bluuebunny/crossref_metadata_2025 huggingface dataset:
The binding of dihydronicotinamide–adenine dinucleotide and pyridine-3-aldehyde–adenine dinucleotide by yeast alcohol dehydrogenase
Authors: Dickinson F. M.
Published: August 2015

1. Yeast alcohol dehydrogenase has been found to react with NADH in the presence of acetamide to form a highly fluorescent ternary complex. Titration of the enzyme to form this complex has provided a method for the estimation of the number of binding sites on the enzyme. 2. The binding of NADH by the enzyme has been studied independently, with a modified form of equilibrium dialysis, by using gel filtration. 3. A third method, depending upon the formation of a ternary complex of enzyme, hydroxylamine and pyridine-3-aldehyde–adenine dinucleotide, has also been used to titrate the enzyme. 4. Values obtained with all three methods are substantially in agreement that only three coenzyme-binding sites are available. This is in contrast with the established fact that the enzyme is composed of four identical subunits.
Selective precipitation of ribonucleic acid from a mixture of total cellular nucleic acids extracted from cultured mammalian cells
Authors: Harrison P. R.
Published: August 2015

A simple and reproducible method is described for precipitating RNA selectively from total mammalian-cell nucleic acids extracted by the phenol–sodium dodecyl sulphate procedure at pH8.0. Under specified conditions bulk RNA is precipitated almost quantitatively whereas bulk DNA remains in solution. Minor components of RNA (detected by pulse-labelling and chromatography on methylated albumin–kieselguhr) and rapidly labelled components of DNA containing single-stranded regions are also precipitated. The usefulness of the method is discussed in the context of isolating separately both RNA and DNA from cultured cells that are difficult to obtain in quantity.
Studies on energy-linked reactions. Energy-linked transhydrogenase reaction in Escherichia coli
Authors: Sweetman A. J. , Griffiths D. E.
Published: August 2015

1. Small particles from Escherichia coli catalyse an ATP-dependent reduction of NADP+ by NADH. 2. The reaction was stimulated by Mg2+ and only ATP and ITP could serve as energy donors. 3. Studies of the stoicheiometry of the reaction indicate that 1–2mol of ATP are utilized/mol of NADH produced. 4. The reaction is inhibited by uncoupling agents such as 2,4-dinitrophenol (200μm) and tetrachlorotrifluorobenzimidazole (2μm) but oligomycin does not inhibit. 5. The reaction is inhibited by relatively high concentrations (200μm) of piericidin A.
The influence on platelet aggregation of drugs that affect the accumulation of adenosine 3′:5′-cyclic monophosphate in platelets
Authors: Mills D. C. B. , Smith J. B.
Published: August 2015

1. The involvement of intracellular 3′:5′-cyclic AMP in the inhibition of platelet aggregation by prostaglandin E1, isoprenaline and adenosine has been examined by a radiochemical technique. Platelet-rich plasma was incubated with radioactive adenine to incorporate 14C radioactivity into platelet nucleotides. Pairs of identically treated samples were taken, one for the photometric measurement of platelet aggregation induced by ADP, the other for estimation of the radioactivity of 3′:5′-cyclic AMP. 2. Theophylline, papaverine, dipyridamole and 2,6-bis-(diethanolamino)-4-piperidinopyrimido[5,4d]pyrimidine (compound RA233) were found to inhibit 3′:5′-cyclic AMP phosphodiesterase from platelets. At concentrations of 3′:5′-cyclic AMP greater than 50μm the most active inhibitor was dipyridamole; at 3′:5′-cyclic AMP concentrations less than 19μm, papaverine and compound RA233 were more active than dipyridamole. 3. In the presence of compound RA233 (50μm), the effectiveness of prostaglandin E1 as an inhibitor of platelet aggregation was increased tenfold. Compound RA233 also increased the stimulation by prostaglandin E1 of the incorporation of radioactivity into 3′:5′-cyclic AMP. 4. Compound RA233 (50μm) increased the effectiveness of both adenosine and 2-chloroadenosine as inhibitors of aggregation by 70–100-fold, and in the presence of compound RA233 both adenosine and 2-chloroadenosine stimulated the incorporation of radioactivity into 3′:5′-cyclic AMP; the extent of the stimulation was proportional to the logarithm of the nucleoside concentration. 5. Compound RA233 (100–500μm) inhibited platelet aggregation by itself and caused small increases in the radioactivity of 3′:5′-cyclic AMP. Partial positive correlations were found between the radioactivity of 3′:5′-cyclic AMP in platelets measured at the time of addition of the aggregating agent (ADP) and the extent to which the aggregation was inhibited. 6. The results are interpreted as indicating that adenosine, 2-chloroadenosine, isoprenaline, prostaglandin E1 and drugs that inhibit platelet 3′:5′-cyclic AMP phosphodiesterase all inhibit aggregation by a common mechanism involving intracellular 3′:5′-cyclic AMP.
Glycopeptides from human χ-chains
Authors: Milstein Celia P. , Milstein C.
Published: August 2015

Glycopeptides have been isolated from tryptic digests of κ-type light chains separated from human myeloma proteins obtained from the serum of two patients, Car and Rai. The glycopeptides are derived from the variable region of the chain in both cases, but from different sections. On the basis of homology it is deduced that glycopeptide from Car, κI type, is derived from position 25–31 whereas that from Rai, κII type, is from position 62–77, their sequences being respectively Ala-Ser-Gln-Asn-Ile-Ser and Phe-Ser-Gly-Ser-Gly-Ser-Gly(Thr,Asp)Phe-Thr-Leu-Asx-Ile-Ser-Arg. The significance of the results is discussed in connexion with the nature of the attachment site of carbohydrate to protein.
Nature of the interaction of chondroitin 4-sulphate and chondroitin sulphate–proteoglycan with collagen
Authors: Öbrink Björn , Wasteson Åke
Published: August 2015

The electrostatic interaction of chondroitin sulphate and the chondroitin sulphate–proteoglycan with collagen was studied by chromatography of the glycosaminoglycan and the proteoglycan on a collagen gel. The observed binding between the macromolecules increased with decreasing pH and ionic strength, and was significant under physiological conditions. A study of the interaction between chondroitin sulphate and a preparation of soluble collagen, with a partition-equilibrium technique, afforded similar results.
Heterogeneity of protein–polysaccharides of porcine articular cartilage. The sequential extraction of chondroitin sulphate–proteins with iso-osmotic neutral sodium acetate
Authors: Brandt Kenneth D. , Muir Helen
Published: August 2015

Protein–polysaccharides of knee-joint cartilage of 9-month-old pigs were extracted sequentially with neutral iso-osmotic sodium acetate after five repeated homogenizations. One-third of the uronic acid originally present in the tissue was brought into solution, about half being in the first extract. The protein–polysaccharides, which were purified by precipitation with 9-aminoacridine, were heterogeneous in size on gel chromatography. The smallest (retarded by 6% agarose) were the most easily extracted since they were most prevalent in the initial extracts and absent from later ones, whereas the proportion of larger molecules increased progressively in successive extracts. Nevertheless a small proportion of the largest molecules (excluded from Sepharose 2B) was present even in the first extract. None of the protein–polysaccharide preparations contained hydroxyproline, and the analyses of their constituent sugars were the same, although there was a progressive increase in the protein content and in the glucosamine/galactosamine molar ratio of successive extracts. In each preparation this molar ratio was invariably greater in larger than in smaller molecules separated by gel filtration. From galactosamine/pentose molar ratios it appeared that the chondroitin sulphate chains were on average about 29 disaccharide units in length in the protein–polysaccharides of each extract, although gel-chromatography and cetylpyridinium chloride elution profiles showed that a somewhat higher proportion of shorter chondroitin sulphate chains occurred in the larger protein–polysaccharides. In the last extract, where the largest molecules predominated, about half could be reversibly dissociated by urea, whereas this had no effect on the protein–polysaccharides of earlier extracts even though these contained some large molecules.
Polyribosomal attachment to rat liver and hepatoma endoplasmic reticulum in vitro. A method for its study
Authors: Ragland W. L. , Shires T. K. , Pitot H. C.
Published: August 2015

A system for study and measurement of the attachment in vitro of exogenous polyribosomes to membranes has been presented. Its main features are use of low temperature, post-microsomal supernatant, pyrophosphate and citric acid to remove ribosomes from the surface of rough endoplasmic reticulum, and a method for quantitative separation of unattached from membrane-associated polyribosomes. The following were found. (1) Rough endoplasmic reticulum, from which ribosomes had been removed by treatment with pyrophosphate and citrate, bound over 50% of added polyribosomes, whereas the untreated (or control) rough and smooth endoplasmic reticulum and the smooth endoplasmic reticulum treated with pyrophosphate–citrate did not bind polyribosomes. (2) The polyribosome-binding capacity of rough endoplasmic reticulum stripped of its ribosomes decayed upon storage of the membranes at 0–4°C. The half-life of this decay was about 6 days whereas that of the polyribosome-binding capacity of hepatoma stripped rough endoplasmic reticulum was about 1.5 days. (3) Preparations of stripped rough endoplasmic reticulum after reassociation with polyribosomes in vitro were quite similar to preparations of native rough endoplasmic reticulum as viewed with the electron microscope. Evidence is presented to support the contention that association of polyribosomes with membranes was the result of polyribosomal reattachment to the membranes rather than trapping of the polyribosomes between vesicles of the membranes.
The cellulase of Fusarium solani. Purification and specificity of the β-(1→4)-glucanase and the β-d-glucosidase components
Authors: Wood T. M.
Published: August 2015

1. Cell-free culture filtrates of the fungus Fusarium solani were examined for homogeneity with respect to β-d-glucosidase and Cx activities. 2. o-Nitrophenyl β-d-glucoside and cellobiose were both used as substrates for β-d-glucosidase activity. 3. No evidence for the non-identity of nitrophenyl β-d-glucosidase and cellobiase activities could be found, either by heat treatment, gel filtration on Sephadex G-100 or by isoelectric focusing. 4. The β-d-glucosidase component was also a feeble exo-β-glucanase: it had a molecular weight of approx. 400000. 5. The fall in viscosity of a solution of CM-cellulose, the formation of reducing sugars in a solution of CM-cellulose and the solubilization of phosphoric acid-swollen cellulose (Walseth cellulose), were all used for the measurement of Cx activity. 6. The ratio of the two types of CM-cellulase activity was not changed after gel filtration on Sephadex G-100 or after chromatography on DEAE-Sephadex. 7. Three peaks of Cx activity were obtained after electrofocusing, but all three possessed the same ratio of the two types of CM-cellulase activity as well as the same CM-cellulase/Walseth activity ratio, as the unfractionated enzyme; all three isoenzymes (isoelectric points, 4.75, 4.80–4.85 and 5.15) acted in synergism with a mixture of the C1 and the β-d-glucosidase components to the same extent in the solubilization of cotton fibre. 8. The molecular weight of the Cx component was approx. 37000.
Metabolism of camphanediols
Authors: Robertson J. S. , Solomon E.
Published: August 2015

1. The metabolism of the camphane-2,3-diols and camphane was investigated in rabbits. All the compounds increased the content of glucuronide in the urine. 2. Both trans- and cis-diols gave rise to ketols; the cis-diols gave rise to trans-diols, but not vice versa; camphorquinone gave cis-diol, ketols and trans-diol; camphane did not give rise to diols. 3. The possibility is discussed that an enediol is the intermediate for ketol interconversions in the present series of compounds and in other series such as hydroxyoestrones and hydroxyindanones.
Isotopic studies of the conversion of oxophlorins and their ferrihaems into bile pigments in the rat
Authors: Kondo T. , Nicholson D. C. , Jackson A. H. , Kenner G. W.
Published: August 2015

1. Tritiated oxymesoporphyrins and their ferrihaems were tested as possible intermediates in the catabolism of haemoglobin. The tritiated compounds were injected into rats with biliary fistulae and the incorporation of the isotope into bile, bile pigment, urine, faeces, liver, kidney and spleen was measured. 2. α-Oxymesoferrihaem was extensively converted into bile pigment and specifically to the expected mesobilirubin. 3. β-Oxymesoferrihaem was poorly converted into bile pigment and was not converted into mesobiliverdin IXβ. The latter was independently shown to be excreted rapidly in bile. 4. The free oxyporphyrins were also poor precursors of bile pigment, and α-oxymesoporphyrin competed with bilirubin for excretion by the liver. 5. By analogy with the results obtained with α-oxymesoferrihaem it is concluded that α-oxyprotoferrihaem is an intermediate in the catabolism of haemoglobin, undergoing further oxidation to bile pigment under the catalysis of an enzyme of definite specificity.
Studies on ribonucleic acid synthesis in the venom glands of Vipera palaestinae (Ophidia, Reptilia)
Authors: Rotenberg D. , Bamberger E. S. , Kochva E.
Published: August 2015

RNA metabolism in the venom glands of Vipera palaestinae was studied at different stages after manual extraction of the venom (milking). The rate of 32P incorporation into gland RNA was found to be maximal at 1–4 days after milking in correlation with the height of the secretory epithelium. Venom production attained a maximum only after 8–16 days, in parallel with the accumulation of stable species of cellular RNA.
Purification and some kinetic properties of rat liver glucosamine synthetase
Authors: Winterburn P. J. , Phelps C. F.
Published: August 2015

1. Glucosamine synthetase (l-glutamine–d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) was purified about 300-fold from rat liver by two techniques. One procedure utilized the protective action of fructose 6-phosphate and gave a relatively stable preparation, the other yielded an unstable enzyme (half-life of about 20h), free of contaminant activities, on which kinetic experiments were performed. Although the properties of the two preparations showed slight differences, the unstabilized form could be converted into the stabilized form. 2. During preparation the enzyme retained its sensitivity to the feedback inhibitor, UDP-N-acetylglucosamine. 3. The reversibility of the enzyme-catalysed reaction could not be demonstrated. There was no apparent requirement for a cofactor. 4. The pH optimum was at 7.5, at which pH the reaction obeyed a Ping Pong Bi Bi rate equation. At pH values outside the range 6.9–7.6 and at temperatures below 29°C the velocity was described by an ordered Bi Bi rate equation. 5. The molecular weight of the enzyme, determined by two procedures, was 360000–400000. 6. The aminotransferase was unable to utilize ammonia as a substrate.
Studies on the control of hexosamine biosynthesis by glucosamine synthetase
Authors: Winterburn P. J. , Phelps C. F.
Published: August 2015

1. The nature of the feedback inhibition of hexosamine biosynthesis on rat liver glucosamine synthetase (l-glutamine–d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) by UDP-N-acetylglucosamine was investigated in detail. 2. Further modifiers of physiological importance are described. Glucose 6-phosphate and AMP potentiated the UDP-N-acetylglucosamine inhibition, and UTP behaved as an activator. These three compounds only exerted their action when the feedback inhibitor was bound to the enzyme. 3. ATP also inhibited the enzyme. 4. The actions of these various effectors are discussed in kinetic terms. 5. An interpretation of these findings with reference to the regulation of hexosamine biosynthesis is presented.
Aspects of glycine and serine biosynthesis during growth of Pseudomonas AM1 on C1 compounds
Authors: Harder W. , Quayle J. R.
Published: August 2015

1. Methanol or formate can replace serine or glycine as supplements for growth on succinate of the auxotrophic mutants 20S and 82G of Pseudomonas AM1, showing that the organism can synthesize glycine and serine in net fashion from C1 units. 2. Double mutants of Pseudomonas 20S and 82G have been prepared (20ST-1 and 82GT-1) that are unable to grow on succinate+1mm-glyoxylate, succinate+2mm-methanol or methanol alone. 3. Mutants 20ST-1 and 82GT-1 lacked serine–glyoxylate aminotransferase activity, and revertants to the phenotype of 20S and 82G regained serine–glyoxylate aminotransferase activity. A total revertant of 82GT-1 to wild-type phenotype regained activities of serine hydroxymethyltransferase and serine–glyoxylate aminotransferase. 4. The activity of serine–glyoxylate aminotransferase in methanol-grown Pseudomonas AM1 is eightfold higher than in the succinate-grown organism. 5. The combined results show that in Pseudomonas AM1 serine–glyoxylate aminotransferase is necessary for growth on C1 compounds and is involved in the conversion of methanol into glycine via glyoxylate. 6. It is suggested that the phosphorylated pathway of serine biosynthesis from phosphoglycerate replenishes the supply of α-amino groups necessary for the flow of glyoxylate through the main assimilatory pathway during growth on C1 compounds.
Metabolism of C19-steroids by homogenates of normal rat and mouse adrenal tissue and of the Snell transplantable rat adrenocortical tumour 494
Authors: Maynard P. V. , Cameron E. H. D.
Published: August 2015

C19-steroid metabolism in homogenates of adrenal tissue from rats and mice has been studied. Production of these compounds from [7α-3H]cholesterol by rat adrenal tissue appeared to follow a route independent of pregnenolone. The major products of [7α-3H]-dehydroepiandrosterone metabolism by rat adrenal tissue were 5α-reduced steroids, principally androsterone, epiandrosterone and 5α-androstanedione. No differences in metabolism of [7α-3H]dehydroepiandrosterone or [4-14C]pregnenolone were detected between adrenal tissue from Sprague–Dawley, Wistar and Osborne–Mendel rats, but experiments with the Snell rat adrenocortical tumour 494 showed that this tissue had low 5α-reductase activity. In contrast, the major products of [7α-3H]dehydroepiandrosterone metabolism by mouse adrenal tissue were 5β-reduced steroids. Differences were observed between LACA and NH strains of mice in that there was a lower metabolism of androstenedione by NH mouse adrenal and a considerable difference in the proportions of aetiocholanolone and epiaetiocholanolone produced.
Role of the essential thiol groups of yeast alcohol dehydrogenase
Authors: Dickinson F. M.
Published: August 2015

1. Yeast alcohol dehydrogenase inactivated by reaction with iodoacetamide retains 85% of the original NADH-binding capacity as measured under conditions of saturating coenzyme concentration. 2. The dissociation constant of the enzyme–NADH complex is unaffected by inactivation of the enzyme with iodoacetamide, and the affinity of the enzyme for NAD+ and pyridine-3-aldehyde–adenine dinucleotide (PAAD+) appears to be similarly unaffected. 3. Enzyme inactivated with iodoacetamide has lost the ability to form normal ternary complexes of the type enzyme–NADH–acetamide and enzyme–PAAD+–hydroxylamine that are characteristic of the native enzyme.
Studies on the biosynthesis of mitochondrial malate dehydrogenase and the location of its synthesis in the liver cell of the rat
Authors: Bingham R. W. , Campbell P. N.
Published: August 2015

A method is described for the isolation of mitochondrial malate dehydrogenase from either the whole tissue homogenate or from the microsomal fraction of rat liver. The procedure involves the treatment of the tissue extract with detergent followed by gel filtration and chromatography on Amberlite CG-50 and DEAE-cellulose. The resulting enzyme was homogeneous by the criterion of gel electrophoresis. Incubation of the microsomal fraction from rat liver under the usual conditions for protein synthesis in the presence of [3H]leucine resulted in the incorporation of 3H into the mitochondrial malate dehydrogenase when purified as described. The results are taken to indicate that the mitochondrial enzyme is synthesized by the cytoplasmic ribosomes. Possible ways in which the cytoplasmic and mitochondrial forms of malate dehydrogenase reach their final locations in the cell are discussed.
Immunochemical studies on the capsular polysaccharides of pneumococcal type IX
Authors: Higginbotham J D. , Das A , Heidelberger M
Published: August 2015

1. In addition to the previously identified components, d-glucose, N-acetyl-d-glucosamine and d-glucuronic acid, the immunologically specific capsular polysaccharide of pneumococcal type IX (polysaccharide S IX) is now found to contain mannosamine and galactosamine, probably as their N-acetyl derivatives. 2. Partial hydrolysis yields a complex mixture of oligosaccharides, several of which have been separated. 3. One of these is α-d-glucuronopyranosyl-(1→3)-d-glucose. It is an inhibitor of the precipitation of antipneumococcal type IX serum by polysaccharide S IX. 4. Two others, d-glucuronopyranosyl-(1→3)-d-glucosamine and d-glucuronopyranosyl-(1→3)-d-galactosamine have been identified and a trisaccharide and pentasaccharide partially characterized.
Subcellular fractionation by differential and zonal centrifugation of aerobically grown glucose-de-repressed Saccharomyces carlsbergensis
Authors: Cartledge T. G. , Lloyd D.
Published: August 2015

1. Homogenates were prepared from sphaeroplasts of aerobically grown glucose-de-repressed Saccharomyces carlsbergensis and the distributions of marker enzymes were investigated after differential centrifugation. Cytochrome c oxidase and cytochrome c were sedimented almost completely at 105g-min, and this fraction also contained 37% of the catalase, 27% of the acid p-nitrophenyl phosphatase, 53 and 54% respectively of the NADH– and NADPH–cytochrome c oxidoreductases. 2. Zonal centrifugation indicated complex density distributions of the sedimentable portions of these enzymes and of adenosine triphosphatases and suggested the presence of two mitochondrial populations, as well as a bimodal distribution of peroxisomes and heterogeneity of the acid p-nitrophenyl phosphatase-containing particles. 3. Several different adenosine triphosphatases were distinguished in a post-mitochondrial supernatant that contained no mitochondrial fragments; these enzymes varied in their sensitivities to oligomycin and ouabain and their distributions were different from those of pyrophosphatase, adenosine phosphatase and adenosine pyrophosphatase. 4. The distribution of NADPH–cytochrome c oxidoreductase demonstrated that it cannot be used in S. carlsbergensis as a specific marker enzyme for the microsomal fraction. Glucose 6-phosphatase, inosine pyrophosphatase, cytochrome P-450 and five other enzymes frequently assigned to microsomal fractions of mammalian origin were not detected in yeast under these growth conditions.
Intermediate reactions in the binding of aminoacyl-transfer ribonucleic acid to rat liver ribosomes. The role of guanosine triphosphate
Authors: Hradec J.
Published: August 2015

1. Transferase I from rat liver binds relatively low quantities of GTP when incubated with this nucleotide in the absence of aminoacyl-tRNA. 2. Transferase I reacts with both aminoacyl-tRNA and GTP to form a relatively stable complex that is retained on cellulose nitrate filters. The ternary complex transferase I–aminoacyl-tRNA–GTP is also formed when the transferase I–aminoacyl-tRNA complex is incubated with GTP or during the incubation of the transferase I–GTP complex with aminoacyl-tRNA. Synthesis of this complex does not require the presence of Mg2+. 3. In the presence of Mg2+ the ternary complex becomes readily bound to ribosomes without requirements for any other cofactors. 4. An extensive cleavage of GTP takes place when aminoacyl-tRNA becomes bound to ribosomes. 5. The low interdependence of reactions leading to the formation of transferase I complexes with aminoacyl-tRNA and GTP indicates that the mechanisms of the binding reaction in mammalian systems may be different from those in bacterial cells.
Utilization and metabolic effects of acetaldehyde and ethanol in the perfused rat liver
Authors: Lindros K. O. , Vihma R. , Forsander O. A.
Published: August 2015

1. Removal of acetaldehyde and ethanol has been studied in perfused rat livers. 2. The maximum rate of ethanol oxidation was 2μmol/min per g of liver, which was less than the calculated capacity of the ethanol-oxidizing system. The lactate/pyruvate ratio of the medium increased with the rate of ethanol removal. At low ethanol concentrations most of the acetaldehyde formed was oxidized further, but at ethanol concentrations above 16mm about 60% of the acetaldehyde left the liver unmetabolized. 3. At lower concentrations the greater part of added acetaldehyde was oxidized, but above 5mm, 50–60% of that removed was recovered as ethanol. 4. When the reduction of acetaldehyde was blocked by pyrazole, removal was strongly diminished. There was no effect on the lactate/pyruvate ratio during oxidation of low concentrations of acetaldehyde, even in the presence of pyrazole, but at higher concentrations a gradual increase occurred. 5. The results indicate that during ethanol oxidation the ethanol/acetaldehyde pair is not in redox equilibrium with the lactate/pyruvate pair. Ethanol oxidation was abolished by addition of acetaldehyde. Under these conditions the lactate/pyruvate ratio was 1.5–1.8 times the ethanol/acetaldehyde ratio, indicating equilibration of the alcohol dehydrogenase and lactate dehydrogenase systems. 6. The results support the view that ultimately the rate of mitochondrial oxidation of NADH limits the removal of ethanol in the liver.
Appendix: Rates of plasma protein synthesis by deconvolution
Authors: Norwich K. H.
Published: August 2015

The plasma specific radioactivity of arginine guanidine 14C-labelled protein has been measured at various times after the injection of labelled protein precursor (Na214CO3). The resulting ‘appearance‘ curve is a balance between the rate of synthesis and delivery of new protein and the rate of destruction and distribution of new plus existing protein. The measured appearance curve can be corrected to give a measure solely of the rate of synthesis of the plasma protein concerned, if the amplitude of the curve at every point is increased by an amount that compensates for the destruction and distribution processes. The decrease of plasma specific radioactivity of labelled plasma protein after an injection of exogenously labelled protein is termed the ‘disappearance’ curve, and the method of correcting the appearance curve by using the disappearance curve is shown to take the form of a deconvolution.
The enzymic degradation of l-serine O-sulphate. Mechanism of the reaction
Authors: Tudball N. , Thomas P.
Published: August 2015

1. During the enzyme-catalysed degradation of l-serine O-sulphate no exchange occurs between the hydrogen atom attached to the α-carbon atom of the substrate and the tritiated water of the incubation medium. 2. The participation of an intermediate carbanion has been demonstrated in the degradation by performing the reaction in the presence of tetranitromethane. 3. Photo-oxidation of the enzyme in the presence of Rose Bengal led to a rapid inactivation of enzyme with the concomitant loss of four histidine residues/molecule. 4. Rose Bengal was also a non-competitive inhibitor of the enzyme.
The complete amino acid sequence of a mouse ϰ light chain
Authors: Svasti J. , Milstein C.
Published: August 2015

The complete amino acid sequence of the κ-chain of the mouse myeloma protein MOPC 21 was established. The protein was reduced and alkylated with iodo[2-14C]acetic acid, and 21 tryptic peptides were isolated, mainly by paper electrophoresis and paper chromatography. Three large tryptic peptides (of 35, 36 and 42 residues), which were difficult to isolate in this manner, were obtained pure and in excellent yields by a combination of Sephadex G-50 gel filtration in 1% (w/v) NH4HCO3 and chromatography on a DEAE-cellulose column in ammonium acetate buffer, pH8.1. Peptides overlapping the tryptic peptides were isolated from a chymotryptic digest. The chain is 214 residues long. Microheterogeneity of two peptides was observed and is believed to be due to deamidation. It was not excluded that such deamidation could occur in serum from which the protein was isolated. The sequence is compared with the sequences of two other mouse κ-chains, and with the human κ-chain basic sequences.
The formation of glucosides of isoflavones and of some other phenols by rabbit liver microsomal fractions
Authors: Labow Rosalind S. , Layne Donald S.
Published: August 2015

1. Rabbit liver microsomal fractions in vitro effected the transfer of glucuronic acid from UDP-glucuronic acid to biochanin A, formononetin, daidzein, genistein and equol. Only monoglucuronides were formed. 2. The same isoflavones were converted into monoglucosides when UDP-[6-3H]glucose was substituted for UDP-glucuronic acid in the incubation medium in vitro. The glucosides were formed in much lesser yield than were the glucuronides. 3. The glucoside of genistein was identified as genistin (genistein 7-glucoside) by Sephadex chromatography and reverse isotope dilution. 4. The specificity of the glucuronyl- and glucosyl-transfer mechanisms was compared for a series of steroids and other phenols in addition to the isoflavones. It was concluded that separate transferases were responsible for the formation of the two types of glycosides.
The preparation and properties of a group of proteins from the high-sulphur fraction of wool
Authors: Lindley H. , Elleman T. C.
Published: August 2015

A method is reported for the preparation of a group of three proteins from the S-carboxymethylated high-sulphur fraction of wool. These proteins have been partially characterized by their tryptic peptides. All have similar structural features and show an interesting homology within the group and some similarities of sequence with a different group of wool high-sulphur proteins. The evidence for the sequence of some of the peptides is given in a supplementary paper that has been deposited as Supplementary Publication 50008 at 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. (1972) 126, 5.
Nucleotide sequences of two fragments from the coat-protein cistron of bacteriophage R17 ribonucleic acid
Authors: Jeppesen P. G. N. , Barrell B. G. , Sanger F. , Coulson A. R.
Published: August 2015

Bacteriophage R17 RNA was labelled with 32P and was subjected to partial digestion with ribonuclease T1. The products were fractionated by ionophoresis on polyacrylamide gel. Two fragments were purified and their nucleotide sequences determined by methods involving complete and further partial digestion with ribonucleases A and T1. Fragment 20 had a sequence that coded for the amino acids in positions 32–53 of the coat protein of the bacteriophage. Fragment 20X, on further purification in 7m-urea, gave rise to two smaller nucleotides whose sequences coded for the amino acids in positions 56–66 and 67–76 of the coat protein. The sequence of the two fragments was such that they could be written in the form of loops stabilized by base-pairing.
Developmental changes in tissue and plasma concentrations of the biologically active species of nerve growth factor in the mouse, by using a two-site radioimmunoassay
Authors: Hendry I A.
Published: August 2015

A sensitive and specific radioimmunoassay has been developed for the biologically active β-subunit of the mouse submaxillary-gland nerve-growth-factor protein. This assay is based on the ‘two-site’ radioimmunoassay method of Addison & Hales (1971), and a detailed description of the experimental method is given. The assay is readily adaptable for the rapid assay of nerve growth factor in partially purified preparations, or can be used as a sensitive method for measuring the low tissue concentrations of this protein, measurement of concentrations as low as 0.25ng/ml in plasma or tissue extracts being possible. This assay was used to determine tissue concentrations of nerve growth factor in adult mice and developmental changes in its content in various tissues. There were significant sex differences in the nerve-growth-factor content of submaxillary glands, plasma and sympathetic ganglia, with males having higher concentrations, whereas the concentrations in all other tissues measured showed no significant difference between male and female animals. The changes in plasma concentration of the factor during development parallel the changes observed in the submaxillary gland, and it was suggested that plasma nerve growth factor may be derived mainly from this source. Circulating nerve growth factor appears to be specifically concentrated in sympathetic ganglia. There is a relatively high concentration of the factor in neonatal ganglia and plasma, and it is suggested that this may be derived from maternal sources.
Biochemical heterogeneity of rat liver mitochondria separated by rate zonal centrifugation
Authors: Wilson M. A. , Cascarano J.
Published: August 2015

1. Rat liver mitochondria were separated on the basis of their sedimentation coefficients in an iso-osmotic gradient of Ficoll–sucrose by rate zonal centrifugation. The fractions (33, each of 40ml) were collected in order of decreasing density. Fractions were analysed by spectral analysis to determine any differences in the concentrations of the cytochromes and by enzyme analyses to ascertain any differences in the activities of NADH dehydrogenase, succinate dehydrogenase and α-glycerophosphate dehydrogenase. 2. When plotted as% of the highest specific concentration, the contents of cytochrome a+a3 and cytochrome c+c1 were constant in all fractions but cytochrome b was only 65% of its maximal concentration in fraction 7 and increased with subsequent fractions. As a result, the cytochrome b/cytochrome a+a3 ratio almost doubled between fractions 7 and 25 whereas the cytochrome c+c1/cytochrome a+a3 ratio was unchanged. 3. Expression of the dehydrogenase activities as% of highest specific activity showed the following for fractions 6–26: NADH dehydrogenase activity remained fairly constant in all fractions; succinate dehydrogenase activity was 62% in fraction 6 and increased steadily to its maximum in fraction 18 and then decreased; the activity of α-glycerophosphate dehydrogenase was only 53% in fraction 6 and increased slowly to its peak in fractions 22 and 24. 4. These differences did not result from damaged or fragmented mitochondria or from microsomal contamination. 5. These results demonstrate that isolated liver mitochondria are biochemically heterogeneous. The importance of using a system for separating biochemically different mitochondria in studies of mitochondrial biogenesis is discussed.
Insulin release from mouse islets. Effect of glucose and hormones on adenylate cyclase
Authors: Davis Brian , Lazarus Norman R.
Published: August 2015

The adenylate cyclase system of normal mouse islets was characterized. The pH optimum of the system was 7.6. The enzyme preparation contained particulate phosphodiesterase activity. This could be removed by treatment with 0.4% (v/v) Triton X-100 or inhibited by 8mm-theophylline in the presence of 2mm-cyclic AMP (adenosine 3′:5′-cyclic monophosphate). ATP at 0.32mm produced one-half maximal enzyme activity. The enzyme was stimulated in the presence of F-and strongly inhibited by Ca2+. The isolated enzyme retained hormonal sensitivity and was stimulated by glucagon, pancreozymin and secretin at physiological concentrations. Glucose at 17mm, 8mm and 2mm had no direct effect on the activity of the enzyme; neither did galactose at the same concentrations. Groups of islets incubated in 17mm- or 2mm-glucose for 5 or 15min and then homogenized and assayed for adenylate cyclase activity showed no differences in adenylate cyclase activity. The results suggest that the mechanism of glucose-mediated insulin release is not via the adenylate cyclase system. Hormones, however, could mediate insulin secretion via their effects on the adenylate cyclase system.
The role of histidine residues in glutamate dehydrogenase
Authors: Tudball N. , Bailey-Wood R. , Thomas P.
Published: August 2015

1. Glutamate dehydrogenase was subject to rapid inactivation when irradiated in the presence of Rose Bengal or incubated in the presence of ethoxyformic anhydride. 2. Inactivation in the presence of Rose Bengal led to the photo-oxidation of four histidine residues. Oxidation of three histidine residues had little effect on enzyme activity, but oxidation of the fourth residue led to the almost total loss of activity. 3. Acylation of glutamate dehydrogenase with ethoxyformic anhydride at pH6.1 led to the modification of three histidine residues with a corresponding loss of half the original activity. Acylation at pH7.5 led to the modification of two histidine residues and a total loss of enzyme activity. 4. One of the histidine residues undergoing reaction at pH6.1 also undergoes reaction at pH7.5. 5. The presence of either glutamate or NAD+in the reaction mixtures at pH6.1 had no appreciable effect. At pH7.5 glutamate caused a marked decrease in both the degree of alkylation and degree of inactivation. NAD+had no effect on the degree of inactivation at pH7.5 but did modify the extent of acylation. 6. The normal response of the enzyme towards ADP was unaffected by acylation at pH6.1 or 7.5. 7. The normal response of the enzyme towards GTP was altered by treatment at both pH6.1 and 7.5.
Fatty acid synthetase activity in the liver and adipose tissue of rats fed with various carbohydrates
Authors: Bruckdorfer K. R. , Khan I. H. , Yudkin John
Published: August 2015

1. The inclusion of sucrose in the diet of rats led to an increase in hepatic fatty acid synthetase activity compared with that of rats fed with starch as the sole carbohydrate. The higher activity occurred within 18h of the introduction of sucrose and persisted with fluctuations for the 30 days of the experiment. Reversal of the diets in some rats after 21 days led to changes in the enzyme activity to values appropriate to the second diet. The plasma triglyceride concentration followed a similar pattern. 2. A comparison of the effects of diets with starch, glucose, maltose, sucrose or fructose showed that fructose gave the highest values of triglyceride content and of fatty acid synthetase activity in liver, but the lowest values of the synthetase activity in adipose tissue and the lowest values of plasma insulin concentration. These effects may perhaps be attributed to the low insulin response to fructose and to the high affinity of the liver for this sugar. 3. When the diet contained fructose or sucrose there was a correlation between hepatic synthetase activity and plasma triglyceride concentration. Neither of these, however, was related to plasma insulin concentration. On the other hand, there was a correlation between plasma insulin concentration and fatty acid synthetase activity in adipose tissue. 4. When rats were starved and then re-fed the differences in enzyme activities induced by fructose or glucose were minimized. This, together with the varying degree of difference during the course of the experiments, may explain why other workers, using the starvation–re-feeding technique and making measurements on one day only, have failed to observe differences in the activities of lipogenic enzymes in animals fed with either fructose or glucose.
The mode of action of adenosine 3′:5′-cyclic monophosphate in mammalian islets of Langerhans. Preparation and properties of islet-cell protein phosphokinase
Authors: Montague W. , Howell S. L.
Published: August 2015

1. A protein was demonstrated in mammalian islets of Langerhans that after purification appeared as a single component possessing both cyclic-AMP (adenosine 3′:5′-cyclic monophosphate)-binding and cyclic-AMP-dependent protein phosphokinase activities. 2. The protein had an intrinsic association constant for cyclic AMP of 1.15×10−8m, which was similar to the Km for cyclic AMP (1.11×10−8m) of the protein phosphokinase activity. 3. Incubation of the protein in the presence of cyclic AMP resulted in its dissociation into cyclic-AMP-independent protein phosphokinase (catalytic) and cyclic-AMP-binding (receptor) subunits, which could be separated on Sephadex G-200. 4. The cyclic-AMP-dependent protein phosphokinase was capable of phosphorylating a variety of proteins, the most readily phosphorylated being histone, casein and protein components of sub-cellular fractions prepared from islets of Langerhans. 5. The cyclic-AMP-dependent phosphorylation of histone had a Km for ATP of 1.1×10−5m. 6. The endogenous protein phosphokinase activity in rat islets incubated with agents that are known to alter the intracellular concentration of cyclic AMP was investigated. Theophylline and 3-isobutyl-1-methylxanthine, agents that raise cyclic AMP concentrations in islets, increased the activity of the protein phosphokinase, whereas adrenaline, which lowers islet cyclic AMP concentrations, decreased its activity. 7. It is suggested that cyclic AMP may exert its effects on insulin release by increasing the activity of a protein phosphokinase and may thereby promote the phosphorylation and activity of a rate-determining component of the secretory mechanism.
Studies of the denaturation and partial renaturation of ovalbumin
Authors: Holt J. C. , Creeth J. M.
Published: August 2015

1. The denaturation of ovalbumin by the reagents sodium dodecyl sulphate and guanidinium chloride was investigated, by following the changes in sedimentation velocity, optical rotatory dispersion and viscosity as a function of denaturant concentration. 2. With sodium dodecyl sulphate both the optical-rotatory-dispersion parameters a0 and b0 become more negative, the sedimentation coefficient decreases and the viscosity increases; significant differences in the denaturation profiles are observed. The change in each parameter is indicative of only limited denaturation. 3. With guanidinium chloride the transition occurs over the concentration range 1–4m: more extensive changes occur in all the physical parameters than with sodium dodecyl sulphate. The values of a0 and b0 are indicative of complete denaturation. Reduction by mercaptoethanol produces only minor further changes. 4. Renaturation was attempted from both denaturants, the removal of reagent being accomplished reversibly by controlled slow dialysis. Partial renaturation was observed, but aggregated or insoluble material was produced in both cases at relatively low concentrations of denaturant. Similar behaviour was observed with fully reduced protein in guanidinium chloride–mercaptoethanol; complete renaturation could not be brought about even at very low protein concentrations.
The binding of ribosomal subunits to endoplasmic reticulum membranes
Authors: Rolleston Francis S.
Published: August 2015

The binding of ribosomes and ribosomal subunits to endoplasmic reticulum preparations of mouse liver was studied. (1) Membranes prepared from rough endoplasmic reticulum by preincubation with 0.5m-KCl and puromycin bound 60–80% of added 60S subunits and 10–15% of added 40S subunits. Membranes prepared with pyrophosphate and citrate showed less clear specificity for 60S subunits particularly when assayed at low ionic strengths. (2) Ribosomal 40S subunits bound efficiently to membranes only in the presence of 60S subunits. The reconstituted membrane–60S subunit–40S subunit complex was active in synthesis of peptide bonds. (3) No differences in binding to membranes were seen between subunits derived from free and from membrane-bound ribosomes. (4) It is concluded that the binding of ribosomes to membranes does not require that they be translating a messenger RNA, and that the mechanism whereby bound and free ribosomes synthesize different groups of proteins does not depend on two groups of ribosomes that differ in their ability to bind to endoplasmic reticulum.
Mechanisms regulating adipose-tissue pyruvate dehydrogenase
Authors: Martin B. R. , Denton R. M. , Pask H. T. , Randle P. J.
Published: August 2015

1. Isolated rat epididymal fat-cell mitochondria showed an inverse relationship between ATP content and pyruvate dehydrogenase activity consistent with competitive inhibition of pyruvate dehydrogenase kinase by ADP. At constant ATP concentration pyruvate rapidly activated pyruvate dehydrogenase in fat-cell mitochondria, an observation consistent with inhibition of fat-cell pyruvate dehydrogenase kinase by pyruvate. Pyruvate dehydrogenase in fat-cell mitochondria was also activated by nicotinate (100μm) and by extramitochondrial Na+ (replacing K+) but not by ouabain or insulin. 2. In rat epididymal fat-pads incubated in vitro pyruvate dehydrogenase was activated by addition of insulin in the absence of substrate or in the presence of glucose (10mm) or fructose (10mm). Glucose and fructose activated the dehydrogenase in the absence or in the presence of insulin, and pyruvate also activated in the absence of insulin. It is concluded that extracellular glucose, fructose and pyruvate may activate the dehydrogenase by raising intracellular pyruvate and that insulin may activate the dehydrogenase by some other mechanism. 3. Ouabain (300μm) and medium in which K+ was replaced by Na+, activated pyruvate dehydrogenase in epididymal fat-pads. Prostaglandin E1 (1μg/ml), 5-methylpyrazole-3-carboxylate (10μm) and nicotinate (10μm), which are as effective as insulin as inhibitors of lipolysis and which like insulin lower tissue concentration of cyclic AMP (adenosine 3′:5′-cyclic monophosphate), did not activate pyruvate dehydrogenase. Higher concentrations of prostaglandin E1 (10μg/ml) and nicotinate (100μm) produced some activation of the dehydrogenase. 4. It is concluded that the activation of pyruvate dehydrogenase by insulin is not due to the antilipolytic effect of the hormone and that the action of insulin in lowering adipose-cell concentrations of cyclic AMP does not afford an obvious explanation for the effect of the hormone on pyruvate dehydrogenase. The possibility that the effects of insulin, ouabain and K+-free medium may be mediated by Ca2+ is discussed.
Fractionation and isolation of the multiple forms of prorennin (prochymosin)
Authors: Asato N. , Rand A. G.
Published: August 2015

The heterogeneity of prorennin was studied by chromatography on DEAE-cellulose and microgranular DEAE-cellulose columns, as well as by polyacrylamide-gel electrophoresis. Prorennin prepared by alum treatment, salting-out and chromatography was resolved into three components by a compound gradient of sodium phosphate on microgranular DEAE-cellulose. Polyacrylamide-gel electrophoresis confirmed the chromatographic results, but crystalline rennin was shown to consist of four bands. When prorennin was isolated directly by chromatography, four zymogen components were resolved on microgranular DEAE-cellulose with a modified compound gradient of sodium phosphate. Polyacrylamide-gel electrophoresis confirmed the existence of four multiple forms of prorennin as well as homogeneity of the chromatographic fractions.