bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of haem synthesis caused by cobalt in rat liver. Evidence for two different sites of action
Authors: De Matteis F , Gibbs A H
Published: August 2015

Cobalt inhibits liver haem synthesis in vivo by acting at least two different sites in the biosynthetic pathway: (1) synthesis of 5-aminolaevulinate and (2) conversion of 5-amino-laevulinate into haem. The first effect is largely, if not entirely, due to inhibition of the activity of 5-aminolaevulinate synthase, rather than to inhibition of the formation of the enzyme. The second effect results from diversion of 5-aminolaevulinate into an unidentified liver pool with solubility properties similar to those of cobalt protoporphyrin.
Compartmentation of free amino acids for protein biosynthesis. Influence of diurnal changes in hepatic amino acid concentrations of the composition of the precursor pool charging aminoacyl-transfer ribonucleic acid
Authors: Vidrich A , Airhart J , Bruno M K , Khairallah E A
Published: August 2015

To investigate further the mechanisms by which amino acids are segregated for protein biosynthesis, the distribution of a pulse of [3H]valine was monitored in hepatic amino acid pools at seven intervals in the diurnal cycle of meal-fed rats. Although each condition was characterized by a unique balance between intracellular and extracellular valine, in every case the specific radioactivity of valyl-tRNA at steady state was higher that that of intracellular valine but below the extracellular value. Further, the specific radioactivity of the valyl-tRNA could be accurately predicted if extracellular and intracellular valine were combined in proportions specified by the transmembrane concentration gradient. These observations not only substantiate our earlier conclusions that the amino acids used for protein synthesis do not originate exclusively from either the intracellular or extracellular pools, but also strengthen our theory that the membrane transport system is the physical basis for such compartmentation. On the basis of these data we present a method for measuring the specific radioactivity of the precursor pool for protein biosynthesis in cases where the actual isolation of the aminoacyl-tRNA is not technically feasible, and also suggest a theoretical basis for interpreting the unequal distribution of both total and [3H]valine between intracellular and extracellular fluids.
Effects of anti-microtubular agents and cycloheximide on the metabolism of chylomicron cholesteryl esters by hepatocyte suspensions
Authors: Nilsson A
Published: August 2015

1. Post-heparin plasma that promoted rapid hydrolysis of about 90% of the triacylglycerol markedly stimulated the uptake or binding of chylomicron cholesteryl ester by suspended hepatocytes. The net hydrolysis of chyle cholesteryl ester after the uptake by the cells was, however, slower than in vivo. 2. The cholesteryl ester uptake in the presence of post-heparin plasma was larger if the cells had been preincubated for 2h. It was inhibited by the presence of colchicine, vinblastine or cycloheximide during the preincubation, and by mild trypsin treatment of the preincubated cells. 3. The results suggested that the anti-microtubular agents, but not cycloheximide, also inhibited the hydrolysis of chyle cholesteryl ester after uptake or binding to the cells. 4. The uptake of isolated chylomicron remnant particles was more efficient than that of native chyle lipoproteins. It was, however, still stimulated by heparin alone and by post-heparin plasma. The heparin-stimulated uptake was markedly decreased if cycloheximide was present during the preincubation period.
The effects of hyperphenylalaninaemia on the concentrations of aminoacyl-transfer ribonucleic acid in vivo. A mechanism for the inhibition of neural protein synthesis by phenylalanine
Authors: Hughes J V , Johnson T C
Published: August 2015

An acute administration of phenylalanine to neonatal animals has been reported to result in large decreases in the intracellular concentrations of several essential amino acids in neural tissue, as well as an inhibition of neural protein synthesis. The present report evaluates the effects of the loss of amino acids on the concentrations of aminoacyl-tRNA in vivo, with the view that an alteration in the concentrations of specific aminoacyl-tRNA molecules could be the rate-limiting step in brain protein metabolism during hyperphenylalaninaemia. tRNA was isolated from saline- and phenylalanine-injected mice 30-45 min after injection, by using a procedure designed to maintain the concentrations of aminoacyl-tRNA present in vivo. Periodate oxidation of the non-acylated tRNA and aminoacylation with radioactively labelled amino acids was used to determine the proportion of tRNA that was present in vivo as aminoacyl-tRNA. Although decreases in the intracellular concentrations of alanine, lysine and leucine were observed after phenylalanine administration, the concentrations of alanyl-tRNA, lysyl-tRNA and leucyl-tRNA actually increased by 15%. Although tryptophan has been suggested to be rate-limiting during hyperphenylalaninaemia, the proportion of tryptophan tRNA that was acylated was maximal in both normal and hyperphenylalaninaemic animals. This unexpected increase in aminoacyl-tRNA concentration is discussed as perhaps a secondary effect resulting from the phenylalanine-induced inhibition of protein synthesis. In contrast, the proportion of methionine tRNA that was acylated in vivo after phenylalanine administration was demonstrated to be decreased by approx. 17%. When the isoaccepting species of methionine tRNA were separated by reverse-phase column chromatography, three species were separated, one of which was demonstrated to be the initiator species, tRNAfMet, by the selective aminoacylation and formylation with Escherichia coli enzymes. After the administration of phenylalanine, the acylation of each of the three methionine tRNA species was decreased, with the initiator species being lowered by 10%. This effect on aminoacylation of tRNAfMet may be the primary step by which phenylalanine affects neural protein synthesis, and this is consistent with previous reports that re-initiation may be inhibited during hyperphenylalaninaemia.
The dissociation of σ-factor from ribonucleic acid polymerase
Authors: Campbell A M , Lowe P A
Published: August 2015

The sigma-factor of Escherichia coli RNA polymerase was shown to dissociate from the core enzyme as a function of absolute concentration. The association constant is in the range 10(6)-10(8) litre/mol. This implies that the amount of holoenzyme, core enzyme and sigma-factor in RNA polymerase assays may vary according to the absolute concentration of the enzyme.
Kinetic properties of the primary inhibitor of plasmin from human plasma
Authors: Christensen U , Clemmensen I
Published: August 2015

The interaction of human plasmin with the newly discovered alpha2-plasmin inhibitor was investigated. It was found from rate measurements that the reaction involves the rapid formation of a first enzyme-inhibitor complex, followed by the slow irreversible transition to another complex. L-Lysine influences the first step, but not the second.
A map of the cleavage sites for endonuclease AvaI in the chromosome of bacteriophage lambda
Authors: Hughes S G
Published: August 2015

The linear order of nine fragments generated by the action of endonuclease AvaI on the DNA of bacteriophage lambda was determined from the altered fragmentation patterns of bacteriophages containing known deletions and of hybrids of bacteriophages lambda and phi80. Digestion of 5′-terminally 32P-labelled bacteriophage-lambda DNA was used to identify the terminal fragments. Measurement of relative fragment lengths permitted rough mapping of the endonuclease-AvaI cleavage sites relative to the ends of the bacteriophage-lambda chromosome. The fragment order was confirmed and the map refined by analysis of the fragmentation of derivative phages containing single cleavage sites for endonuclease EcoRI.
Activation of selenate by adenosine 5′-triphosphate sulphurylase from Saccharomyces cerevisiae
Authors: Dilworth G L , Bandurski R S
Published: August 2015

In the presence of ATP and Mg2+, ATP sulphurylase from Saccharomyces cerevisiae catalysed the conversion of selenate into a compound with the electrophoretic and acid-lability properties of adenosine 5′-sulphatophosphate. Structural characterization, involving extensive purification of adenosine 5′-selenophosphate, proved impossible. However, we showed ATP-, Mg2+- and ATP sulphurylase-dependent, and inorganic pyrophosphatase-stimulated, production of elemental selenium from selenate in the presence of GSH (reduced glutathione). Since selenate was not reduced by GSH, this reaction proved that ATP sulphurylase had formed an active selenate. The enzyme catalysed formation of elemental selenium had the same kinetics and GSH-dependency as the non-enzymic reduction of selenite to elemental selenium by GSH. In the presence of inorganic pyrophosphatase, 2 mol of Pi was released for each mol of ‘active selenate’ formed. This was shown by a spectrophotometric assay for elemental selenium. The observed reactivity with thiols and the instability of the enzymic product were those predicted for selenium anhydrides. By analogy with the chemistry of sulphur, the product of the thiolytic cleavage of a selenium anhydride would be converted into selenite. The selenite would then be reduced by the thiol to elemental selenium. We conclude that ATP sulphurylase can catalyse the formation of adenosine 5′-selenophosphate. The anhydride can be reduced by thiols in a manner similar to the reduction of selenite. These results probably explain the ability of mammals, lacking a sulphate reductase system, to incorporate selenium from selenate into seleno-amino acids.
Effects of dolichol monophosphate on galactose incorporation into glycoconjugates of cell cultures
Authors: McEvoy F A , Ellis D E , Shall S
Published: August 2015

Cultured-cell homogenates catalysed the incorporation of galactose from UDP-galactose into protein and sphingolipid acceptors. Dolichol monophosphate stimulated the incorporation of galactose into glycoproteins, but it did not affect the rate of glycosylation of either exogenous or endogenous glycosphingolipids. It is proposed that, under certain conditions, galactose may be incorporated into glycoproteins via polyisoprenol intermediates, as is the case with N-acetylglucosamine and mannose.
Characterization of a photoproduct of 7,12-dimethylbenz[a]anthracene and its effects on chick-embryo cells in culture
Authors: Warshawsky D , Kerns E , Bissell M J , Calvin M
Published: August 2015

A common impurity of 7,12-dimethylbenz[alpha]anthracene was more effective than 7,12-dimethylbenz[alpha]anthracene in inducing morphological alterations, and in causing an increase in glucose uptake, DNA synthesis and cell number in chick-embryo fibroblasts. Gradual morphological transformation follows the increase in DNA synthesis after 2 days when either primary or secondary cultures are treated with 3 microgram of the compound/ml. The compound, isolated from 7,12-dimethylbenz[alpha]anthracene by alumina column chromatography, was characterized by t.l.c., mass spectroscopy, carbon-hydrogen analysis, u.v. and nuclear-magnetic-resonance spectroscopy and thermal decomposition. It was the photo-oxidation product of 7,12-dimethylbenz[alpha]anthracene, 7,12-epidioxy-7,12-dimethylbenz[alpha]anthracene. It is suggested that some of the biological effects observed after treatment of cultures with 7,12-dimethylbenz[alpha]anthracene may be due in part to the presence of the photo-oxidation product.
A new method for assaying rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and its application in a study of the effect of dietary cholesterol on this effect of dietary cholesterol on this enzyme
Authors: Baqir Y A , Booth R
Published: August 2015

A new method suitable for measuring rat liver 3-hydroxy-3-methylglutaryl-CoA reductase activity is described and its advantages over methods previously available are discussed. An accurate time course was measured for the inhibition of liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase activity by dietary cholesterol; this enzyme was affected 1 1/4 h after the rats began to consume a cholesterol-rich diet. In this experiment there was no correlation between concentrations of microsomal cholesterol ester and the activity of 3-hydroxy-3-methylglutary-CoA reductase.
Effects of acetoacetate administration on glucose metabolism in mammary gland of fed lactating rats
Authors: Robinson A M , Williamson D H
Published: August 2015

Administration of acetoactate to fed lactating rats rapidly decreases glucose uptake by the mammary gland, and causes an output of pyruvate, whereas lactate uptake remains unchanged. Similar changes, though not identical, occur in starved lactating rats, which suggests that the increased acetoacetate concentration in this situation may be one of the factors responsible for the alterations in glucose metabolism.
The covalent linkage of protein to carbohydrate in the extracellular protein-polysaccharide from the red alga Porphyridium cruentum
Authors: Heaney-Kieras J , Rodén L , Chapman D J
Published: August 2015

The extracellular anionic polysaccharide isolated from cultures of a unicellular red alga, Porphyridium cruentum, contains a small amount of protein after extensive purification. The polysaccharide and protein are recovered in the same fraction after isopycnic CsCl-density-gradient centrifugation in 4M-guanidinium chloride, under conditions designed to separate proteins from polysaccharide. The peptide portion of the protein-polysaccharide is released from the polysaccharide by alkali under conditions for beta-elimination. The released peptide is non-diffusible, but in can be separated from the polysaccharide by precipitation of the polysaccharide as the cetylpyridinium complex. Under conditions for beta-elimination of certain O-glycosidic carbohydrate-protein linkages, selective destruction of serine and threonine occurs. The addition of a reducing agent to the alkali mixture produces a selective increase in alanine and alpha-aminobutyric acid. Addition of a tritiated reducing agent to the alkali mixture produces radioactive alanine and alpha-aminobutyric acid, and xylitol as the only sugar alcohol. Similar results are obtained from glycopeptides isolated from partial acid hydrolysates. A macromolecular structure of the protein-polysaccharide is suggested by a comparison of the intrinsic viscosity of material before and after treatment with alkali and proteolytic enzymes.
Subcellular distribution of superoxide dismutases in human neutrophils. Influence of myeloperoxidase on the measurement of superoxide dismutase activity
Authors: Rest Richard F. , Spitznagel John K.
Published: August 2015

We have identified two distinct pools of superoxide dismutase in fractions of human peripheral neutrophils obtained by the isopycnic fractionation of homogenates of the latter with linear sucrose gradients. Superoxide dismutase activity, observed with polyacrylamide gels impregnated with Nitro Blue Tetrazolium, was present in: (1) the mitochondrial fraction [density (ρ) 1.169g/ml], containing the high-molecular-weight KCN-resistant enzyme, and (2) the cytoplasm fraction, containing the low-molecular-weight KCN-sensitive enzyme. Superoxide dismutase activity, observed with a quantitative assay involving cytochrome c, was present in: (1) the mitochondria, (2) the cytoplasm, and (3) the azurophil-granule fractions (ρ=1.206 and 1.222g/ml). No substantial enzyme activity was observed in specific-granule fractions (ρ=1.187g/ml) or in the membranous fraction (ρ=1.136g/ml) in either assay. The apparent superoxide dismutase activity observed in the azurophil granules with the cytochrome c assay was attributable not to true superoxide dismutase but to myeloperoxidase, an enzyme found solely in the azurophil granules. In the presence of H2O2, human neutrophil myeloperoxidase oxidized ferrocytochrome c. Thus, in the cytochrome c assay for superoxide dismutase, the oxidation of ferrocytochrome c by myeloperoxidase mimicked the inhibition of reduction of ferricytochrome c by superoxide dismutase. When myeloperoxidase was removed from azurophilgranule fractions by specific immuno-affinity chromatography, both myeloperoxidase and apparent superoxide dismutase activities were removed. It is concluded that there is no detectable superoxide dismutase in either the azurophil or specific granules of human neutrophils. Mitochondrial superoxide dismutase, 15% of the total dismutase activity of the cells, occurred only in fractions of density 1.160g/ml, where isocitrate dehydrogenase and cytochrome oxidase were also observed.
The degradation and turnover of fucosylated glycoproteins in the plasma membrane of a neuroblastoma-cell line
Authors: Hudson James E. , Johnson Terry C.
Published: August 2015

When monolayer cultures of neuroblastoma N2a cells were prelabelled with [3H]fucose to steady state, and then reincubated in complete medium in the presence of unlabelled 40mm-l-fucose, there was a rapid metabolism of fucosylated cellular macromolecules and the specific radioactivity of the acid-insoluble material decreased by 22% within 2h. After this period of time the remaining radioactive glycoproteins appeared to be more stable and the rate of loss of specific radioactivity markedly decreased. Since fucose is known to be associated predominantly with plasma-membrane components, the analysis of fucosylated glycoproteins was characterized in plasma-membrane fractions by polyacrylamide-gel electrophoresis. Two experimental approaches were used to measure glycoprotein degradation and turnover in the cell-surface membranes. In one set of experiments, with a similar incubation procedure to that used with intact cells, three membrane components were rapidly degraded (150000, 130000 and 48000 daltons), but another surface glycoprotein (68000 daltons) appeared to be more slowly metabolized than the mean rate of glycoprotein degradation. The relationship of the degradation of membrane glycoproteins to their turnover was analysed by dual-label experiments that used both [14C]fucose and [3H]fucose. Glycoproteins of the surface membrane of neuroblastoma cells were found to turn over at heterogeneous rates. The components mentioned above that exhibited significantly rapid rates of degradation, were also shown to turn over more rapidly than the average surface component. In addition to the membrane components detected by the use of only [3H]fucose, dual-label experiments illustrated that numerous surface glycoproteins were metabolized more rapidly or slowly than most of the cell-surface constituents.
Characteristics and postnatal development of the acid lipase activity of the rat small intestine
Authors: Coates P M , Brown S A , Jumawan J , Koldovský O
Published: August 2015

Acid lipase was identified in the rat small intestine by using esters of 4-methylumbelliferone as substrates. Maximum activity towards the oleate ester was found at pH 4.0. In adult animals, the activity of acid lipase exhibited both latency and sedimentability, indicating a lyosomal localization. The activity of acid lipase was practically the same along the height of the villus, thus paralleling the distribution of acid beta-galactosidase. In adult rats, the activity of acid lipase in proximal (jejunum) and middle (mid-jejunum) sections of the small intestine was practically the same and exceeded the activity in the distal (ileum) section by a factor of 2. In suckling rats, the activity of the enzyme in the mid-jejunum exceeded that in the jejunum and ileum by 2.5- and 1.5-fold respectively. During postnatal development, the acid lipase activity of the mid-jejunum showed a peak between days 10 and 15, at which time it exceeded the adult mid-jejunum activity by 5–6-fold.
Loss of haem from cytochrome P-450 caused by lipid peroxidation and 2-allyl-2-isoprophylacetamide. An abnormal pathway not involving production of carbon monoxide
Authors: De Matteis F , Gibbs A H , Unseld A
Published: August 2015

1. Microsomal preparations undergoing lipid peroxidation produce CO and lose haem from cytochrome P-450. 2. The amount of CO produced does not correlate with the amount of haem lost and, after pre-labelling of microsomal haem in its bridges with 5-amino[5-14C]laevulinate, the radioactivity lost from haem is not recorved as CO. 3. Similarly, when pre-labelled microsomal haem is destroyed by the action of 2-allyl-2-isopropylacetamide, no radioactivity is recovered as CO. In clear contrast, on degradation of haem by the haem oxygenase system, CO is produced in an amount equimolar to the haem lost. 4. It is concluded that (a) the CO produced during lipid peroxidation originates from a source different from haem and (b) the degradations of haem caused by lipid peroxidation and 2-allyl-2-isopropylacetamide do not involve to any significant extent evolution of the methene-bridge carbon of haem as CO.
The uptake and release of calcium by heart mitochondria
Authors: Harris Eric J.
Published: August 2015

The kinetics of uptake of Ca2+ by rat heart mitochondria were studied by a spectrophotometric method with Arsenazo III indicator. The exponential rate coefficients measured with or without added phosphate increase with the amount of Ca2+ added up to about 24μm. Evidence is given that the effect is attributable to a combination of formation of chelates at low concentrations to act as Ca2+ buffers, with co-transport of substrate to provide more respiratory fuel. The inhibitory effect of Mg2+ depends on the Ca2+ concentration, so with a constant [Mg2+] the low concentrations of Ca2+ are most inhibited, and the rate coefficients are still more Ca2+-dependent. Ca2+ uptake is slowed by local anaesthetics such as butacaine and dibucaine, and also by propranolol and palmitoyl-CoA. After an uptake, the release of Ca2+ was investigated. The spontaneous release involves an initially slow and small appearance of free Ca2+ and is followed by an auto-accelerated phase. The release is accompanied by a gradual decrease in internal ATP; it is initiated by palmitoyl-CoA (reversed by carnitine), by lysophosphatidylcholine, by Na+ salts (reversed by oligomycin) and by K+ salts added to a K+-free medium containing valinomycin. The process is probably a response to an increased energy load imposed on the mitochondria by the various conditions, which include the spontaneous action of phospholipase activated by traces of Ca2+. The problem of how much mitochondrial activity is participating in normal heart Ca2+ turnover is discussed, and experiments showing only 7–14% exchange of the mitochondrial Ca2+ occurring in vivo in 10 or 20min are reported.
Vitamin A receptors. Retinoic acid binding in ocular tissues
Authors: Wiggert B , Bergsma D R , Helmsen R , Chader G J
Published: August 2015

Analysis of the sucrose-density-gradient patterns of the 110 000g supernatant fractions of adult and foetal retina and pigment epithelium showed them to contain a limited number of highly specific binding sites (‘receptors’) for [3H]retinoic acid that sediment at approx. 2S. Binding in pigment epithelium is higher than in any tissue yet reported. A 5S binding component is also observed and is probably due to serum contamination. Fractionation studies indicate that [3H]retinoic acid binding in the retina is lower in the photoreceptor units than in the retinal inner layers. This is in contrast with previous results that show greater [3H]retinol binding in photoreceptors. Studies with dystrophic human and rat retinas, which lack the photoreceptor layers, confirm that [3H]retinoic acid binding is greater in the non-photoreceptor layers of the retina. No specific [3H]retinoic acid binding is found in corneal epithelium, although endothelium and the conjunctiva demonstrate specific 2S binding. Such differences in retinol and retinoic acid binding may indicate different roles for the two compounds in ocular tissues.
Oestrogen receptor of mammary gland. Inhibition of aggregation and characterization of receptor from lactating gland in the presence of sodium bromide
Authors: Auricchio Ferdinando , Rotondi Andrea , Schiavone Ettore , Bresciani Francesco
Published: August 2015

1. When NaBr, a chaotropic salt, is added, in concentrations ranging from 0.5m to 2m, to low-salt mammary cytosol, (i) age-dependent aggregation of oestrogen receptor is inhibited, (ii) the receptor sediments as a sharp peak at 4.2S on sucrose-gradient centrifugation, with complete disappearance of heavier forms, and (iii) on gel filtration with Sephadex G-200, the receptor is included in the gel matrix. On a calibrated column, the receptor has a Stokes radius of 3.7nm (±6%). 2. Because NaBr inhibits interaction of receptor with other components of cytosol, the values of the sedimentation coefficient, measured by sucrose-gradient sedimentation, and of the Stokes radius, measured by gel filtration, can be accepted with confidence. From these values, it can be computed that the oestrogen-receptor form in NaBr has a mol.wt. of 64000, with a frictional ratio of 1.4. 3. Also, inhibition of aggregation by NaBr allows a 30–90-fold purification of oestrogen receptor. Analysis of this partially purified receptor by sucrose-gradient sedimentation and gel filtration in NaBr gives the same results as for receptor in crude cytosol. On electrofocusing on a pH5–8 gradient, the partially purified oestrogen receptor focuses at pH6.2. On removal of NaBr, receptor aggregates even in this partially purified state. It seems likely that at the protein and ionic concentrations of cytoplasm in vivo, the 64000-mol.wt. receptor form is part of higher states of self- and/or hetero-association with other cytoplasmic components. 4. NaBr up to a concentration of 2m does not inhibit binding of oestrogen by receptor, nor does it decrease the affinity of the interaction (KD≃8.9×10−10m). The total number of binding sites in cytosol, however, decreases by approx. 10%, but this decrease may actually be the result of elimination of lower-affinity binding by non-receptor components of cytosol. 5. NaSCN, another chaotropic salt, was also tested but gave less satisfactory results with the mammary cytosol than with uterine cytosol. EDTA was omitted from the buffers because it favours aggregation of mammary oestrogen receptor. KCl (0.4m), sucrose (15%) and ZnSO4 (3mm) did not prevent aggregation of receptor.
The preparation and purification of individual human pepsins by using diethylaminoethyl-cellulose
Authors: Roberts N B , Taylor W H
Published: August 2015

A procedure was devised for isolating human pepsins 1, 2, 3 and 5 from gastric juice by repetitive column chromatography on DEAE-cellulose. The combined yields in four different experiments varied from 14% to 90% of the total peptic activity of the starting material. The isolated individual pepsins were shown to behave as single homogeneous proteins on agar-gel electrophoresis at pH 5.0 and on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. There is preliminary evidence, requiring further study, of two other pepsins, one migrating between pepsins 1 and 2 and the other a pepsin-3 component associating closely with pepsin 5 on chromatography.
Rapid and convenient method for the assay of aminopropyltransferases
Authors: Hibasami H , Pegg A E
Published: August 2015

A new method for the assay of aminopropyltransferase activity is described. The method measures the formation of [methyl-14C]methylthioadenosine from decarboxylated S-adenosyl[methyl-14C]methionine in the presence of an amine acceptor. When used with extracts from rat ventral prostate, kidney, liver or brain, and with putrescine or spermidine as amines, the method gave results in excellent agreement with those obtained by the much more time-consuming conventional method. It was found that 1,3-diamino-propane and 1,8-diamino-octane were not acceptors for the prostatic enzyme fraction, but 1,5-diaminopentane (cadaverine) was active and 1,9-diaminononane and 1,12-diaminododecane also lead to the production of [methyl-14C]methylthioadenosine.
Fatty acid synthesis in the regenerating liver of the rat
Authors: Gove C D , Hems D A
Published: August 2015

1. Synthesis de novo of fatty acids in the rat liver, measured per g wet wt. of tissue, was increased by a factor of about two, between 1 and 4 days after partial hepatectomy, compared with rates in sham-operated control rat livers. 2. There were no associated changes in the rates of liver cholesterol synthesis or of adipose-tissue fatty acid synthesis in rats after partial hepatectomy, compared with rates in sham-operated rats. 3. In regenerating livers, perfused under three different conditions, there was no alteration in the capacity for fatty acid synthesis compared with that of control rats. 4. The increased synthesis of fatty acids in regenerating liver was associated with insignificant increases in plasma concentrations of tricylglycerols and free fatty acids, with a decrease in content of liver glycogen, and with no change in hepatic activity of acetyl-CoA carboxylase. 5. The accelerated rate of synthesis of fatty adids in regenerating liver appears not to be due to any intrinsic alteration in hepatic capacity for fatty acid synthesis, but it may be caused by the continuous action on liver of unidentified circulating factors.
Calcium-ion transport by intact Ehrlich ascites-tumour cells. Role of respiratory substrates, Pi and temperature
Authors: Charlton R R , Wenner C E
Published: August 2015

1. The interaction of intact Ehrlich ascites-tumour cells with Ca2+ at 37 degrees C consists of Ca2+ uptake followed by efflux from the cells. Under optimum conditions, two or three cycles of uptake and efflux are observed in the first 15 min after Ca2+ addition. 2. The respiratory substrates malate, succinate and ascorbate plus p-phenylenediamine support Ca2+ uptake. Ca2+ uptake at 37 degrees C is sensitive to the respiratory inhibitors rotenone and antimycin A when appropriate substrates are present. Ca2+ uptake and retention are inhibited by the uncoupler S-13. 3. Increasing extracellular Pi (12 to 30 mM) stimulates uncoupler-sensitive Ca2+ uptake, which reaches a maximum extent of 15 nmol/mg of protein when supported by succinate respiration. Ca2+ efflux is partially inhibited at 30 mM-Pi. 4. Optimum Ca2+ uptake occurs in the presence of succinate and Pi, suggesting that availability of substrate and Pi are rate-limiting. K. Ca2+ uptake occurs at 4 degrees C and is sensitive to uncouplers and oligomycin. Ca2+ efflux at this temperature is minimal. These data are consistent with a model in which passive diffusion of Ca2+ through the plasma membrane is followed by active uptake by the mitochondria. Ca2+ uptake is supported by substrates entering respiration at all three energy-coupling sites. Ca2+ efflux appears to be an active process with a high temperature coefficient.
Haemichrome formation from haemoglobin subunits by hydrogen peroxide
Authors: Tomoda A , Sugimoto K , Suhara M , Takeshita M , Yoneyama Y
Published: August 2015

The effect of H2O2 on ferrous human haemoglobin subunits (alphash-, betash-, alphapmb- and betapmb-chains) was studied. These chains were easily transformed to haemichrome by the addition of H2O2 or H2O2-generating systems, including glucose oxidase (EC 1.1.3.4) AND XANTHINE OXIDASE (EC 1.2.3.2), and this was ascertained by e.p.r. measurements and by absorption spectra. The changes in these haemoglobin subunits were not inhibited by superoxide dismutase (EC 1.15.1.1), but were decreased by catalase (EC 1.11.1.6). The rate of oxidation of alphapmb-chains was higher than that of alphash-chains, and the rate of oxidation of betapmb-chains was higher than that of betash-chains. Haemichrome was demonstrated to be formed directly from these ferrous chains by the attack by H2O2, and this process did not involve formation of methaemoglobin. On the basis of these findings the kinetics of the reaction between the haemoglobin subunits and H2O2 was studied, and the pathological significance of H2O2 in disorders of erythrocytes such as thalassaemia was discussed.
Boundary refinement of the lysozyme antigenic site around the disulphide bond 6–127 (site 1) by ‘surface-simulation’ synthesis
Authors: Atassi M. Zouhair , Lee Ching-Li
Published: August 2015

1. We have previously shown that an antigenic site (site 1) in native lysozyme resides around the disulphide bond 6–127 and, by classical synthesis of nine disulphide peptides, the antigenic site was accurately narrowed down to the structure Cys(6)–Arg(14)–[Cys(6)–Cys(127)] –Gly(126)–Arg(128). Only a few residues on this disulphide peptide were proposed to be involved in the reactivity with antibody. However, this lacked direct verification and the role of Arg-128 remained uncertain. 2. In the present work, several peptides were designed and synthesized by the surface-simulation concept devised in our laboratory. These enabled the precise definition of the site as well as the investigation of its conformational and directional requirements. 3. The results showed that the antigenic site (site 1) is made up of the spatially contiguous surface residues: Arg-125, Arg-5, Glu-7, Arg-14, Lys-13. The surface-simulation synthetic peptide Arg-Gly-Gly-Arg-Gly-Glu-Gly-Gly-Arg-Lys (which does not exist in native lysozyme, but copies a surface region of it) accounted entirely for the maximum expected reactivity of the site (i.e. about one-third of the total antigenic reactivity of lysozyme). An immunoadsorbent of the peptide also removed about one-third of the total lysozyme antibodies. 4. The antigenic site exhibited restricted conformational freedom. The achievement of the full reactivity of the site by surface-simulation synthesis requires the appropriate choice of spacer separation between its reactive residues. The surface-simulation synthetic site exhibits the same mono-directional preference (Arg-125 to Lys-13) for the rabbit and goat antisera so far tested. The site describes a line which encircles a part (3.01 nm in C(α)-to-C(α) distance from Arg-125 to Lys-13) of the surface of the molecule.
Studies of an acid-induced species of purple membrane from Halobacterium halobium
Authors: Moore T A , Edgerton M E , Parr G , Greenwood C , Perham R N
Published: August 2015

A new spectral species of the purple membrane of Halobacterium halobium has been observed below pH 3.2. The formation of this new species is temperature-dependent and is favoured by increasing temperature up to the physiological range of the organism. The rate of formation at pH 3.0 and 22 degrees C is 7.9 × 10-3s-1. The spectral distribution and temperature-dependence of the new species suggest that it may be phototransiet O, stabilized by low pH. Flash-photolytic experiments in the pH range 7.2-2.7 show a pH-dependence corresponding to the static events and are consistent with a single protonation of bacteriorhodopsin below pH 3.22. These results can also be interpreted in terms of the stabilization of phototransient O at low pH. The temperature-dependence of the formation of the acid-induced species may reflect a relationship with the phase transition of the membrane.
Simplified preparation of a phosphatase inhibitor and further studies of its action
Authors: Coburn S P , Schaltenbrand W E
Published: August 2015

1-Pyrrolidinecarbothioic acid (2-pyridylmethylene) hydrazide chelates Zn2+ but not Mg2+. This compound is about twice as effective as EDTA for inhibiting alkaline phosphatase from calf mucosa, and approx. 1000-fold more effective than EDTA for inhibiting acid phosphatase from wheat germ. The compound did not inhibit pyridoxine kinase activity in human leucocytes at the highest concentration tested (33 micron). Therefore it may be a useful tool for either examining or eliminating the effects of phosphatases in complex enzyme systems.
The effects of acridine orange on deoxyribonucleic acid in Escherichia coli
Authors: Barker G R , Hardman N
Published: August 2015

1. Acridine Orange inhibits growth of Escherichia coli K12 when incubated at pH 7.9, but not at pH 7.4.2. At a non-permissive temperature for DNA polymerase I, Acridine Orange inhibits growth of a temperature-sensitive strain and also increases the rate of elimination of the F'-Lac plasmid. 3. DNA isolated from cells treated with Acridine Orange under conditions that inhibit growth contains material of low molecular weight, which is absent from DNA isolated from cells treated under conditions in which growth is not impaired. 4. Cells incubated with Acridine Orange at both pH 7.4 and 7.9 suffer degradation of DNA, as shown by loss of labelled DNA from the acid-insoluble fraction, which is not observed with untreated cells at either pH. 5. The results suggest that elimination of the F'-Lac plasmid by Acridine Orange requires inactivation of repair processes.
Intramolecular labelling of sucrose made by leaves from [14C)carbon dioxide or [3-14C]serine
Authors: Bird I F , Cornelius M J , Keys A J , Whittingham C P
Published: August 2015

Pea leaves were illuminated in air containing 150 or 1000p.p.m. of 14CO2 for various times. Alternatively, segments of wheat leaves were supplied with [3-14C]serine for 40 min in the light in air with 145, 326 or 944p.p.m. of 12CO2. Sucrose was extracted from the leaf material, hydrolysed with invertase, and 14C in the pairs of carbon atoms C-3+C-4, C-2+C-5 and C-1+C-6 in the glucose moiety was measured. The results obtained after metabolism of 14CO2 were consistent with the operation of the photosynthetic carbon-reduction cycle; the effects of CO2 concentration on distribution of 14C in the carbon chain of glucose after metabolism of [3-14C]serine is more easily explained by metabolism through the glycollate pathway than by the carbon-reduction cycle.
The effects of diphosphonates on the growth and glycolysis of connective-tissue cells in culture
Authors: Fast D K , Felix R , Dowse C , Neuman W F , Fleisch H
Published: August 2015

1. The effects of two diphosphonates (compounds containing a P-C-P bond), disodium dichloromethanediphosphonate and disodium 1-hydroxyethane-1,1-diphosphonate, on the metabolism of cultured rat calvaria cells, rabbit ear cartilage cells and rat skin fibroblasts were investigated. 2. The diphosphonates had no effect on the growth of cartilage cells and on the exponential growth of the calvaria cells and the fibroblasts. However, dichloromethanediphosphonate stopped the growth of the calvaria cells and the fibroblasts after the beginning of confluence, whereas the untreated cells were still growing to a certain extent. This inhibition was dose-dependent. After the drug was withdrawn, the cells recovered slowly. 1-Hydroxyethane-1,1-diphosphonate had no detectable effect on the growth of any of the cell types studied. Both diphosphonates decreased the cloning efficiency of calvaria cells and fibroblasts. 3. The K+ content of cartilage, calvaria and skin cells was diminished only by the highest (0.25 mM) concentration of dichloromethanediphosphonate. 4. Radioactive dichloromethanediphosphonate and 1-hydroxyethane-1,1-diphosphonate were taken up linearly with time for at least 48 h by calvaria cells and fibroblasts. The diphosphonate concentration in the cells depended on its concentration in the medium. 5. Both diphosphonates, in a dose-dependent fashion, markedly inhibited glycolysis, dichloromethanediphosphonate being more effective than 1-hydroxyethane-1,1-diphosphonate, at drug doses that had no effect on cell growth or cellular K+ content. Calvaria cells were much more sensitive than cartilage cells. When cartilage cells were cultured in an N2 atmosphere, these effects on glucose and lactate metabolism disappeared. 6. As increased acid production appears to be associated with resorption of bone, this decrease in lactate may explain why diphosphonates are effective inhibitors of bone resorption in vivo.
Comparative studies on two ferredoxins from the cyanobacterium Nostoc strain MAC
Authors: Hutson K G , Rogers L J , Haslett B G , Boulter D , Cammack R
Published: August 2015

Two ferredoxins were isolated from the cyanobacterium Nostoc strain MAC grown autotrophically in the light or heterotrophically in the dark. In either case approximately three times as much ferredoxin I as ferredoxin II was obtained. Both ferredoxins had absorption maxima at 276, 282 (shoulder), 330, 423 and 465 nm in the oxidized state, and each possessed a single 2 Fe-2S active centre. Their isoelectric points were approx. 3.2. The midpoint redox potentials of the ferredoxins differed markedly; that of ferredoxin I was −350mV and that of ferredoxin II was −445mV, at pH 8.0. The midpoint potential of ferredoxin II was unusual in being pH dependent. Ferredoxin I was most active in supporting NADP+ photoreduction by chloroplasts, whereas ferredoxin II was somewhat more active in pyruvate decarboxylation by the phosphoroclastic system of Clostridum pasteurianum. Though the molecular weights of the ferredoxins determined by ultracentrifugation were the same within experimetnal error, the amino acid compositions showed marked differences. The N-terminal amino acid sequences of ferredoxins I and II were determined by means of an automatic sequencer. There are 11–12 differences between the sequences of the first 32 residues. It appears that the two ferredoxins have evolved separately to fulfil different roles in the organism.
The amino acid sequence of the tryptic peptides from actinidin, a proteolytic enzyme from the fruit of Actinidia chinensis
Authors: Carne A , Moore C H
Published: August 2015

The amino acid sequences of the tryptic peptides of the thiol proteinase actinidin from Actinidia chinensis were determined by the manual dansyl–Edman procedure. There are 12 tryptic peptides, which give a polypeptide chain of 220 residues with a mol.wt. of 23500. An alignment of the tryptic peptides was made by using the X-ray-crystallographic data of Baker [(1977) J. Mol. Biol. 115, 263–277] determined at 0.28 nm resolution on crystalline actinidin. Detailed evidence for the amino acid sequences of the tryptic peptides has been deposited as Supplementary Publication SUP 50083 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.
Subcellular distribution of glycosidases in human polymorphonuclear leucocytes
Authors: Rest R F , Cooney M H , Spitznagel J K
Published: August 2015

The subcellular distribution of nine glycosidases were studied in fractions of homogenized human polymorphonuclear leucocytes (neutrophils) obtained by isopycnic centrifugation through linear sucrose density gradients. The substrates were 4-methylumbelliferyl glycosides. All nine glycosides were hydrolysed by enzymes in neutrophil cytosol fractions, and by enzymes in at least one granule population. alpha-Glucosidase activity sedimented in sucrose density gradients to a point (p = 1.180 g/ml) just above the specific granules, possibly the ‘tertiary’ granule population. The peak corresponding to alpha-glucosidase did not co-sediment with, but considerably overlapped, the peak corresponding to lactoferrin, a marker for specific granules (p = 1.187 g/ml). alpha-Galactosidase activity was found primarily in heavy azurophil granules (p = 1.222 g/ml). alpha-Mannosidase activity was found primarily in light azurophil granules (p = 1.206 g/ml), following the distribution of myeloperoxidase, the commonly used azurophil granule marker. beta-Glucosidase activity was concentrated in mitochondrial fractions (p = 1.160 g/ml). All other glycosidases presented complex distributions, with activities not restricted to one granule class. Granule-associated glycosidase activities were increased 2–38 times when measured in the presence of 0.05% Triton X-100, indicating latency of the enzymes within granules.
Alterations in response of rat white adipocytes to insulin, noradrenaline, corticotropin and glucagon after adrenalectomy. Correction of these changes by adenosine deaminase
Authors: Fernandez B M , Saggerson E D
Published: August 2015

1. Adipocytes isolated from rats 6–9 days after adrenalectomy had significantly increased sensitivity to insulin action against noradrenaline-stimulated lipolysis. In the presence of adenosine deaminase there was no significant difference in insulin sensitivity between cells from adrenalectomized and sham-operated rats. 2. Adipocytes from adrenalectomized rats had decreased lipolytic responses to all concentrations of noradrenaline and glucagon tested and a decreased lipolytic response to low but not high concentrations of corticotropin. There was no difference in lipolytic response to theophylline after adrenalectomy. Adenosine deaminase corrected the differences in response to noradrenaline and glucagon resulting from adrenalectomy. 3. In the presence of adenosine deaminase rates of lipolysis, after stimulation by high concentrations of noradrenaline, glucagon, corticotropin or theophylline, were the same in cells from adrenalectomized or sham-operated rats. 4. These findings and previously reported effects of adenosine and adrenalectomy on adipocyte function are discussed. It is proposed that changes in adipocyte hormone responsiveness after adrenalectomy may result from changes in adenosine metabolism or release.
The protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans. Magnitude, sites of generation and comparison with the phosphorylation potential
Authors: Kell Douglas B. , John Philip , Ferguson Stuart J.
Published: August 2015

1. The magnitude of the protonmotive force in phosphorylating membrane vesicles from Paracoccus denitrificans was estimated. The membrane potential component was determined from the uptake of S14CN−, and the transmembrane pH gradient component from the uptake of [14C]methylamine. In each case a flow-dialysis technique was used to monitor uptake. 2. With NADH as substrate, the membrane potential was about 145mV and the pH gradient was below 0.5 pH unit. The membrane potential was decreased by approx. 15mV during ATP synthesis, and was abolished on addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the presence of KCl plus valinomycin the membrane potential was replaced by a pH gradient of 1.5 units. 3. Succinate oxidation generated a membrane potential of approx. 125mV and the pH gradient was below 0.5 pH unit. Oxidation of ascorbate (in the presence of antimycin) with either 2,3,5,6-tetramethyl-p-phenylenediamine or NNN′N′-tetramethyl-p-phenylenediamine as electron mediator usually generated a membrane potential of approx. 90mV. On occasion, ascorbate oxidation did not generate a membrane potential, suggesting that the presence of a third energy-coupling site in P. denitrificans vesicles is variable. 4. With NADH or succinate as substrate, the phosphorylation potential (ΔGp=ΔG0′+RTln[ATP]/ [ADP][Pi]) was approx. 53.6kJ/mol (12.8kcal/mol). Comparison of this value with the protonmotive force indicates that more than 3 protons need to be translocated via the adenosine triphosphatase of P. denitrificans for each molecule of ATP synthesized by a chemiosmotic mechanism. In the presence of 10mm-KNO3 the protonmotive force was not detectable (<60mV) but ΔGp was not altered. This result may indicate either that there is no relationship between the protonmotive force and ΔGp, or that for an unidentified reason the equilibration of SCN− or methylamine with the membrane potential and the pH gradient is prevented by NO3− in this system.