bluuebunny/crossref_metadata_2025 huggingface dataset:
Modification of diamine oxidase activity in vitro by metabolites of asparagine and differences in asparagine decarboxylation in normal and virus-transformed baby hamster kidney cells
Authors: Quash G , Calogero H , Fossar N , Ferdinand A , Taylor D
Published: August 2015

1. The oxidation of putrescine in vitro by pig kidney diamine oxidase (EC 1.4.3.6) was increased in the presence of 2-oxosuccinamic acid and malonamic acid. 2. It was inhibited by 3-aminopropionamide, oxaloacetate and pyruvate. 3. 2-Oxosuccinamate was derived from asparagine in virus-transformed baby hamster kidney (BHK) cells growing in tissue culture. 4. Asparagine was decarboxylated more efficiently by transformed than by normal BHK cells. 5. In BHK cells transformed by polyoma virus (Py BHK), 2-oxosuccinamate is the most likely immediate precursor of the 14CO2 arising from [U-14C]asparagine, and there was some evidence for its formation in an asparagine-dependent clone of BHK cells before and after their transformation by hamster sarcoma virus (respectively Asn- and HSV Asn-). 6. The relationship between 2-oxosuccinamate and pyruvate and the possible roles of these two substances in controlling cellular diamine oxidase activity are discussed.
Organ distribution of rat histidine-pyruvate aminotransferase isoenzymes
Authors: Noguchi T , Minatogawa Y , Okuno E , Kido R
Published: August 2015

The organ distribution of rat histidine-pyruvate aminotransferase isoenzymes 1 and 2 was examined by using an isoelectric-focusing technique. Isoenzyme 1 (pI8.0) is present only in the liver and its activity is increased by the injection of glucagon, whereas isoenzyme 2 (pI5.2) is distributed in all tissues (liver, kidney, brain and heart) tested, and is not affected by glucagon injection. Isoenzyme 2 of the liver, kidney, brain and heart was purified by the same procedure and characterized. Isoenzyme 2 preparations from these four tissues were nearly identical in physical and enzymic properties. These properties differed from those previously found for the highly purified isoenzyme 1 preparation of rat liver. Isoenzyme 2 was active with pyruvate but not with 2-oxoglutarate as amino acceptor. Amino donors were effective in the following order of activity: tyrosine greater than histidine greater than phenylalanine greater than kynurenine greater than tryptophan. Very little activity was found with 5-hydroxytryptophan. The apparent Km for histidine was about 0.45 mM. The Km for pyruvate was about 4.5 mM with histidine as amino donor. The amino-transferase activities of isoenzyme 2 towards phenylalanine and tyrosine were inhibited by histidine. The ratio of aminotransferase activities towards these three amino acids was constant through gel filtration, electrophoresis, isoelectric focusing and sucrose-density-gradient centrifugation of the purified isoenzyme 2 preparations. These results suggest that these three activities are properties of the same enzyme protein. Sephadex G-150 gel filtration and sucrose-density-gradient centrifugation yielded mol.wts. of approx. 95000 and 92000 respectively. The pH optimum was between 9.0 and 9.3.
Characterization of the binding of human growth hormone to microsomal membranes from rat liver
Authors: Herington A C , Veith N , Burger H G
Published: August 2015

The binding of 125I-labelled human growth hormone to the 100000g microsomal membrane fraction prepared from the livers of normal female rats was dependent on time, temperature, pH, membrane concentration and concentration of 125I-labelled human growth hormone. At 22 degrees C binding reached a steady state after 16h, with the mean maximal specific binding being 20% of the tracer initially added. Dissociation of 125I-labelled human growth hormone from the membranes, after addition of excess of unlabelled hormone, was relatively slow with a half-time greater than 24h. Only minor degradation of the 125I-labelled human growth hormone was observed during incubation with membranes for 16 or 25h at 22 degrees C. Similarly, no significant change in the ability of membranes to bind human growth hormone was evident after preincubation of the membranes for 16 or 25h. Specificity studies showed that up to 90% of the 125I-labelled human growth hormone bound could be displaced by 1 mug of unlabelled hormone. Ovine prolactin also showed considerable competition for the binding site. Non-primate growth-hormone preparations (ovine, bovine, porcine and rat) and non-related hormones (insulin, thyrotropin, lutropin and follitropin) all showed negligible competition. Scatchard analysis of the binding data was consistent with two classes of binding site with binding affinities of 0.64 × 10(10) +/- 0.2 × 10(10)M-1 and 0.03 × 10(10) +/- 0.007 × 10(10)M-1 and corresponding binding capacities of 98.4 +/- 10 fmol/mg of protein and 314.6 +/- 46.3 fmol/mg of protein. These studies provide data which, in general, are consistent with the criteria required for hormone-receptor interaction. However, proof of the thesis that the human-growth-hormone-binding sites in female rat liver represent physiological receptors must await the demonstration of a correlation between hormone binding and a biological response.
Basal and thyroliberin-stimulated prolactin synthesis in single-cell cultures and in populations of rat pituitary cells
Authors: Gautvik K M , Fossum S
Published: August 2015

1. Newly synthesized prolactin was obtained from cultures of rat pituitary tumour cells (GH4C1 cells) after incubation with [35S]methionine. 2. Radioactive synthesized and secreted prolactin was quantified by an immunoprecipitation method by using disc-gel electrophoresis of the dissolved immunoprecipitate in the presence of sodium dodecyl sulphate. By using a microanalytical modification, hormone synthesis and secretion could also be studied in single-cell cultures. This technique was combined with a cytoimmunofluorescence method in which rhodamine-conjugated antibodies were used for studying intracellular prolactin. 3. The presence of radioactive synthesized and secreted prolactin was demonstrated in nine out of 13 single-cell cultures. Cell cultures containing 10 cells or more and clonal populations originating from one cell always secreted radioactive prolactin. 4. Thyroliberin treatment (2 muM) for 24h increased the extracellular accumulation of radioactive prolactin in five out of seven single-cell cultures and always in populations of cells. 5. The number of cells showing prolactin specific fluorescence increased from 20 to 50% and the intensity of this fluorescence became greater after thyroliberin treatment. 6. Studies of [35S]prolactin secretion from single cells and immunochemical detection of intracellular prolactin showed that some cells in an unsynchronized population did not secret radioactive prolactin or show prolactin specific fluorescence. 7. The quantitative effect of thyroliberin as studied in single-cell cultures suggested that the main if not the only effect was to increase prolactin synthesis in cells already producing hormone.
Activities and some properties of adenylate cyclase and phosphodiesterase in muscle, liver and nervous tissues from vertebrates and invertebrates in relation to the control of the concentration of adenosine 3′:5′-cyclic monophosphate
Authors: Arch J R S , Newsholme E A
Published: August 2015

1. The basal and fluoride-stimulated activities of adenylate cyclase, and the maximal activities of 3′:5′-cyclic AMP phosphodiesterase and 3′:5′-cyclic GMP phosphodiesterase, together with the Km values for their respective substrates, were measured in muscle, liver and nervous tissues from a large range of animals to provide information on the mechanism of control of cyclic AMP concentrations in these tissues. High activities of adenylate cyclase and cyclic AMP diesterase are found in nervous tissues and in the more aerobic muscles (e.g. insect flight muscles, cardiac muscle and some vertebrate skeletal muscles). The activities of these enzymes in liver are similar to those in the heart of the same animal. The Km values for the enzymes from different tissues and animals are remarkably similar. 2. The comparison of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase activities suggests that in vertebrate tissues only one enzyme (the high-Km enzyme), which possesses dual specificity, exists, whereas in invertebrate tissues there are at least two phosphodiesterases with separate specificities. 3. A simple quantitative model to explain the control of the steady-state concentrations of cyclic AMP is proposed. The maximum increase in cyclic AMP concentration predicted by comparison of basal with fluoride-stimulated activities of adenylate cyclase is compared with the maximum increases in concentration produced in the intact tissue by hormonal stimulation: reasonable agreement is obtained. The model is also used to predict the actual concentrations and the rates of turnover of cyclic AMP in different tissues and, where possible, these values are compared with reported values. Reasonable agreement is found between predicted and reported values. The possible physiological significances of different rates of turnover of cyclic AMP and the different ratios of high- and low-Km phosphodiesterases in different tissues are discussed.
Selective resistance to desiccation of nuclear ribonucleic acid synthesis in isolated nuclei of Artemia salina embryos during pre-emergence development
Authors: Chaffoy D D , Kondo M
Published: August 2015

The developing gastrula embryos of Artemia salina are resistant to a complete redesiccation during a period of pre-emergence development. Isolated nuclei from these dehydrated embryos could retain a transcriptional activity in vitro comparable with that of non-desiccated controls. On the other hand, redesiccation of both prenauplii and nauplii completely destroys their viability as well as the nuclear transcriptional activity. However, those gastrula embryos that did not develop in a first incubation period could remain viable and develop with a considerable time-lag after a subsequent second incubation.
Mouse haemoglobin beta chains. Comparative sequence data on adult major and minor β chains from two species, Mus musculus and Mus cervicolor
Authors: Gilman J G
Published: August 2015

In inbred Mus musculus several different beta chains are known. In certain strains two beta chains are produced in unequal amounts by the two closely linked genes of the doublet breeding unit allele Hbb(d): Betadmaj and betadmin. One strain has a variant doublet allele, Hbb(p), which produces a variant minor beta chain, betapmin (the major beta chain, betapmaj, may not differ from betadmaj chain). Certain other strains have a singlet allele, Hbb(s), that produces only one beta chain, betas. Other species have different beta-chain patterns. In M. cervicolor two variant major beta chains are found, betacmaj (d-like) and betacmaj (s-like), both of which were found associated with minor beta chains. M. caroli has only one, ‘Leporelike’ beta chain, with structural features characteristic predominantly of betadmin chain in the N-terminal half and of betadmaj chain in the C-terminal half. The present paper presents sequence data on betas, betadmaj, betadmin, betapmin and betacmaj (d-like) chains. The data on betadmin chain cover almost the whole of that chain and show a minimum of nine differences from betadmaj chain and two from betapmin chain. It is suggested that the data on the beta chains of the various species show evidence for the past occurrence of double crossovers over regions within a gene coding for only one or a few amino acids, which events can be explained by the ‘hybrid DNA’ models of genetic recombination. Supplementary information on the amino acid sequence of the proteins has been deposited as Supplementary Publication SUP 50067 (36 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1976) 153, 5.
Subsite mapping of enzymes. Depolymerase computer modelling
Authors: Allen J D , Thoma J A
Published: August 2015

We have developed a depolymerase computer model that uses a minimization routine. The model is designed so that, given experimental bond-cleavage frequencies for oligomeric substrates and experimental Michaelis parameters as a function of substrate chain length, the optimum subsite map is generated. The minimized sum of the weighted-squared residuals of the experimental and calculated data is used as a criterion of the goodness-of-fit for the optimized subsite map. The application of the minimization procedure to subsite mapping is explored through the use of simulated data. A procedure is developed whereby the minimization model can be used to determine the number of subsites in the enzymic binding region and to locate the position of the catalytic amino acids among these subsites. The degree of propagation of experimental variance into the subsite-binding energies is estimated. The question of whether hydrolytic rate coefficients are constant or a function of the number of filled subsites is examined.
Location of nucleotide pyrophosphatase and alkaline phosphodiesterase activities on the lymphocyte surface membrane
Authors: Abney E R , Evans W H , Parkhouse R M E.
Published: August 2015

1. Isolated mouse spleen lymphocytes hydrolysed UDP-galactose added to the medium. Nucleotide pyrophosphatase activity that accounted for this hydrolysis was enriched to a similar extent as alkaline phosphodiesterase and 5'-nucleotidase in a lymphocyte plasma-membrane fraction. 2. The cell surfaces of mouse spleen and thymus lymphocytes were iodinated with 125I by using the lactoperoxidase-catalysis method. Detergent extracts of the cells were mixed with a purified anti-(mouse liver plasma-membrane nucleotide pyrophosphatase) antiserum and the immunoprecipitates analysed by polyacrylamide-gel electrophoresis. Only one major radioactive component, similar in size (apparent mol.wt 110000-130000) to the liver enzyme, was observed. 3. Electrophoresis of an iodinated spleen plasma-membrane fraction indicated peaks of radioactivity, including one of apparent mol.wt 110000-130000. 4. When detergent extracts of spleen lymphocytes were passed through a Sepharose-bead column containing covalently attached anti-(nucleotide pyrophosphatase) antiserum, the nucleotide pyrophosphatase activity was retained by the beads, whereas protein and leucine naphthylamidase activity were eluted. 5. The results indicate that nucleotide pyrophosphatase and alkaline phosphodiesterase activities are due to the location of the same or similar enzymes at the outer aspect of the lymphocyte plasma membrane. Some possible functions of enzymes at this location are discussed.
The accessibility of protein-bound dinitrophenyl groups to univalent fragments of anti-dinitrophenyl antibody
Authors: Knight C G , Green N M
Published: August 2015

A series of N-(N-dinitrophenylaminoalkyl)maleimides were sythesized with alkyl-chain lengths of two, four and six carbon atoms. When these compounds reacted with the thiol group of mercaptalbumin, the tryptophan fluorescence of the protein was quenched. This change in fluorescence was used to determine the rate of reaction of the Dnp (dinitrophenyl)-maleimides with mercaptalbumin. The second-order rate constants were similar to those observed in reactions between low-molecular-weight thiol compounds and maleimides. When N-(N-Dnp-aminoalkyl)succinimidomercaptalbumins were added to univalent fragments of anti-Dnp antibody the antibody fluorescence was quenched. Florescence-quenching titrations showed that the protein-bound Dnp groups were fully available to the antibody even when the alkyl chain was short. The apparent dissociation constants were significantly > that of the interaction between anti-Dnp antibody and the free hapten, 6-(N-Dnp)-aminohexanoate. The antibody fluorescence was quenched efficienty by [dnp-Lys41]ribonuclease A, also with an increased dissociation constant. It could be concluded from the increase in dissociation constant that the Dnp group spent no more than 0.1% of its time in the dissociated state, available to antibody. The second-order rate constants for the association between the Dnp-mercaptablumins and the antibody were determined and were similar in magnitude to those observed in other interactions between protein and anti-protein antibody.
Identity of isoenzyme 1 of histidine-pyruvate aminotransferase with serine-pyruvate aminotransferase
Authors: Noguchi T , Okuno E , Kido R
Published: August 2015

After glucagon injection, rats showed virtually identical percentage increases in hepatic histidine-pyruvate aminotransferase and serine-pyruvate aminotransferase activities, both in the mitochondria and in the cytosol. Histidine-pyruvate aminotransferase isoenzyme 1, with pI8.0, was purified to homogeneity from the mitochondrial fraction of liver from glucagon-injected rats. The purified enzyme catalysed transamination between a number of amino acids and pyruvate or phenylpyruvate. For transamination with pyruvate, the activity with serine reached a constant ratio to that with histidine during purification, which was unchanged by a variety of treatments of the purified enzyme. Serine was found to act as a competitive inhibitor of histidine transamination, and histidine of serine transamination. These results suggest that histidine-pyruvate amino-transferase isoenzymes 1 is identical with serine-pyruvate aminotransferase. The enzyme is probably composed of two identical subunits with mol. wt. approx. 38000. The absorbance maximum at 410 nm and the inhibition by carbonyl reagents strongly indicate the presence of pyridoxal phosphate.
Similarities between a dipeptide hydrolase from brush-border and cytosol fractions of guinea-pig intestinal mucosa
Authors: Piggott C O , O'Cuinn G , Fottrell P F
Published: August 2015

A dipeptide hydrolase from the brush border of guinea-pig intestinal mucosa was purified. The enzyme resembles another dipeptide hydrolase isolated from the cytosol fraction of intestinal mucosa. Studies on the binding of cytosol peptide hydrolase to brush-border membranes indicate that the enzyme found in the brush border may be a cytoplasmic contaminant.
Role of histone kinases as mediators of corticotropin-induced steroidogenesis
Authors: Moyle W R , MacDonald G J , Garfink J E
Published: August 2015

In an attempt to determine the role of protein (histone) kinases as mediators of corticotropin-induced corticosterone formation, the ability of homogenates, prepared from adrenals treated with various doses of corticotropin to catalyse the phosphorylation of calf thymus histones was measured. Although corticotropin promoted an increase in histone kinase activity, much more of the hormone was required to induce this response than to stimulate steroidogenesis maximally. In addition, a derivative, nitrophenylsulphenyl-corticotropin, which inhibits the stimulatory effect of corticotropin on cyclic AMP accumulation, stimulated corticosterone synthesis without altering histone kinase activity. Very high doses of nitrophenylsulphenyl-corticotropin were capable of stimulating histone kinase activity. In contrast, when dibutyryl cyclic AMP was used to stimulate steroidogenesis under the same conditions, any dose of the nucleotide which increased adrenal corticosteroid content also increased histone kinase activity. Assuming that histones serve as useful substrates for measurement of total adrenal protein kinase activity, the role of protein kinases as mediators of steroidogenesis is not supported by these studies.
Comparison of amphibian whole-cell and cell-free activity towards different transferrins
Authors: James G T
Published: August 2015

1. A cell-free preparation from immature-erythrocytes of the bullfrog (Rana catesbeiana) was about equally active towards iron bound to homologous, rabbit or human transferrin. 2. In contrast with the cell-free system, whole immature erythrocytes exhibited optimum, intermediate or low uptake of iron from the above and from other transferrins.
Changes in the activities of the enzymes of urea synthesis caused by dexamethasone and dibutyryladenosine 3′:5′-cyclic monophosphate in foetal rat liver maintained in organ culture
Authors: Edkins E , Rïhä N C R
Published: August 2015

Liver explants from 19-day foetal rats were maintained in organ culture, in a defined medium, for up to 48h. Both 6-N,2′-O-dibutyryl cyclic AMP, in the presence of theophylline, and dexamethasone caused an increase in the activities of carbamoyl phosphate synthase, argininosuccinate synthetase, argininosuccinate lyase and arginase. These increases could be abolished by simultaneously incubating the explants with cycloheximide. No change in the activity of ornithine transcarbamoylase was found with either hormone. Previous work has shown that injection of corticosteroids into 19.5-day foetal rats in utero did not cause an increase in the arginine synthetase system. Present results suggest that this lack of effect is not due to any incompetence of the foetal rat liver at this stage to respond to this agent. The observations on ornithine transcarbamoylase activity suggest that this enzyme is induced in the liver of the perinatal rat by neither corticosteroids nor hormones acting via cyclic AMP, and it may be that all the enzymes of the urea cycle are induced physiologically by an agent or agents as yet unidentified.
A sex difference in hepatic glutathione S-transferase B and the effect of hypophysectomy
Authors: Hales B F , Neims A H
Published: August 2015

The glutathione S-transferases are a group of proteins with overlapping substrate specificities and ligand-binding capacities. This report examines certain approaches to the measurement of transferase B (ligandin) in the rat liver. The ratio of catalytic activities toward 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene gives some indication of the relative proportions of the various transferases present in 100 000 g supernatants. The fraction of catalytic activity towards 1-chloro-2,4-dinitrobenzene, due to transferase B, was best measured by immunoprecipitation with anti-(transferase B). Male rat liver exhibited three times more activity towards 1,2-dichloro-4-nitrobenzene than female tissue; however, the activities towards 1-chloro-2,4-dinitrobenzene were almost identical. By assuming a specific activity of 11 mumol/min per mg, immunoprecipitable transferase B comprised 4.5 +/- 0.2% of total protein in the 100 000 g supernatant of female rat liver, and 70% of the transferase activity towards 1-chloro-2,4-dinitrobenzene. The amount of transferase B in the 100 000 g supernatant from male rat liver is significantly lower with respect to both fraction of total protein (3.3 +/- 0.2%) and overall transferase activity towards 1-chloro-2,4-dinitrobenzene (48%). Hypophysectomy eliminated this sex difference in the hepatic concentration of glutathione S-transferase B.
The selective isolation of the uterine oestradiol-receptor complex by binding to oligo(dT)-cellulose. The mediation of an essential activator in the transformation of cytosol receptor
Authors: Thrower S , Hall C , Lim L , Davison A N
Published: August 2015

The [3H]oestradiol-receptor complex was selectively isolated from rat uterus cytosol by column chromatography on oligo(dT)-cellulose. Optimal conditions are described for the binding of the complex to oligo(dT)-cellulose, which is shown to be similar to its binding to DNA-cellulose. The cytosol complex has an apparent mol. wt. of 50,000-60,000 in high salt concentrations, as determined by Sephadex G-100 chromatography. This corresponds to the 4S cytoplasmic oestradiol receptor. In binding to oligo(dT)-cellulose the receptor is transformed into a form with an apparent mol.wt. of 100,000-120,000, corresponding to the 5S nuclear receptor complex. This transformation mimics the conversion in vivo of the cytoplasmic oestradiol receptor into the nuclear form. The binding of the complex to oligo(dT)-cellulose as a 5S nuclear form is unequivocally demonstrated to require the mediation of an activating present in the cytosol. The requirement for an activating factor is discussed in relation to reports that nuclear binding of the oestradiol-receptor complex is not dictated solely by the availability of the cytoplasmic oestradiol receptor.
Orotate decreases the inhibitory effect of ethanol on galactose elimination in the perfused rat liver
Authors: Keiding S , Vinterby A
Published: August 2015

1. The galactose-elimination rate in perfused livers from starved rats was decreased in the presence of ethanol (2-28mM) to one-third of the control values. Orotate injections partly reversed the effect of ethanol, so that the galactose-elimination rate was about two-thirds of the control values. Orotate alone had no effect on the galactose-elimination rate. 2. Ethanol increased [galactose 1-phosphate] and [UDP-galactose], and decreased (UDP-glucose] and [UTP], both with and without orotate. Orotate increased [UTP], [UDP-galactose], both with and without ethanol. The increase of [galactose 1-phosphate] in the presence of ethanol was inhibited by orotate. Orotate alone had no appreciable effect on [galactose 1-phosphate]. 3. Both the effect of ethanol and that of orotate on the galactose-elimination rate can be accounted for by assuming inhibition of galactokinase by galactose 1-phosphate with Ki about 0.2mM, the inhibition being either non-competitive or uncompetitive. 4. The primary effect of ethanol seems to be inhibition of UDP-glucose epimerase (EC 5.1.3.2), followed by accumulation of UDP-galactose, trapping of UDP-glucose and increase of [galactose 1-phosphate]. Orotate decreased the effect of ethanol, probably by increasing [UDP-glucose].
A study of the ionic properties of the essential histidine residue of yeast alcohol dehydrogenase in complexes of the enzyme with its coenzymes and substrates
Authors: Dickenson C J , Dickinson F M
Published: August 2015

1. Initial-rate studies of the reduction of acetaldehyde by NADH, catalysed by yeast alcohol dehydrogenase, were performed at pH 4.9 and 9.9, in various buffers, at 25 degrees C. The results are discussed in terms of the mechanism previously proposed for the pH range 5.9-8.9 [Dickenson & Dickinson (1975) Biochem. J. 147, 303-311]. 2. Acetaldehyde forms a u.v.-absorbing complex with glycine. This was shown not to affect the results of kinetic experiments under the conditions used in this and earlier work. 3. The variation with pH of the dissociation constant for the enzyme-NADH complex, calculated from the initial-rate data, indicates that the enzyme possesses a group with pK7.1 in the free enzyme and pK8.7 in the complex. 4. The pH-dependences of the second-order rate constants for inactivation of the enzyme by diethyl pyrocarbonate were determined for the free enzymes (pK7.1), the enzyme-NAD+ complex (pK approx. 7.1) and the enzyme-NADH complex (pK approx. 8.4). The essential histidine residue may therefore be the group involved in formation and dissociation of the enzyme-NADH complex. 5. Estimates of the rate constant for reaction of acetaldehyde with the enzyme-NADH complex indicate that acetaldehyde may combine only when the essential histidine residue is protonated. The dissociation constants for butan-1-ol and propan-2-ol, calculated on the basis of earlier kinetic data, are, however, independent of pH. 6. The results obtained are discussed in relation to the role of the essential histidine residue in the mechanism of formation of binary and ternary complexes of the enzyme with its coenzymes and substrates.
The isolation of minimally degraded hyaluronate from rat skin
Authors: Mathieson J M , Pearce R H
Published: August 2015

Hyaluronate was isolated quantitatively from fresh rat skin by homogenization in an Edebo [J. Biochem. Microbiol. Technol. Eng. 2, (1960) 453-479] press, extraction with buffered saline, selective elution from DEAE-cellulose, and gel chromatography on Sephadex G-200. Traces of a protein contaminant remained.
Purification and properties of an α-d-galactoside galactohydrolase from the seeds of Trifolium repens (white clover)
Authors: Williams J , Villarroya H , Petek F
Published: August 2015

Five alpha-D-galactosidases (alpha-D-galactoside galactohydrolase; EC 3.2.1.22) have been identified by chromatography and polyacrylamide-disc-gel electrophoresis in the germinated seeds of Trifolium repens (white clover). alpha-Galactosidase I has been purified to homogeneity with an approx. 2000-fold increase in specific activity. The enzyme was purified by a procedure which included precipitation by dialysis against citrate/phosphate buffer, pH3.5; (NH4)2SO4 precipitation; hydroxyapatite, DEAE-cellulose and ECTEOLA-cellulose column chromatography. Each stage of purification was controlled by polyacrylamide-disc-gel electrophoresis; the purified enzyme showed a single protein band that corresponded to the alpha-D-galactosidic activity. The pH optimum was found to be between pH 3.8 and 4.2; the enzyme is highly thermolabile. Hydrolysis of oligosaccharides and galactomannans has been examined, and it has been found that alpha-galactosidase I exhibits two enzymic activities, namely alpha-D-galactoside galactohydrolase and galactosyltransferase. By the polyacrylamide-gel-electrophoresis method of Hendrick & Smith (1968), and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the mol.wt. has been estimated to be 43 000 and 41 000 respectively. These results indicate that alpha-galactosidase I is a monomeric protein and that both enzymic activities associated with the enzyme reside on the same polypeptide chain.
An examination of potential matrices for the affinity chromatography of NADP+-linked dehydrogenases with special reference to 6-phosphogluconate dehydrogenase
Authors: Griffiths P , Houlton A , Adams M J , Harvey M J , Dean P D G
Published: August 2015

1. 6-phosphogluconate dehydrogenase from sheep liver has been purified 350-fold by affinity chromatography with a final specific activity of 18 micronmol of NADP+/reduced min per mg of protein and an overall yield of greater than 40%. 2. A systematic investigation of potential ligands has been carried out: these included 6-phosphogluconate and NADP+, pyridoxal phosphate and several immobilized nucleotides. The results indicate that NADP+ is the most suitable ligand for the purification of 6-phosphogluconate dehydrogenase. 3. The effects of pH and alternative eluents have been examined in relation to the parameters known to affect the desorption phase of affinity chromatography; careful manipulation of the elution conditions permitted the separation of glucose 6-phosphate dehydrogenase, glutathione reductase and 6-phosphogluconate dehydrogenase from sheep liver on NADP+-Sepharose 4B. 4. A large-scale purification scheme for 6-phosphogluconate dehydrogenase is presented that uses the competitive inhibitors inorganic pyrophosphate and citrate as specific eluents.
Deoxyribonucleic acid and polyamine synthesis in rat ventral prostate. Effects of age of the intact rat and androgen stimulation of the castrated rat with testosterone, 5α-dihydrotestosterone and 5α-androstane-3β, 17β-diol
Authors: Takyi E E K , Fuller D J M , Donaldson L J , Thomas G H
Published: August 2015

The relationship between polyamine synthesis, growth and secretion in vivo was examined in ventral prostates from: (a) intact rats aged 3-60 weeks; (b) animals castrated for 7 days before injection with 5 alpha-dihydrotestosterone (17 beta-hydroxy-5-alpha-androstan-3-one), testosterone and 5 alpha-androstane-3 beta, 17 beta-diol for up to 10 days; (c) rats injected with the 3 beta, 17 beta-diol immediately after castration. Ornithine decarboxylase activity and the concentrations of putrescine, spermidine and spermine were measured. DNA-synthetic activity was monitored by measuring [125I]iododoxyuridine incorporation. An enhanced spermidine/spermine molar ratio reflected increased activity of the prostate. The ratio was higher (greater than 2) in prostates from sexually immature animals, than in the intact adult (1.5), suggesting that the ratio was indicative of the proliferative activity of the tissue. However, in the androgen-stimulated castrated rat, enhanced spermidine/spermine ratios tended to correlate with hypertrophy and secretion. In both sets of experiments there was a linear relationship between protein and spermidine content. High spermidine/spermine molar ratios were the consequence of a relatively low rate of accumulation of spermine relative to spermidine and protein. The relationship between polyamine synthesis and DNA-synthetic activity was investigated in cultured prostate. A combination of insulin (3 mug/ml) and testosterone (0.1 muM caused a stimulatory response in the incorporation of [125I]iododeoxyuridine and in cell division, despite a depleted polyamine content and low ornithine decarboxylase activity in the cultured tissue.
Studies on the biosynthesis, assembly and secretion of vitellogenin, an oestrogen-induced multicomponent protein
Authors: Penning T M , Merry A H , Munday K A , Akhtar M
Published: August 2015

1. The process by which the egg-yolk protein precursor vitellogenin is biosynthesized, assembled and secreted by Xenopus laevis (South African clawed toad) liver was studied. It was previously shown in other laboratories that vitellogenin contains the two egg-yolk proteins lipovitellin (mol.wt. 140 000) and phosvitin (mol.wt. 35 000). 2. Evidence is presented which shows that Xenopus liver microsomal fractions synthesize precursors of vitellogenin. These precursors were solubilized from the membranes with detergent and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This analysis indicated that there is only one precursor polypeptide, and this has mol.wt. approx. 200 000 +/- 20 000. This demonstrates that the egg-yolk proteins are translated as part of this larger polypeptide. 3. Experiments also demonstrate the existence of a microsomal proteinase which is able to cleave the precursor into smaller fragments. The nature of these fragments provided some indirect evidence that phosvitin and lipovitellin light chains are situated together within the precursor molecule. 4. These precursor data fit in well with structural studies on serum vitellogenin, since it has been shown that the latter protein consists of two identical subunits each with a mobility on sodium dodecyl sulphate/polyacrylamide gels identical with that shown by the microsomal precursor. This indicates that both the intracellular precursor and subunit of vitellogenin have similar (but not necessarily identical) molecular weights. 5. It was also shown that trypsin or chymotrypsin can cleave the serum vitellogenin into leucine- and serine-rich fragments which resemble lipovitellin and phosvitin respectively. Attention is, however, drawn to the fact that the serine-rich fragment is not identical with phosvitin, since it contains eight times more leucine than that expected for the authentic phosvitin molecule [Penning (1976) Ph.D. Thesis, University of Southampton].
Occurrence of ornithine decarboxylase and polyamines in cartilage
Authors: Conroy P D , Simms D M , Pointon J J
Published: August 2015

The activity of ornithine decarboxylase was investigated in cartilage from chick embryos, rabbits, rats and human foetuses. The enzyme activity in these cartilages was of the same order as the detected in other body tissues. Ornithine decarboxylase activity in chick-embryo cartilage and liver was the same when compared on the basis of total soluble tissue protein. The cartilage enzyme exhibited a pH optimum of 6.5 and a Km for ornithine of 0.16mM. Ornithine decarboxylase activity in chick-embryo pelvic leaflets was maintained at the value in vivo for up to 22h when the isolated tissue was incubated in a modified Waymouth's medium (MB 752/1) at 37 degrees C. After addition of cycloheximide to the incubation medium, ornithine decarboxylase activity declined, with a half-life of 40 min. The concentrations of the polyamines spermidine and spermine in chick-embryo pelvic cartilage and rabbit costal cartilage were of the same order as the concentrations detected in other tissues.
Lipoprotein lipase activity at onset of development of white adipose tissue in newborn rats
Authors: Péquignot-Planche E , De Gasquet P , Boulangé A , Tonnu N T
Published: August 2015

The low triacylglycerol concentration in inguinal tissue of newborn rats did not change during the first 6h after birth, despite the relatively high lipoprotein lipase activity in the tissue. Subsequently triacylglycerol concentration and enzyme activity rose in parallel. The results show that lipoprotein lipase activity was present in the tissue before fat accumulation.
Studies of energy-linked reactions. Localization of the site of action of trialkyltin in yeast mitochondria
Authors: Cain K , Griffiths D E
Published: August 2015

Ligand-binding studies with labelled triethyltin on yeast mitochondrial membranes showed the presence of high-affinity sites (KD = 0.6 micronM; 1.2 +/- 0.3 nmol/mg of protein) and low-affinity sites (KD less than 45 micronM; 70 +/- 20 nmol/mg of protein). The dissociation constant of the high-affinity site is in good agreement with the concentration of triethyltin required for inhibition of mitochondrial ATPase (adenosine triphosphatase) and oxidative phosphorylation. The high-affinity site is not competed for by oligomycin or venturicidin, indicating that triethyltin reacts at a different site from these inhibitors of oxidative phosphorylation. Fractionation of the mitochondrial membrane shows a specific association of the high-affinity sites with the ATP synthase complex. During purification of ATP synthase (oligomycin-sensitive ATPase) there is a 5-6-fold purification of oligomycin- and triethyltin-sensitive ATPase activity concomitant with a 7-9-fold increase in high-affinity triethyltin-binding sites. The purified yeast oligomycin-sensitive ATPase complex contains approximately six binding sites for triethyltin/mol of enzyme complex. It is concluded that specific triethyltin-binding sites are components of the ATP synthase complex, which accounts for the specific inhibition of ATPase and oxidative phosphorylation by triethyltin.
The appearance of gluconeogenesis at birth in sheep. Activation of the pathway associated with blood oxygenation
Authors: Warnes D M , Seamark R F , Ballard F J
Published: August 2015

1. Measurements of pyruvate carboxylase, mitochondrial phosphoenolpyruvate carboxykinase (GTP), hexose bisphosphatase and glucose 6-phosphatase in developing sheep liver showed substantial activities of all enzymes in the foetus, especially towards the end of gestation. Cytosol phosphoenolpyruvate carboxykinase (GTP) in livers of mid-term foetuses was only 10% of the activity at birth. 2. All enzymes except pyruvate carboxylase showed 1.5-2-fold increases after birth. 3. Gluconeogenesis form [14C]actate could not be detected in chronically cannulated sheep foetuses at any developmental stage and was not initiated by the infusion of adrenaline or glucagon. 4. An active pathway of gluconeogenesis was evident in vivo within 2 min after natural birth or within 4 min after Caesarian delivery of term lambs, and was delayed in prematurely delivered lambs until breathing was established and the blood fully oxygenated. 5. It is proposed that oxygen availability initiates gluconeogenesis in the newborn lamb.
Rates of glucose utilization and glucogenesis in rats in the basal state induced by halothane anaesthesia
Authors: Heath D F , Frayn K N , Rose J G
Published: August 2015

1. Rates and rate coefficients of glucose utilization and replacement were determined with [5-3H]- and [U-14C]-glucose in rats starved for 24h, either conscious or under halothane anaesthesia, in a thermoneutral environment. Plasma insulin concentrations were also measured. 2. Halothane anaesthesia decreased the turnover rate by 20%, which was similar to previously reported decreases in metabolic rates caused by natural sleep. 3. Fractional recycling of glucose carbon was little affected by halothane. 4. Comparison of values in one rat with those in another, among both conscious rats and those under halothane anaesthesia, showed that rate coefficients were inversely correlated with plasma glucose concentrations. 5. These findings indicated that halothane, in the concentration used (1.25%, v/v), had little specific effect on glucose metabolism. 6. Although equilibrium plasma glucose concentrations in different rats under halothane were widely different (4-8 mmol/l) the rates of utilization were very similar (2.5-3.1 micronmol/min per 100 g), indicating that these rates were determined by the production of glucose from gluconeogenic precursors released by basal metabolism, the rate of which is necessarily similar in different rats. 7. Among rats under halothane anaesthesia plasma insulin concentrations were negatively correlated with rate coefficients, showing that the differences between rate coefficients were mostly accounted for by differences between rats in tissue sensitivities to insulin. Thus in each 24h-starved rat, sleeping or resting, the main regulators of the plasma glucose concentrations were the rate of supply of gluconeogenic substrates from energy metabolism and the intrinsic sensitivity of the tissues to insulin. 8. We found that a commonly used deionization method of purifying glucose for determination of its specific radioactivity was inadequate.
Binding of bile acids by 100 000g supernatants from rat liver
Authors: Strange R C , Nimmo I A , Percy-Robb I W
Published: August 2015

1. The binding of glycocholic acid, chenodeoxycholic acid and lithocholic acid to rat liver 1000 000g supernatants was studied by equilibrium dialysis. 2. The binding characteristics of the bile acids suggest that the binding components are involved in bile acid transport. 3. When mixtures of [14C]lithocholic acid and liver supernatants were eluted from columns of Sephadex G-75, a prominent peak of [14C]lithocholic acid appeared with proteins of mol.wt. approx. 40000. A second, smaller, peak of [14C]lithocholic acid was eluted with proteins of mol.wt. approx. 100000. 4. The inclusion of cholic acid, glycocholic acid or chenodeoxycholic acid in the eluting buffer decreased the amount of [14C]lithocholic acid that was eluted with the higher-molecular-weight component.
Partial diploids of Escherichia coli carrying normal and mutant alleles affecting oxidative phosphorylation
Authors: Gibson F , Cox G B , Downie J A , Radik J
Published: August 2015

A plasmid was isolated which included the region of the Escherichia coli chromosome carrying the known genes concerned with oxidative phosphorylation (unc genes). This plasmid was used to prepare partial diploids carrying normal unc alleles on the episome and one of the three mutant alleles (unc A401, uncB402 or unc-405) on the chromosome. These strains were compared with segregants from which the plasmid had been lost. Dominance of either normal ormutant unc alleles was determined by growth on succinate, growth yields on glucose, Mg-ATPase (Mg2+-stimulated adenosine triphosphatase) activity, atebrin-fluorescence quenching, ATP-dependent transhydrogenase activity and oxidative phosphorylation. In all the above tests, dominance of the normal allele was observed. However, in membranes from the diploid strains which carried a normal allele and either of the mutant alleles affecting Mg-ATPase activity (uncA401 or unc-405), the energy-linked functions were only partially restored.
Ribonucleic acid stimulation of mammalian liver nuclear-envelope nucleoside triphosphatase. A possible enzymic marker for the nuclear envelope
Authors: Agutter P S , Harris J R , Stevenson I
Published: August 2015

1. The specific activity of rat and pig liver nuclear-envelope nucleoside triphosphatase (EC 3.6.1.3) decreases when the system is depleted of RNA. The activity can be restored by adding high concentrations of yeast RNA to the assay medium. 2. Exogenous RNA also increases the activity of the enzyme in control envelopes (not RNA-depleted). The effect appears to be largely specific for poly(A) and poly(G); it is not stimulated by rRNA or tRNA preparations, ribonuclease-hydrolysed RNA, AMP, or double- or single-stranded DNA. 3. Inhibitors of the enzyme, in concentrations at which half-maximal inhibition of the enzyme is achieved, do not affect the percentage stimulation of the enzyme by yeast RNA. 4. The simulation is abolished by the inclusion of 150 mM-KCl or -NaCl in the assay medium, but not by increasing the assay pH to 8.5. 5. The results are discussed in the light of the possible role of the nucleoside triphosphatase in vivo in nucleo-cytoplasmic ribonucleoprotein translocation. 6. It is proposed that poly(G)-stimulated Mg2+-activated adenosine triphosphatase activity should be adopted as an enzymic marker for the nuclear envelope.
The activation of sodium-plus-potassium ion-dependent adenosine triphosphatase from marine teleost gills by univalent cations
Authors: Bell M V , Tondeur F , Sargent J R
Published: August 2015

The apparent affinity constants for the binding of Cs+, Rb+, K+, Li+, Tl+ and NH4+ to (Na+ + K+)-dependent adenosine triphosphatase from teleost gills were measured and the values discussed in terms of the ion-selectivity isotherm described by Eisenman & Krasne (1975) [in MTP International Review of Science: Biochemistry Series One (Fox, C.F., ed.), vol. 2, pp. 27--59, Butterworths University Park Press, Baltimore]. The ion selectivity of the present enzyme is remarkably similar to that from nerve and brain.
The binding of calcium to a salivary phosphoprotein, protein C, and comparison with calcium binding to protein A, a related salivary phosphoprotein
Authors: Bennick A
Published: August 2015

The binding of Ca2+ to a salivary phosphoprotein, protein C, was studied by equilibrium dialysis. In 5mM-Tris/HCl buffer, pH 7.5, protein C bound 190 nmol of Ca2+/mg of protein. The apparent dissociation constant, K, was determined to be 1.9 x 10(-4)M and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ to protein C apparently depends on groups which ionize above pH 5.0. Ca2+ binding decreased with increased concentration of the dialysis buffer and on addition of SrCL2, MgCl2 and MnCl2 to the dialysis buffer. Digestion of protein C with trypsin or collagenase or heating of the protein to 60 degrees or 100 degrees C had little or no effect on the Ca2+ binding. Digestion of protein C with alkaline phosphatase caused a decrease in the amount of protein-bound Ca2+. This was also found for another salivary phosphoprotein, protein A. In the absence of Ca2+ the S020,w for protein C was 1.29 S and in the presence of Ca2+ it was 1.46S. Ca2+ may cause a conformational change in the protein or an aggregation of the protein molecules. No conformational changes of protein C in the presence of Ca2+ could be detected by circular dichroism or nuclear magnetic resonance.
Calcium ion-dependent myosin from decapod-crustacean muscles
Authors: Lehman W
Published: August 2015

Ca2+ regulation of arthropod actomyosin adenosine triphosphatase is associated with both the thin filaments, as in vertebrates, and with the myosin, as in molluscs. The actomyosin of decapod-crustacean fast muscles was previously considered to be an exception, displaying only a Ca2+-regulatory system linked to the thin filaments and not a myosin-linked regulatory system. In the present study, myosin regulation is demonstrated in a variety of decapod muscles when they are tested under more physiological ionic conditions. Myosin regulation is shown by using mixtures of pure rabbit actin with myofibrils, with actomyosin and with purified myosin, and in each case the adenosine triphosphatase is Ca2+ dependent. Myosin regulation may also occur in vertebrate striated muscle, but seemingly is lost during purification of the myosin.
The separation of oligonucleotides of baker's-yeast valine transfer ribonucleic acid 2b by high-voltage electrophoresis on DEAE-paper and by thin-layer chromatography
Authors: Gorbulev V G , Kutateladze T V , Barciszewski J , Axel'rod V D
Published: August 2015

A modified procedure for the separation of oligoribonucleotides is described that is based on the combination of t.l.c. on cellulose and electrophoresis on DEAE-paper at 4000 V on a cooling plate. The technique is relatively rapid and allows the analysis of larger quantities than is possible by electrophoresis on cellulose acetate.