is suggested that early myelin is extruded glial plasma membrane, which only later becomes mature myelin.
284 citations
TL;DR: It is concluded that, though unphysiological concentrations of K(+) may influence the secretion of insulin, fluxes ofK(+) in the islets do not appear to be important in the initiation of insulin secretion.
Abstract: 1. A method was devised for the isolation of islets of Langerhans from rabbit pancreas by collagenase digestion in order to study the influx and efflux of K(+) in islets during insulin secretion. 2. Glucose-induced insulin release was accompanied by an increased rate of uptake of (42)K(+) by the islets of Langerhans, though this was not the case for secretion in response to tolbutamide. Ouabain significantly inhibited the uptake of (42)K(+) by islet tissue. 3. No significant increase in the rate of efflux of (42)K(+) was demonstrated during active insulin secretion. 4. Slices of rabbit pancreas were incubated in media of different K(+) content, and rates of insulin release were determined. Alteration of the K(+) concentration of the medium between 3 and 8mm had no effect on the rate of insulin release by pancreas slices. However, decrease of the K(+) concentration to 1mm resulted in inhibition of secretion in response to both glucose and to tolbutamide. Conversely, an increase in K(+) concentration increased rates of insulin release in response to both these stimuli. 5. It is concluded that, though unphysiological concentrations of K(+) may influence the secretion of insulin, fluxes of K(+) in the islets do not appear to be important in the initiation of insulin secretion.
281 citations
TL;DR: Binding of a transition metal ion to specific sites in concanavalin A induces the formation of specific Ca(2+) ion-binding sites.
Abstract: Binding of a transition metal ion to specific sites in concanavalin A induces the formation of specific Ca2+ ion-binding sites. Sites for binding α-methyl d-glucopyranoside exist only when a transition metal ion and Ca2+ ion are bound.
TL;DR: Chromatographic analysis suggests that bilirubin monoglucuronide is the product of the enzyme systems studied, and this method has been used in the development of assays for UDP-glucuronyltransferase.
Abstract: 1. Conjugated bilirubin is conveniently determined by coupling with the diazonium salt of ethyl anthranilate. 2. This method has been used in the development of assays for UDP-glucuronyltransferase (EC 2.4.1.17), with bilirubin as substrate, in rat liver homogenates, microsomal preparations and partly purified fractions. 3. Chromatographic analysis suggests that bilirubin monoglucuronide is the product of the enzyme systems studied.
TL;DR: The subcellular distributions of glutamate decarboxylase and aspartate transaminase were studied in rat and guinea-pig brain as two isoenzymes with different kinetic properties and the release and binding of the enzyme to membrane fractions by Ca(2+) were investigated.
Abstract: 1. The subcellular distributions of glutamate decarboxylase and aspartate transaminase were studied in rat and guinea-pig brain. 2. Glutamate decarboxylase is localized in the synaptosome fraction. The mean density of the particles containing the enzyme is slightly greater than those derived from cholinergic neurones, though overlap is substantial. 3. The enzyme is readily released from synaptosomes by hypo-osmotic treatment, but in the presence of Ca2+, Na+ and K+ it sediments with particulate material. 4. The release and binding of the enzyme to membrane fractions by Ca2+ were investigated. 5. Aspartate transaminase is present in brain as two isoenzymes with different kinetic properties. One isoenzyme is associated with the cytoplasm and the other with mitochondria.
TL;DR: It is brought out that the light-scattering method fails to distinguish between changes in sucrose space and in Sucrose-inaccessible space, which in the present experiments could be altered respectively by phosphate (in high K(+) solution) and by cation uptake induced by antibiotic.
Abstract: 1. Total water exchangeable with tritiated water and sucrose space were measured in rat liver mitochondria during the uptake of K+ induced by valinomycin and the release caused by nigericin. The K+ content and the sucrose-inaccessible water rose and fell together. 2. Swelling resulting from phosphate addition in a medium of high K+ concentration was associated mainly with increased sucrose-accessible water, which carried dissolved K+. This change was reversed by addition of ATP. 3. The response of the sucrose-inaccessible space to changed osmolarity was qualitatively that expected if the mitochondrial K+ is assumed to be present in this space with a univalent anion. 4. It is brought out that the light-scattering method fails to distinguish between changes in sucrose space and in sucrose-inaccessible space, which in the present experiments could be altered respectively by phosphate (in high K+ solution) and by cation uptake induced by antibiotic.
TL;DR: The enzyme was isolated from leaves of tropical grasses that catalyses the reversible conversion of pyruvate, ATP and orthophosphate into phosphopyruvates, AMP and pyrophosphate and its role in photosynthesis is discussed.
Abstract: 1. An enzyme was isolated from leaves of tropical grasses that catalyses the reversible conversion of pyruvate, ATP and orthophosphate into phosphopyruvate, AMP and pyrophosphate. A requirement for Mg(2+) could not be replaced by Mn(2+) or Ca(2+). 2. By replacing orthophosphate with [(32)P]orthophosphate or with arsenate, evidence was provided that the orthophosphate consumed appears in pyrophosphate. 3. Without Mg(2+) or 2-mercaptoethanol the enzyme was rapidly and irreversibly inactivated. EDTA only partially replaced the requirement for the thiol compound. The enzyme was considerably more unstable at 0 degrees or when frozen than at 22 degrees . Even with the best conditions devised the enzyme lost about 25% of its activity every 3hr. 4. The activities of the enzyme in leaves of the tropical grasses sugar cane (Saccharum hybrid var. Pindar), maize (Zea mays) and sorghum (Sorghum vulgare) were comparable with their maximum photosynthesis rates. The enzyme was not detectable in leaf extracts from several other plants. 5. Its role in photosynthesis is discussed.
TL;DR: The binding of non-occluded choline acetyltransferase to synaptosome membranes is a reversible process that is primarily dependent on the pH and ionic strength of the suspending medium and Varying the temperature or prolonged hypo-osmotic treatment does not release the membrane-bound enzyme.
Abstract: 1. The binding of non-occluded choline acetyltransferase to synaptosome membranes is a reversible process that is primarily dependent on the pH and ionic strength of the suspending medium. 2. The distribution of soluble enzyme bound to synaptosome membranes was studied by density-gradient centrifuging. 3. Choline acetyltransferase shows enzyme activity both in the free and in the membrane-bound form. 4. Varying the temperature or prolonged hypo-osmotic treatment does not release the membrane-bound enzyme. 5. The release of choline acetyltransferase from membranes by different anions, thiols, adenosine nucleotides and enzyme substrates was studied.
TL;DR: Diquat can stimulate NADH oxidase and NADPH oxidase activity in the postmicrosomal soluble fraction and their significance in relation to the toxicity of the dipyridilium compounds are discussed.
Abstract: 1. Paraquat and diquat produce only a slight increase in the oxygen uptake of rat liver mitochondria, and it is likely that they do not penetrate the mitochondrial membrane. 2. In mitochondrial fragments inhibited by antimycin A or by Amytal, both substances stimulate oxygen uptake with NADH or β-hydroxybutyrate as substrate but not with succinate. The NADH dehydrogenase of the respiratory chain appears to be involved, at a site only partially inhibited by Amytal. 3. An NADPH oxidase activity is stimulated in rat liver microsomes by diquat, and to a smaller extent by paraquat; diquat also causes an NADH oxidase activity to develop. The effect is not inhibited by carbon monoxide or p-chloromercuribenzoate, and it is probable that a flavoprotein is involved by a mechanism not requiring thiol groups. 4. One molecule of oxygen can oxidize two molecules of NADPH in the stimulated microsomal system, the hydrogen peroxide produced being broken down by a catalase activity in the microsomes. 5. Diquat can stimulate NADH oxidase and NADPH oxidase activity in the postmicrosomal soluble fraction; the enzyme involved may be DT-diaphorase. 6. The mechanism of these reactions and their significance in relation to the toxicity of the dipyridilium compounds are discussed.
TL;DR: C-substance from seven other strains of Pneumococcus possesses a structure common to that described for the strain 1-192R, and in the remaining seven cases the contaminating C- Substance was probably similarly constituted.
Abstract: 1 Pneumococcal C-substance was isolated from the non-capsulated Pneumococcus 1–192R, ATCC 12213, by extraction with trichloroacetic acid solution followed by chromatography on DEAE-cellulose (HCO3− form) 2 The polymer contains 7·0% of phosphorus and 6·0% of nitrogen and is composed of phosphate, N-acetyl-d-galactosamine, d-glucose, N-acetyldiaminotrideoxyhexose, ribitol and choline in the molecular proportions 2:1:1:1:1:1 3 After acid hydrolysis, d-galactosamine hydrochloride and galactosamine 6-phosphate were isolated in crystalline form and crystalline derivatives of d-glucose and anhydroribitol were obtained A product of partial acid hydrolysis was provisionally characterized as 6′-O-phosphoryl-[O-β-d-galactosaminyl-(1′→6)-d-glucose] 4 C-substance contains free amino groups accessible to attack by 1-fluoro-2,4-dinitrobenzene and nitrous acid 5 Choline phosphate and ribitol phosphate are units in the polymer 6 Treatment with hot alkali gave a fragment comprising phosphate, d-galactosamine, d-glucose, diaminotrideoxyhexose and ribitol in the molecular proportions 2:1:1:1:1 7 After selective N-acetylation, the fragment contained one of its phosphate groups as a phosphomonoester and one as a phosphodiester, shown by potentiometric titration and by treatment with a phosphomonoesterase 8 C-substance from seven other strains of Pneumococcus possesses a structure common to that described for the strain 1–192R 9 Capsular materials from 26 different strains of Pneumococcus were analysed for suspected contamination by C-substance In 19 cases the presence of C-substance with the normal structure was demonstrated, and in the remaining seven cases the contaminating C-substance was probably similarly constituted 10 F-substance was isolated and the associated fatty acid material analysed
TL;DR: The biosynthesis of the mannan polymer in the yeast cell wall is discussed with regard to the two types of carbohydrate-amino acid linkages found in the glycoprotein.
Abstract: 1. Glycopeptides containing mannose were extracted from isolated yeast cell walls by ethylenediamine and purified by treatment with Pronase and fractionation on a Sephadex column. 2. A glycopeptide that appeared homogeneous on electrophoresis and ultracentrifugation had a molecular weight of 76000, and contained a high-molecular-weight mannan and approx. 4% of amino acids. 3. The amino acid composition of the peptide was determined. It was rich in serine and threonine and also contained glucosamine. No cystine and methionine were detected. 4. The glycopeptide underwent a β-elimination reaction when treated with dilute alkali at low temperatures. The reaction resulted in the release of mannose, mannose disaccharides and possibly other low-molecular-weight mannose oligosaccharides. During the β-elimination reaction the dehydro derivatives of serine and threonine were formed. One of the linkages between carbohydrate and amino acids in the glycopeptide is an O-mannosyl bond from mannose and mannose oligosaccharides to serine and threonine. 5. After the β-elimination reaction the bulk of the mannose in the form of the large mannan component was still covalently linked to the peptide. This polysaccharide was therefore attached to the amino acids by a linkage different from the O-mannosyl bonds to serine and threonine that attach the low-molecular-weight sugars. 6. Mannan was prepared from the glycopeptide and from the yeast cell wall by treatment of the fractions with hot solutions of alkali. The mannan contained aspartic acid and glucosamine and some other amino acids. The aspartic acid and glucosamine were present in equimolar amounts; the aspartic acid was the only amino acid present in an amount equivalent to that of glucosamine. Thus there is the possibility of a linkage between the mannan and the peptide via glucosamine and aspartic acid. 7. Mannose 6-phosphate was shown to be part of the mannan structure. Information about the structure of the mannan and the linkage of the glucosamine was obtained by periodate oxidation studies. 8. The glucosamine present in the glycopeptide could not be released by treatment with an enzyme preparation obtained from the gut of Helix pomatia. This enzyme released glucosamine from the intact cell wall. Thus there are probably at least two polymers containing glucosamine in the cell wall. 9. The biosynthesis of the mannan polymer in the yeast cell wall is discussed with regard to the two types of carbohydrate–amino acid linkages found in the glycoprotein.
TL;DR: The similarity of the choline-uptake process in synaptosomes to that in erythrocytes and cortex slices indicates that thesynaptosome limiting membrane is functionally competent in this respect.
Abstract: 1. The uptake of [(14)C]choline into synaptosomes in vitro was investigated by a gel-filtration method. Synaptosomes incubated in a medium fortified with glucose and succinate rapidly take up [(14)C]choline. 2. A substantial proportion of the radioactivity taken up can be released by osmotic shock, and is recoverable as choline on a thin-layer chromatogram. This suggests that choline is taken up across the limiting membrane into the cytoplasmic compartment of the synaptosome. 3. The concentration of choline in the synaptosome has a dependence on the external concentration of choline that is similar to that in erythrocytes and mouse cerebral-cortex slices. The choline influx has two components, one that is linear and one that is saturable with increasing choline concentration. 4. Omission of Na(+) from the incubation medium, or addition of 100mm-K(+), inhibits choline uptake. Hemicholinium no. 3 is a powerful inhibitor of the choline uptake. 5. The similarity of the choline-uptake process in synaptosomes to that in erythrocytes and cortex slices indicates that the synaptosome limiting membrane is functionally competent in this respect.
TL;DR: It is concluded that variations in the concentrations of enzymes involved in acetoacetate synthesis play no major role in the regulation of ketone-body formation in starvation and alloxan-diabetes.
Abstract: 1. The activities of hydroxymethylglutaryl-CoA synthase and lyase in rat liver were found to be two- to 15-fold greater than those reported by other authors under similar conditions. 2. When expressed on the basis of body weight, no appreciable differences were found between the activities of hydroxymethylglutaryl-CoA synthase in whole homogenates of livers from normal and starved rats. The synthase activity increased by 70% and 140% in livers of alloxan-diabetic rats and rats fed on a high-fat diet respectively. 3. Hydroxymethylglutaryl-CoA lyase activity showed no significant increases in starvation or alloxan-diabetes, but a 40% increase was found in fat-fed rats. 4. Less than 12% of the activities of both enzymes were found in the cytoplasmic fraction of normal liver. The cytoplasmic activity doubled in alloxan-diabetes and starvation; on feeding with a high-fat diet the increase, though significant, was less marked. 6. The intracellular distribution of glutamate dehydrogenase indicated that the changes in the cytoplasmic activities observed were not due to leakage from the mitochondria. 7. Feeding with a normal or high-fat diet after 48hr. starvation caused within 24hr. a decrease in the cytoplasmic activity of hydroxymethylglutaryl-CoA synthase to values lower than those found in rats fed on a corresponding diet for a longer period of time. 8. Acetoacetyl-CoA deacylase activity in liver was about 20% of that of hydroxymethylglutaryl-CoA synthase and was primarily located in the cytoplasm. Starvation or alloxan-diabetes did not alter the acetoacetyl-CoA deacylase activity. 9. It is concluded that variations in the concentrations of enzymes involved in acetoacetate synthesis play no major role in the regulation of ketone-body formation in starvation and alloxan-diabetes. The changes in the cytoplasmic activities of hydroxymethylglutaryl-CoA synthase and lyase suggest that acetoacetate synthesis can occur in the cytoplasm. This may play a role in the disposal of surplus acetyl-CoA arising in the cytoplasm when lipogenesis is inhibited.
TL;DR: A sequence of events for collagen fibrogenesis in vivo is proposed based on kinetic experiments and results found that aggregates of chondroitin sulphate-protein and tropocollagen form instantaneously and that these act as sites for the second stage of precipitation of fibrils.
Abstract: 1. It was found that the precipitation of collagen fibrils at 37° from mixtures of chondroitin sulphate–protein and tropocollagen at physiological ionic strength and pH takes place in two distinct phases. The first occurs immediately on mixing either at 4° or at 37°, and the second occurs only at 37° and after a lag phase whose magnitude depends on the proportions of components. 2. When the second stage of precipitation was inhibited by mixing the reactants at 4°, the initial precipitate was found to contain `native-type' collagen fibrils and chondroitin sulphate–protein. 3. On the basis of kinetic experiments it was concluded that aggregates of chondroitin sulphate–protein and tropocollagen form instantaneously and that these act as sites for the second stage of precipitation of fibrils. 4. The gels that result after continued incubation at 37° are fibrous in appearance if formed in the presence of the initial precipitate of chondroitin sulphate–protein and tropocollagen. 5. On the basis of these experiments in vitro the authors propose a sequence of events for collagen fibrogenesis in vivo.
TL;DR: Removal of a low-molecular-weight carboxymethylcellulase (CM- cellulase) component by chromatography on Sephadex G-75 had no effect on the ability of the enzyme complex to solubilize cotton.
Abstract: 1 Cell-free culture filtrates from Trichoderma koningii were concentrated by precipitation with ammonium sulphate between the limits of 20% and 80% saturation 2 Removal of a low-molecular-weight carboxymethylcellulase (CM-cellulase) component by chromatography on Sephadex G-75 had no effect on the ability of the enzyme complex to solubilize cotton 3 Further chromatography on DEAE-Sephadex separated a component (C1) from the Cx (CM-cellulase) and β-glucosidase activities Separately these components had little ability to produce soluble sugars from cotton, but when recombined in their original proportions this capacity was almost completely recovered 4 The Cx component was further fractionated on SE-Sephadex into a fraction containing only CM-cellulase and a fraction showing CM-cellulase and β-glucosidase activities: the latter two components could be separated by heat treatment 5 The C1 component had no swelling factor (S-factor) activity (Marsh, Merola & Simpson, 1953; Reese & Gilligan, 1954) on its own, but it had a synergistic effect on the S-factor activity associated with the CM-cellulase and β-glucosidase components
TL;DR: It is concluded that enzyme-substrate and enzyme-modulator interactions are important factors in short-term and in evolutionary adaptations by poikilotherms to changes in temperature.
Abstract: 1. The effects of temperature on the catalytic and regulatory properties of pyruvate kinases from the temperate-zone rainbow trout and the Antarctic fish Trematomus bernacchii were examined. 2. The K(m) value of pyruvate kinase for one of its two substrates, phosphoenolpyruvate, is temperature-dependent, and is lowest at temperatures that closely coincide with the habitat temperatures of the two fishes. 3. Two regulatory functions of pyruvate kinase, feedforward activation by fructose diphosphate and feedback inhibition by ATP, are temperature-independent. Enzyme-ADP interaction is also temperature-independent. 4. It is concluded that enzyme-substrate and enzyme-modulator interactions are important factors in short-term and in evolutionary adaptations by poikilotherms to changes in temperature. Though the K(m) for substrate may vary in apparently adaptive manners, the regulatory functions of an enzyme appear to be unchanged over the range of temperatures experienced by the organism in Nature.
TL;DR: After growth on glucose, deacetylaseless mutants have a repressed glucosamine 6-phosphate synthetase and a super-induced glucosamines 6- phosphate deaminase; this may be related to an intracellular accumulation of acetylamino sugar that also occurs under these conditions.
Abstract: 1. Growth of Escherichia coli on glucosamine results in an induction of glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] and a repression of glucosamine 6-phosphate synthetase (l-glutamine–d-fructose 6-phosphate aminotransferase, EC 2.6.1.16); glucose abolishes these control effects. 2. Growth of E. coli on N -acetylglucosamine results in an induction of N -acetylglucosamine 6-phosphate deacetylase and glucosamine 6-phosphate deaminase, and in a repression of glucosamine 6-phosphate synthetase; glucose diminishes these control effects. 3. The synthesis of amino sugar kinases (EC 2.7.1.8 and 2.7.1.9) is unaffected by growth on amino sugars. 4. Glucosamine 6-phosphate synthetase is inhibited by glucosamine 6-phosphate. 5. Mutants of E. coli that are unable to grow on N -acetylglucosamine have been isolated, and lack either N -acetylglucosamine 6-phosphate deacetylase (deacetylaseless) or glucosamine 6-phosphate deaminase (deaminaseless). Deacetylaseless mutants can grow on glucosamine but deaminaseless mutants cannot. 6. After growth on glucose, deacetylaseless mutants have a repressed glucosamine 6-phosphate synthetase and a super-induced glucosamine 6-phosphate deaminase; this may be related to an intracellular accumulation of acetylamino sugar that also occurs under these conditions. In one mutant the acetylamino sugar was shown to be partly as N -acetylglucosamine 6-phosphate. Deaminaseless mutants have no abnormal control effects after growth on glucose. 7. Addition of N -acetylglucosamine or glucosamine to cultures of a deaminaseless mutant caused inhibition of growth. Addition of N -acetylglucosamine to cultures of a deacetylaseless mutant caused lysis, and secondary mutants were isolated that did not lyse; most of these secondary mutants had lost glucosamine 6-phosphate deaminase and an uptake mechanism for N -acetylglucosamine. 8. Similar amounts of 14 C were incorporated from [1− 14 C]-glucosamine by cells of mutants and wild-type growing on broth. Cells of wild-type and a deaminaseless mutant incorporated 14 C from N -acetyl[1− 14 C]glucosamine more efficiently than from N [1− 14 C]-acetylglucosamine, incorporation from the latter being further decreased by acetate; cells of a deacetylaseless mutant showed a poor incorporation of both types of labelled N -acetylglucosamine.
TL;DR: Details of an improved method for starch-gel electrophoresis of water-soluble muscle proteins are given, and a detailed comparison of the mobilities of various enzymes in extracts of muscles from a wide variety of species was made.
Abstract: 1. Details of an improved method for starch-gel electrophoresis of water-soluble muscle proteins are given. 2. Methods are described for detecting enzyme activities on the starch gel after electrophoresis, by using pieces of filter paper. 3. Compositions of incubation mixtures suitable for detecting any of the enzymes of glycolysis, and certain other enzymes, are given. 4. A comparison of the various enzymes in extracts of several muscles from one rabbit was made; most differences are quantitative only. 5. A detailed comparison of the mobilities of various enzymes in extracts of muscles from a wide variety of species was made. Each species was found to have a characteristic pattern of proteins on the starch gel, and the mobilities of individual enzymes varied considerably. 6. Potential uses and extensions of the methods are discussed.
TL;DR: The results suggest that, in the rabbit, the rate-limiting step is the reduction of cytochrome P-450, and in the rat the difference in activities can be explained by differences in the concentration of cy tochromeP-450.
Abstract: Mixed-function oxidase activity, when measured by the N-demethylation of ethylmorphine or the hydroxylation of aniline, is significantly higher in the smooth hepatic endoplasmic reticulum than in the rough. In the rabbit the smooth membrane/rough membrane activity ratios are significantly greater than 1 whether the activities are expressed per g. of liver (ratio 5), per mg. of protein (ratio 3-5), per mug. of phospholipid phosphorus (ratio 2), per unit of cytochrome P-450 (ratio 1.7) or per unit of NADPH-cytochrome c reductase activity (ratio 2). On the other hand, if the activities are normalized to the NADPH-cytochrome P-450 reductase, there is no significant difference between the rough and smooth membranes. These results suggest that, in the rabbit, the rate-limiting step is the reduction of cytochrome P-450. In contrast, in the rat the difference in activities can be explained by differences in the concentration of cytochrome P-450.
TL;DR: The metabolic pathways of aromatic-ring fission were examined in a range of fungal genera that utilize several compounds related to lignin, suggesting the existence of a ;meta' type of ring cleavage among fungi.
Abstract: 1. The metabolic pathways of aromatic-ring fission were examined in a range of fungal genera that utilize several compounds related to lignin. 2. Most of the genera, after growth on p-hydroxybenzoate, protocatechuate or compounds that are degraded to the latter (e.g. caffeate, ferulate or vanillate), rapidly oxidized these compounds, but not catechol. 3. Such genera possessed a protocatechuate 3,4-oxygenase and accumulated beta-carboxymuconate as the product of protocatechuate oxidation. This enzyme had a high pH optimum in most organisms; the Rhodotorula enzyme was competitively inhibited by catechol. 4. beta-Carboxymuconate was converted by all competent fungi into beta-carboxymuconolactone, which was isolated and characterized. None of the fungi produced or utilized at significant rates the corresponding bacterial intermediate gamma-carboxymuconolactone. 5. The lactonizing enzymes of Rhodotorula and Neurospora crassa had a pH optimum near 5.5 and approximate molecular weights of 19000 and 190000 respectively. 6. The fungi did not degrade the isomeric (+)-muconolactone, gamma-carboxymethylenebutanolide or beta-oxoadipate enol lactone at significant rates, and thus differ radically from bacteria, where beta-oxoadipate enol lactone is the precursor of beta-oxoadipate in all strains examined. 7. The end product of beta-carboxymuconolactone metabolism by extracts was beta-oxoadipate. 8. Evidence for a coenzyme A derivative of beta-oxoadipate was found during further metabolism of this keto acid. 9. A few anomalous fungi, after growth on p-hydroxybenzoate, had no protocatechuate 3,4-oxygenase, but possessed all the enzymes of the catechol pathway. Catechol was detected in the growth medium in one instance. 10. A strain of Penicillium sp. formed pyruvate but no beta-oxoadipate from protocatechuate, suggesting the existence also of a ;meta' type of ring cleavage among fungi.
TL;DR: Electrostatic binding of acid mucopolysaccharide-proteins to collagen may be an important factor in the organization and functioning of connective tissues at all stages of growth and development.
Abstract: 1. The effects of acid mucopolysaccharides and acid mucopolysaccharide-proteins on the size and rate of formation of fibril aggregates from collagen solutions in pH7.6 buffers were studied by turbidimetric and light-scattering methods. 2. Serum albumin, orosomucoid, methylated cellulose, chondroitin sulphate A and chondroitin sulphate C of molecular weight less than 20000, and hyaluronate of molecular weight less than 40000 did not influence rates of fibril formation. Chondroitin sulphate A, chondroitin sulphate C and hyaluronate of high molecular weight retarded the rate of fibril formation. This effect of high-molecular-weight chondroitin sulphate C decreased with increasing ionic strength. Heparin, though of low molecular weight (13000), was highly effective, as was also heparitin sulphate. The chondroitin sulphate-proteins of very high molecular weight were highly effective, despite the fact that for some preparations the component chondroitin sulphate chains had molecular weights much less than 20000. 3. Agents that had delayed fibril formation were also effective in producing an increase in degree of aggregation of fibrillar collagen, as indicated by dissymmetry changes observed in light-scattering experiments at low collagen concentrations. Methylated cellulose and heparin at 2.5mug./ml. were unusual in decreasing aggregation, but heparin at 0.25mug./ml. increased aggregation. Electron microscopy of gels showed fibrils and fibril aggregates with ;normal' collagen spacing and dimensions consistent with the light-scattering results. 4. The rates of electrical transport of agents and of solvent (electro-osmosis) through collagen gels indicated a contribution of molecular entanglement that increased with increase in molecular size of the agents. Electrostatic binding of heparin to collagen was noted. Binding to collagen during fibril formation was also found for heparitin sulphate and a chondroitin sulphate with extra sulphate groups. 5. Electrostatic binding of acid mucopolysaccharide-proteins to collagen may be an important factor in the organization and functioning of connective tissues at all stages of growth and development. Excluded-volume (molecular-entanglement) effects may also be important. These factors operate simultaneously and interact mutually so that precise assessment of their relative importance is difficult.