bluuebunny/crossref_metadata_2025 huggingface dataset:
Complement-mediated lysis of pigeon erythrocyte ghosts analysed by flow cytometry. Evidence for the involvement of a ‘threshold’ phenomenon
Authors: Edwards S W , Morgan B P , Hoy T G , Luzio J P , Campbell A K
Published: August 2015

Flow-cytometric analysis of complement-mediated lysis of antibody-coated pigeon erythrocyte ghosts containing fluorescein was carried out to determine whether lysis involved a gradual release of fluorescein or a ‘threshold’ release from individual cells. Antibody-coated ghosts were comprised of three subpopulations identified by fluorescence and scatter (size). These were: (a) highly fluorescent, medium scatter, (b) medium fluorescence, high scatter, and (c) low (or zero) fluorescence, low scatter. Lysed ghosts and isolated nuclei were identified by fluorescence microscopy and scanning electron microscopy. Fluorescence distributions analysed by flow cytometry indicated that, after complement attack, those ghosts remaining intact retained all their fluorescent label. A time course of changes in ratios of the three subpopulations indicated that once lysis of an individual ghost was initiated, release of label was complete within 1 min; no stages of intermediary fluorescence appeared, and those ghosts remaining at the end of the experiment retained the same fluorescence intensity as control ghosts. The results supported the hypothesis that complement-mediated cell lysis is a ‘threshold’ phenomenon; a submaximal response by a cell population representing a complete response by only some of the cells rather than a partial response by all of the cells.
Red-cell amino acid transport. Evidence for the presence of system ASC in mature human red blood cells
Authors: Young J D , Wolowyk M W , Jones S M , Ellory J C
Published: August 2015

The properties of Na+-dependent L-alanine transport in human erythrocytes were investigated using K+ as the Na+ substitute. Initial rates of Na+-dependent L-alanine uptake (0.2 mM extracellular amino acid) for erythrocytes from 22 donors ranged from 40 to 180 mumol/litre of cells per h at 37 degrees C. Amino acid uptake over the concentration range 0.1-8 mM was consistent with a single saturable component of Na+-dependent L-alanine transport. Apparent Km and Vmax. values at 37 and 5 degrees C measured in erythrocytes from the same donor were 0.27 and 0.085 mM respectively, and 270 and 8.5 mumol/litre of cells per h respectively. The transporter responsible for this uptake was identified as system ASC on the basis of cross-inhibition studies with a series of 42 amino acids and amino acid analogues. Apparent Ki values for glycine, L-alpha-amino-n-butyrate, L-serine and L-leucine as inhibitors of Na+-dependent L-alanine uptake at 37 degrees C were 4.2, 0.12, 0.16 and 0.70 mM respectively. Reticulocytes from a patient with inherited pyruvate kinase deficiency were found to have a 10-fold elevated activity of Na+-dependent L-alanine uptake compared with erythrocytes from normal donors. Separation of erythrocytes according to cell density (cell age) established that even the oldest mature erythrocytes retained significant Na+-dependent L-alanine transport activity. Amino acid transport was, however, a more sensitive indicator of cell age than acetylcholinesterase activity. Erythrocytes were found to accumulate L-alanine against its concentration gradient (distribution ratio approx. 1.5 after 4 h incubation), an effect that was abolished in Na+-free media. Na+-dependent L-alanine uptake was shown to be associated with L-alanine-dependent Na+ influx, the measured coupling ratio being 1:1.
Metabolic effects of bacitracin in isolated rat hepatocytes
Authors: Agius L , Wilding C , Alberti K G M M
Published: August 2015

Bacitracin is a proteolytic inhibitor which interacts with the intracellular processing of insulin. Its effects on pyruvate, fatty acid and amino acid metabolism were examined in rat hepatocyte suspensions. Bacitracin (0.25-1.0 mM) increased the oxidation of [1-14C]pyruvate by 50-70% and presumably therefore increased the flux through pyruvate dehydrogenase. This was found both in the presence of extracellular Ca2+ and in its absence, but not in the presence of 2 mM-2-chloropropionate, which inhibits pyruvate dehydrogenase kinase. Insulin did not further stimulate [1-14C]pyruvate oxidation in the presence of 1 mM-bacitracin. Bacitracin decreased 14CO2 formation from [2-14C]pyruvate (20-40%) and [U-14C]palmitate (30-70%), suggesting a decreased flux through the tricarboxylic acid cycle. Fatty acid oxidation before acetyl-CoA formation was also decreased. Bacitracin decreased the incorporation of label from [3H]leucine into protein in the absence of insulin, but not in its presence. Bacitracin is commonly used in studies on insulin action. Our results suggest that in such studies the effects noted may be related not only to an interaction of bacitracin with the intracellular processing of insulin but also to direct metabolic effects of bacitracin independent of insulin.
The binding of monoclonal antibodies to cell-surface molecules. A quantitative analysis with immunoglobulin G against two alloantigenic determinants of the human transplantation antigen HLA-A2
Authors: Ways J P , Parham P
Published: August 2015

Monoclonal IgG1 (immunoglobulin G1) PA2.1 and MA2.1 antibodies recognize polymorphic sites of the human transplantation antigen HLA-A2. They are distinguishable because MA2.1 binds HLA-A2 and HLA-B17, whereas PA2.1 binds HLA-A2 and HLA-A28. The affinities of PA2.1-Fab for HLA-A2, three HLA-A2 variants and HLA-A28 are similar and relatively low (1.9 × 10(7) M-1). The affinities of MA2.1-Fab for HLA-A2, three HLA-A2 variants and HLA-B17 are similar and high (1.2 × 10(9) M-1). The difference in affinity is due to the rates of dissociation, which give half-times of dissociation of 290 min for MA2.1-Fab and 4 min for PA2.1-Fab. For both Fab, equilibrium measurements and kinetic determinations gave consistent estimates for affinity. When PA2.1-F(ab)2 or IgG is incubated with cells it reaches equilibrium within 3 h, with most molecules bound bivalently to the cell. Under similar conditions, MA2.1-F(ab)2 does not reach equilibrium and a significant proportion of molecules bound with one and two sites are found. For the lower-affinity antibody (PA2.1), estimates of the binding constants for one- and two-site interactions could be made. By simple Scatchard analysis the avidity of F(ab)2 or IgG is 1.3 × 10(9) M-1, giving an enhancement factor of 68 between bivalent and univalent binding. This is a measure of the equilibrium constant for the interchange between bivalent and univalent binding. Analysis of the results with more realistic models indicates that the actual value is larger (10(3)-10(4) M-1) than 68 M-1. The avidities of F(ab)2 and IgG for HLA-A2 are identical, showing the Fc does not interfere with bivalent binding to cells.
Phosphatidylinositol hydrolysis in isolated guinea-pig islets of Langerhans
Authors: Schrey M P , Montague W
Published: August 2015

Previous studies have reported an increased turnover of phospholipid in isolated islets of Langerhans in response to raised glucose concentrations. The present investigation was thus undertaken to determine the nature of any phospholipases that may be implicated in this phenomenon by employing various radiolabelled exogenous phospholipids. Hydrolysis of 1-acyl-2-[14C]arachidonoylglycerophosphoinositol by a sonicated preparation of islets optimally released radiolabelled lysophosphatidylinositol, arachidonic acid and 1,2-diacylglycerol at pH 5,7 and 9 respectively. This indicates the presence of a phospholipase A1 and a phospholipase C. However, the lack of any labelled lysophosphatidylinositol production when 2-acyl-1-[14C]stearoylglycerophosphoinositol was hydrolysed argues against a role for phospholipase A2 in the release of arachidonic acid. Phospholipase C activity as measured by phosphatidyl-myo-[3H]inositol hydrolysis was optimal around pH8, required Ca2+ for activity and was predominantly cytosolic in origin. The time course of phosphatidylinositol hydrolysis at pH 6 indicated a precursor-product relationship for 1,2-diacylglycerol and arachidonic acid respectively. The release of these two products when phosphatidylinositol was hydrolysed by either islet or acinar tissue was similar. However, phospholipase A1 activity was 20-fold higher in acinar tissue. Substrate specificity studies with islet tissue revealed that arachidonic acid release from phosphatidylethanolamine and phosphatidylcholine was only 8% and 2.5% respectively of that from phosphatidylinositol. Diacylglycerol lipase was also demonstrated in islet tissue being predominantly membrane bound and stimulated by Ca2+. The availability of non-esterified arachidonic acid in islet cells could be regulated by changes in the activity of a phosphatidylinositol-specific phospholipase C acting in concert with a diacylglycerol lipase.
Dependence of fluid-phase pinocytosis in arterial smooth-muscle cells on temperature, cellular ATP concentration and the cytoskeletal system
Authors: Muir E M , Bowyer D E
Published: August 2015

125I-labelled poly(vinylpyrrolidone) was used as a marker of fluid-phase pinocytosis in cultured pig arterial smooth-muscle cells. The rate of pinocytosis was temperature-dependent. A decrease in cellular ATP concentrations as a result of inhibition of either glycolysis or oxidative phosphorylation was associated with a similar decrease in pinocytosis. A microfibrillar-disruptive agent, cytochalasin B, caused a concentration-dependent stimulation of pinocytosis, whereas the microtubular-disruptive agents colchicine and vinblastine decreased pinocytosis to approximately half of control values at all concentrations used. These results indicate that fluid-phase pinocytosis in smooth-muscle cells is dependent on a continuing supply of energy and the integrity of the microtubules. Furthermore, microfilaments appear to exert a certain degree of constraint on pinocytosis, possibly by restricting invagination of the plasma membrane.
Ornithine decarboxylase activity in embryos depends on temperature of development rather than on the stage of development. Molecular adaptation to temperature changes in poikilothermic animals
Authors: Neyfakh A A , Yarygin K N , Gorgolyuk S I
Published: August 2015

The activity of ornithine decarboxylase (ODC) (the key enzyme of polyamine synthesis) in different poikilothermic animals depends on the temperatures at which they were kept just before the enzyme assay. With an increase in temperature (within physiological limits) ODC activity rises 5-25-fold within several hours. With a decrease in temperature it falls at the same rate. This effect, studied on loach (Misgurnus fossilis) embryos in detail, was also shown for embryos, larvae and some adult tissues of many species. It is not, however, observed in homoiothermic animals (chick embryos and mammalian cells), nor in bacteria and plants. Changes in polyamine concentrations follow those in ODC activity, but more slowly and to a lesser extent. It is assumed that modulation of ODC activity changes as a result of its synthesis and degradation. We suggest that the temperature-dependence of ODC activity is a mechanism of adaptation which maintains the optimal cellular concentration of polyamines for each temperature.
The preparation and characterization of vitellogenin messenger RNA from rainbow trout (Salmo gairdneri)
Authors: Valotaire Y , Tenniswood M , Le Guellec C , Tata J R
Published: August 2015

Agarose-gel electrophoresis of polyadenylated RNA from livers of oestrogen-treated male rainbow trout revealed a major high-Mr species (7200 nucleotides), which is absent from the polyadenylated RNA isolated from hormonally unstimulated male trout liver. Translation in vitro of the RNA from oestrogen-treated males in a mRNA-dependent rabbit reticulocyte lysate produced a protein (Mr 200 000) that could be immunoprecipitated with antibodies against trout serum vitellogenin, but no immunoprecipitable protein was synthesized with RNA from control animals. DNA complementary to the RNA from oestrogen-stimulated and control male trout liver was synthesized and back-hybridized, with R0t1/2 of 3.8 × 10(-2) and 1 × 10(-1) mol X litre-1 X s for RNA from hormone-treated and control animals respectively. The 9% increase in the abundant mRNA after oestrogen stimulation is due to the induction of vitellogenin mRNA.
Abnormal type I collagen metabolism by cultured fibroblasts in lethal perinatal osteogenesis imperfecta
Authors: Bateman J F , Mascara T , Chan D , Cole W G
Published: August 2015

Cultured skin fibroblasts from seven consecutive cases of lethal perinatal osteogenesis imperfecta (OI) expressed defects of type I collagen metabolism. The secretion of [14C]proline-labelled collagen by the OI cells was specifically reduced (51-79% of control), and collagen degradation was increased to twice that of control cells in five cases and increased by approx. 30% in the other two cases. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that four of the OI cell lines produced two forms of type I collagen consisting of both normally and slowly migrating forms of the alpha 1(I)- and alpha 2(I)-chains. In the other three OI cell lines only the ‘slow’ alpha (I)′- and alpha 2(I)′-chains were detected. In both groups inhibition of the post-translational modifications of proline and lysine resulted in the production of a single species of type I collagen with normal electrophoretic migration. Proline hydroxylation was normal, but the hydroxylysine contents of alpha 1(I)′- and alpha 2(I)′-chains purified by h.p.l.c. were greater than in control alpha-chains. The glucosylgalactosylhydroxylysine content was increased approx. 3-fold while the galactosylhydroxylysine content was only slightly increased in the alpha 1(I)′-chains relative to control alpha 1(I)-chains. Peptide mapping of the CNBr-cleavage peptides provided evidence that the increased post-translational modifications were distributed throughout the alpha 1(I)′- and alpha 2(I)′-chains. It is postulated that the greater modification of these chains was due to structural defects of the alpha-chains leading to delayed helix formation. The abnormal charge heterogeneity observed in the alpha 1 CB8 peptide of one patient may reflect such a structural defect in the type I collagen molecule.
Biosynthesis of the chromophore of phycobiliproteins. A study of mesohaem and mesobiliverdin as possible intermediates and further evidence for an algal haem oxygenase
Authors: Brown S B , Holroyd J A
Published: August 2015

The possible roles of mesohaem and mesobiliverdin as metabolic precursors of phycocyanobilin, the chromophore of phycocyanin, were studied in the unicellular rhodophyte Cyanidium caldarium. Dark-grown cells of this organism, which had been exposed to mesohaem, were either incubated in the dark with 5-aminolaevulinate, which results in excretion of bilins into the suspending medium, or incubated in the light, which results in synthesis of phycocyanin within the cells. By using 14C-labelling, either in the mesohaem or in the 5-aminolaevulinate administered, it was shown that mesohaem is not a precursor of phycocyanobilin in either dark or light systems. However, mesohaem was converted into mesobiliverdin in both systems, a phenomenon that is further evidence for the existence of an algal haem oxygenase. The data also showed that mesobiliverdin is not a precursor of phycocyanobilin. These results suggest that algal bilins are formed via haem degradation to biliverdin in the same way as mammalian bile pigments.
Proton-nuclear-magnetic-resonance studies of serum, plasma and urine from fasting normal and diabetic subjects
Authors: Nicholson J K , O'Flynn M P , Sadler P J , Macleod A F , Juul S M , Sönksen P H
Published: August 2015

Resonances for the ketone bodies 3-D-hydroxybutyrate, acetone and acetoacetate are readily detected in serum, plasma and urine samples from fasting and diabetic subjects by 1H n.m.r. spectroscopy at 400 MHz. Besides the simultaneous observation of metabolites, the major advantage of n.m.r. is that little or no pretreatment of samples is required. N.m.r. determinations of 3-D-hydroxybutyrate, acetoacetate, lactate, valine and alanine were compared with determinations made with conventional assays at six 2-hourly intervals after insulin withdrawal from a diabetic subject. The n.m.r. results closely paralleled those of other assays although, by n.m.r., acetoacetate levels continued to rise rather than reaching a plateau 4h after insulin withdrawal. The 3-D-hydroxybutyrate/acetoacetate ratio in urine during withdrawal gradually increased to the value observed in plasma (3.0 +/- 0.2) as determined by n.m.r. The acetoacetate/acetone ratio in urine (17 +/- 6) was much higher than in plasma (2.5 +/- 0.7). Depletion of a mobile pool of fatty acids in plasma during fasting, as seen by n.m.r., paralleled that seen during insulin withdrawal. These fatty acids were thought to be largely in chylomicrons, acylglycerols and lipoproteins, and were grossly elevated in plasma samples from a non-insulin-dependent diabetic and in cases of known hyperlipidaemia.
Organization of a multifunctional protein in pyrimidine biosynthesis. A domain hypersensitive to proteolysis
Authors: Rumsby P C , Campbell P C , Niswander L A , Davidson J N
Published: August 2015

When the multifunctional protein that catalyses the first three steps of pyrimidine biosynthesis in hamster cells is treated with staphylococcal V8 proteinase, a single cleavage takes place. The activities of carbamoyl-phosphate synthetase (EC 6.3.5.5), aspartate carbamoyltransferase (EC 2.1.3.2) and dihydro-orotase (EC 3.5.2.3) and the allosteric inhibition by UTP are unaffected. One fragment, of Mr 182000, has the first and third enzyme activities, whereas the other fragment, of Mr 42000, has aspartate carbamoyltransferase activity and an aggregation site. A similar small fragment is observed in protein digested with low concentrations of trypsin. A similar large fragment is seen after digestion with trypsin and as the predominating form of this protein in certain mutants defective in pyrimidine biosynthesis. These results indicate that a region located adjacent to the aspartate carbamoyltransferase domain is hypersensitive to proteinase action in vitro and may also be sensitive to proteolysis in vivo.
Purification of human adult and foetal intestinal alkaline phosphatases by monoclonal antibody immunoaffinity chromatography
Authors: Vockley J , Harris H
Published: August 2015

We have used the technique of monoclonal antibody immunoaffinity chromatography to purify adult and foetal intestinal alkaline phosphatases. Pure adult intestinal enzyme was obtained from a crude tissue extract with a single immunoaffinity chromatographic step in yields exceeding 95%. An additional ion-exchange chromatographic step was necessary for purification of the foetal enzyme, but yields still exceeded 70%. Experiments to optimize the efficiency of the monoclonal antibody immunoaffinity chromatography procedure suggest that the relative strength of binding of an antibody to its antigen is the most important factor to consider when constructing such columns. A column made from an antibody of too low an avidity will not retain the enzyme, while one of too high an avidity will make elution of enzyme in the active state difficult. A scheme is suggested for the application of this technique to a general approach to enzyme purification.
Isolation of tropoelastin a from lathyritic chick aortae
Authors: Rich C B , Foster J A
Published: August 2015

Tropoelastin a was isolated from lathyritic chick aortae by using severe denaturing conditions for the initial extraction. The amino acid composition of this new species of tropoelastin is elastin-like in its high proportion of proline, glycine, alanine and valine. However, it differs from authentic tropoelastin b in containing a higher percentage of polar amino acids and cysteine residues. In addition, the amount of proline hydroxylation is 3 times higher than that found in chick tropoelastin b.
Modulation of the alkaline transition in cytochrome c and cytochrome c-T by full or specific partial acetimidylation
Authors: Wallace C J A
Published: August 2015

Acetimidylated horse cytochrome c and related derivatives exhibit more or less marked changes, both upscale and downscale, in apparent pK of the alkaline transition. This transition occurs when the normal methionine-80 residue is replaced at the sixth haem co-ordination position by another strong-field ligand. Analysis of the relationship between structural change and pK shift in these derivatives supports the view that the replacement ligand is a lysine residue, probably 72 or 79, and contradicts an alternative hypothesis. The results add further detail to a comprehensive view of the mechanism of this isomerization.
Characterization of the multiple forms of hydroxymethylbilane synthase from rat spleen
Authors: Williams D C
Published: August 2015

Phenylhydrazine treatment induced hydroxymethylbilane synthase activity (EC 4.3.1.8) in rat spleen, erythrocytes and liver by 40-fold, 7.5-fold and 6-fold respectively. Five multiple forms of the enzyme were resolved by DEAE-cellulose chromatography. In the presence of phenylmethanesulphonyl fluoride only three forms, two major and one minor, were resolved by the fractionation, suggesting that two of the original forms arose by proteolytic modification. Heat treatment (70 degrees C) in the presence of proteinase inhibitor converted one of the major forms into the other major form. Product isomer analysis suggested that this heat-labile form represented an enzyme-substrate covalent intermediate and not a hydroxymethylbilane synthase-uroporphyrinogen III synthase complex. Identical elution profiles and kinetic properties of the enzymes from rat spleen and erythrocytes suggested that the enzyme isolated from spleen was possibly from stored erythrocytes. Sephadex G-75 chromatography of the heat-stable DEAE-cellulose-purified form yielded pure enzyme as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Mr was found to be 43000 +/- 1500. Initial-velocity studies on all enzyme forms showed a hyperbolic dependence of velocity on substrate concentration, demonstrating the existence of a displacement-type mechanism. For the heat-stable form Vmax, varied with pH as a typical bell-shaped curve, indicating that two ionizable groups with pK values of 7.4 and 8.8 are important for catalysis. Km decreased with decreasing pH on the acid side of the pH optimum, suggesting the absence of ionization of a group with pK 7.4 in free enzyme or substrate.
d-glucosamine-induced changes in nucleotide metabolism and growth of colon-carcinoma cells in culture
Authors: Krug E , Zweibaum A , Schulz-Holstege C , Keppler D
Published: August 2015

Human colon-carcinoma cells were exposed to D-glucosamine at 2.5, 5 and 10 mM, concentrations that were growth-inhibitory but not cytocidal in the presence of a physiological glucose concentration. Labelling of these HT-29 cells with D-[14C]-glucosamine, followed by nucleotide analyses, demonstrated that UDP-N-acetyl-hexosamines represented the major intracellular nucleotide pool and the predominant metabolite of the amino sugar. D-[14C]Glucosamine was not a precursor of UDP-glucosamine. After 4h exposure to D-glucosamine (2.5 mM), the pool of UDP-N-acetylhexosamines was increased more than 6-fold, whereas UTP and CTP were markedly decreased. UDP-glucuronate content increased by more than 2-fold, whereas purine nucleotide content was little altered. Uridine (0.1 mM) largely reversed the decrease in UTP, CTP, UDP-glucose and UDP-galactose, while intensifying the expansion of the UDP-N-acetylhexosamine pool. Uridine did not reverse the D-glucosamine-induced retardation of growth in culture. A 50% decrease in growth also persisted when uridine and cytidine, cytidine alone, or UDP, were added together with D-glucosamine. The growth-inhibitory effect of the amino sugar could therefore be best correlated with the quantitative change in the pattern of sugar nucleotides, and, in particular, with the many-fold increase in UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine.
Mechanism of hydroxylation at C-2 during the biosynthesis of ecdysone in ovaries of the locust, Schistocerca gregaria
Authors: Greenwood D R , Dinan L N , Rees H H
Published: August 2015

The stereochemistry of hydroxylation at C-2 during the biosynthesis of ecdysone in the ovaries of Schistocerca gregaria was investigated by incorporation of [1 alpha,2 alpha-3H(n)]cholesterol in admixture with [4-14C]cholesterol into oöcyte 2-deoxyecdysone and ecdysone conjugates in maturing adult female S. gregaria. Extraction of the eggs followed by enzymic hydrolysis of the ecdysteroid conjugate fraction yielded free ecdysteroids, from which 2-deoxyecdysone and ecdysone were purified. The 3H/14C ratios in the 2-deoxyecdysone and ecdysone were similar, suggesting that the 2 alpha hydrogen of cholesterol was retained during hydroxylation at C-2. This was corroborated by oxidation at C-2 of the 3,22-diacetate derivative of the ecdysone, yielding the corresponding 2-oxo compound with removal of essentially all the 3H originally present at the 2 alpha position of cholesterol. The results indicate that the 2 beta hydrogen of cholesterol has been eliminated during the hydroxylation at C-2. Thus, during ecdysone biosynthesis, hydroxylation at C-2 is direct and occurs with retention of configuration.
Assay of adenosine 5′-P1-tetraphospho-P4-5′′′-adenosine and adenosine 5′-P1-tetraphospho-P4-5′′′-guanosine in Physarum polycephalum and other eukaryotes. An isocratic high-pressure liquid-chromatography method
Authors: Garrison P N , Barnes L D
Published: August 2015

A5′pppp5′A has been proposed to serve as a molecular signal that triggers DNA replication. When published methods proved to be inadequate for the assay of A5′pppp5′A in Physarum polycephalum by h.p.l.c. (high-pressure liquid chromatography), a set of purification procedures was developed that allowed assay of as little as 2pmol of A5′pppp5′A. A5′pppp5′A was purified from cellular extract by covalent boronate chromatography, treated with alkaline phosphatase to hydrolyse residual mononucleotides and analysed by isocratic ion-exchange h.p.l.c. The analysis was facilitated by a pre-column switching procedure that allowed early-eluted species to be diverted from the analytical column. By using this procedure A5′pppp5′A has been detected in Physarum polycephalum (1.4 pmol/mg of protein), Saccharomyces cerevisiae (3.6 pmol/mg of protein) and rat liver (3.3 pmol/mg of protein). In each case a minor peak was also seen, which was identified as A5′pppp5′G. The identity of both peaks was confirmed by co-elution with standards on isocratic and gradient h.p.l.c. and treatment with enzymes, including a dinucleoside polyphosphate pyrophosphohydrolase from Physarum polycephalum.
γ-Glutamyltransferase is not involved in the bulk uptake of amino acids, peptides or γ-glutamyl-amino acids in yeast (Saccharomyces cerevisiae)
Authors: Payne G M , Payne J W
Published: August 2015

gamma-Glutamyltransferase activity has been measured in yeast (Saccharomyces cerevisiae) and shown to be associated mainly with the membrane fraction. A similar level of activity is found in a wild-type strain and in gap and gpp strains, the latter mutants being defective in the general amino acid and peptide permeases respectively. The activity is inhibited in whole cells by 6-diazo-5-oxo-L-norleucine (N2O-Nle), azaserine and serine-borate complex; this inactivation seemingly acts from without, for it is similar in (i) control and dicyclohexylcarbodi-imide-treated cells and in (ii) the wild-type and a gap mutant, a treatment and a mutation that it has been shown prevents uptake of the inhibitors. Thus a major portion of the gamma-glutamyltransferase activity appears to exist in a membrane-bound form that is orientated with its gamma-glutamyl-binding site facing the outside. Yeast cells in which gamma-glutamyltransferase has been inactivated by N2O-Nle show no significant change in their rates of uptake of a variety of amino acids, dipeptides and gamma-glutamyl-amino acids. The results preclude a major, direct role for gamma-glutamyltransferase in the transport of these substrates.
The thiol groups of the Folch-Pi protein from bovine white matter. Exposure, reactivity and significance
Authors: Vacher M , Waks M , Nicot C
Published: August 2015

The number and the reactivity of accessible thiol groups of the Folch-Pi apoprotein and proteolipid (50% of myelin proteins) were studied, by using a specific thiol-disulphide interchange reaction, in connection with the known solubility of this protein in organic and aqueous solvents. The high reactivity of 2,2′-dipyridyl disulphide towards thiol groups leads to the titration of 4.8 mol of SH groups/mol of protein (Mr 30000) in alkaline and acidic chloroform/methanol (2:1, v/v). Unlike previous findings, this value was consistently found from batch to batch and remained stable with time. In the proteolipid 1 mol of SH groups/mol was not accessible as compared with the apoprotein. In aqueous solvents, a similar number of 4.4 mol of SH groups/mol was also found. For the first time, kinetic studies carried out in chloroform/methanol discriminated between two classes of thiol groups. The reaction of 2 mol of SH groups/mol was characterized by apparent second-order rate constants whose values were 5-10-fold higher than those of the other class. Kinetic studies and cyanylation experiments in aqueous solvents also indicated the high reactivity of these thiol groups with Ellman's reagent. Together with kinetic results, studies on the stoichiometry of the interchange reaction of equimolar solutions of protein and disulphide indicate that these highly reactive thiol groups are near to each other in the amino acid sequence. The location of the thiol groups at the boundary between hydrophilic and hydrophobic domains of the Folch-Pi protein is suggested in connection with their possible structural and biological significance.
Monosaccharide transport in the mammary gland of the intact lactating rat
Authors: Threadgold L C , Kuhn N J
Published: August 2015

The Michaelis-Menten equation for the utilization of competing substrates was applied to the uptake of 2-deoxy[3H]glucose into the mammary gland of anaesthetized lactating rats. Intracellular water was calculated from total tissue water and sucrose space. Fed rats had a mean transport capacity of 2.2 mumol/min per g of tissue, giving an actual glucose transport in vivo of 1.1 mumol/min per g. Transport decreased by 90% on overnight starvation and returned to normal by 2 h of re-feeding. Similar changes were observed in the 1 min or 5 min transport of circulating 3-O-methylglucose. Transport of 3-O-methylglucose in starved rats was restored towards normal by insulin. In fed rats it increased between parturition and day 12 of lactation. The findings support the proposal that transport is a rate-limiting factor in the mammary utilization of carbohydrate.
Characterization of the protein kinase activities of human platelet supernatant and particulate fractions
Authors: Salama S E , Haslam R J
Published: August 2015

After human platelets were lysed by freezing and thawing in the presence of EDTA, about 35% of the total cyclic AMP-dependent protein kinase activity was specifically associated with the particulate fraction. In contrast, Ca2+-activated phospholipid-dependent protein kinase was found exclusively in the soluble fraction. Photoaffinity labelling of the regulatory subunits of cyclic AMP-dependent protein kinase with 8-azido-cyclic [32P]AMP indicated that platelet lysate contained a 4-fold excess of 49 000-Da RI subunits over 55 000-Da RII subunits. The RI and RII subunits were found almost entirely in the particulate and soluble fractions respectively. Chromatography of the soluble fraction on DEAE-cellulose demonstrated a single peak of cyclic AMP-dependent activity with the elution characteristics and regulatory subunits characteristic of the type-II enzyme. A major enzyme peak containing Ca2+-activated phospholipid-dependent protein kinase was eluted before the type-II enzyme, but no type-I cyclic AMP-dependent activity was normally observed in the soluble fraction. The particulate cyclic AMP-dependent protein kinase and associated RI subunits were solubilized by buffers containing 0.1 or 0.5% (w/v) Triton X-100, but not by extraction with 0.5 M-NaCl, indicating that this enzyme is firmly membrane-bound, either as an integral membrane protein or via an anchor protein. DEAE-cellulose chromatography of the Triton X-100 extracts demonstrated the presence of both type-I cyclic AMP-dependent holoenzyme and free RI subunits. These results show that platelets contain three main protein kinase activities detectable with histone substrates, namely a membrane-bound type-I cyclic AMP-dependent enzyme, a soluble type-II cyclic AMP-dependent enzyme and Ca2+-activated phospholipid-dependent protein kinase, which was soluble in lysates containing EDTA.
Membrane retrieval in the guinea-pig neurohypophysis. Isolation and characterization of secretory vesicles and coated microvesicles after radiolabel incorporation in vivo
Authors: Saermark T , Jones P M , Robinson I C A F
Published: August 2015

We have developed small-scale methods for the isolation and biochemical characterization of subcellular fractions from single guinea-pig posterior-pituitary glands. Secretory vesicles and coated microvesicles produced in this way were of similar purity to those isolated from large amounts of tissue by conventional ultracentrifugation. [35S]Cysteine injected into the hypothalamus was found in the soluble contents of secretory vesicles isolated from the neural lobes 24 h later. High-pressure liquid-chromatographic analysis revealed that the radiolabel was incorporated into the expected neurosecretory products (oxytocin, vasopressin and neurophysin) and also into a biosynthetic intermediate in the vasopressin system. The membranes of secretory vesicles were labelled with [3H]choline 24 h after its hypothalamic injection. Little or no [3H]choline could be demonstrated in coated microvesicles at this time, although these structures were labelled 5 days after injection. Stimulating hormone secretion by chronic dehydration produced a significant fall in [3H]choline content of the secretory-vesicle membranes without any transfer of label into coated microvesicles, suggesting that coated microvesicles are not involved in membrane retrieval in the neurohypophysis.
Inhibition of phospholipid methylation by a cytosolic factor
Authors: Alvarez Chiva V , Mato J M
Published: August 2015

Rat liver cytosol contains a heat-stable factor which inhibits phospholipid methylation by rat liver microsomes. The effect of this factor on lipid methylation was dose- and pH-dependent. This factor has an Mr of approx. 3200 as estimated by gel filtration. It could not be extracted by chloroform/methanol (2:1, v/v), and its action was inhibited by incubation with subtilisin.
Transfer of intestine-derived diamines into tumour cells during treatment of Ehrlich-ascites—carcinoma-bearing mice with polyamine anti-metabolites
Authors: Kallio A , Nikula P , Jänne J
Published: August 2015

Treatment of Ehrlich-ascites-carcinoma-bearing mice with methylglyoxal bis(guanylhydrazone) alone or in combination with 2-difluoromethylornithine greatly enhanced the transfer of intragastrically administered radioactive putrescine and cadaverine into the carcinoma cells. Difluoromethylornithine alone did not have any effect on the accumulation of intestine-derived diamines in the tumour cells. The frequently reported restoration of difluoromethylornithine-induced polyamine depletion on administration of methylglyoxal bis(guanylhydrazone) is in all likelihood attributable to a profound inhibition of intestinal diamine oxidase (EC 1.4.3.6), resulting in an enhanced entry of intestinal (bacterial) diamines into general circulation and finally into tumour cells.
Adsorbed soluble [3H]elastin as substrate for proteinase activity. A new microassay technique
Authors: Jensen H S
Published: August 2015

A microassay for elastase activity has been designed. Microtubes are coated with soluble elastin substrate by passive adsorption to the tube walls. On contact with enzymes in test samples, radioactivity is released into the sample buffer, which is then transferred to vials containing scintillation fluid without further processing. Porcine pancreatic elastase (EC 3.4.21.11) is easily detected in concentrations down to 8 micrograms/litre. The interassay coefficient of variation (CV) was below 10% for values above 50 micrograms/litre. The assay is rapid, precise, economical and sensitive.
The antiheparin effect of a heparinoid, pentosan polysulphate. Investigation of a mechanism
Authors: Scully M F , Kakkar V V
Published: August 2015

A pentosan polysulphate [a fully sulphated (1-4)-beta-D-xylopyranose with a single laterally positioned 4-O-methyl-alpha-D-glucuronic acid] has been shown to inhibit the anticoagulant activity of high-affinity heparin as observed in plasma and when using purified enzyme and inhibitor. The activity was shown to be concentration-dependent with an apparent Ki of approx. 2 microM. The antiheparin property was not shown by a number of other anionic carbohydrates when tested. The rate of thrombin inhibition at 0.33 microM-heparin was reduced from 7.1 × 10(8) M-1 X min-1 in the absence of pentosan polysulphate to 2.3 × 10(8) M-1 X min-1 at 2 microM-pentosan polysulphate and to 0.3 × 10(8)M-1 X min-1 at 20 microM. Using the random bireactant model of heparin action [Griffiths (1982) J. Biol. Chem. 257, 13899-13902] it was observed that the pentosan polysulphate had no effect on the Km for antithrombin III (150 nM) but increased the Km for thrombin from 25 nM to 450 nM. A reduction in the inhibition rate by 17.3-fold predicted by substitution of these values into the general two-substrate reaction-rate equation was confirmed experimentally.
Cross-linking in type IV collagen
Authors: Bailey A J , Sims T J , Light N
Published: August 2015

Type IV collagen could not be extracted from human placenta using 6M-urea containing 10mM-dithiothreitol, indicating that the type IV molecule is stabilized within the basement membrane by covalent cross-links. However, various forms of type IV collagen molecule were extractable by means of increasingly severe proteolytic conditions. Type IV collagen tetramers (‘spiders’) lacking only the C-terminal globular region (NC1) were further digested to the ‘long-form’ 7S fragment and an assortment of helical rod-like molecules derived from the ‘leg’ region. These molecules were separated by salt fractionation and examined by rotary-shadowing electron microscopy. Isolation of these fractions from placenta which had been reduced with NaB3H4 revealed that both the 7S (N-terminal) and C-terminal regions contained significant proportions of reducible lysine-derived cross-links. These cross-links were shown to be exclusively the stable oxo-imine hydroxylysino-5-oxonorleucine. The number of the cross-links per molecule was significantly lower than might be expected in order to fully stabilize the molecule. These results suggest that the keto-imine cross-links in type IV collagen have been stabilized in part by conversion into an unknown non-reducible form, although a sensitive immunoassay failed to show the presence of any pyridinoline. Comparison with the fibrous collagen from placenta suggested that the rate of this conversion is much greater in basement-membrane collagen.
Effect of creatine on contents of myosin heavy chain and myosin-heavy-chain mRNA in steady-state chicken muscle-cell cultures
Authors: Young R B , Denome R M
Published: August 2015

Embryonic-chick muscle cells reach a steady state with respect to protein metabolism after approx. 1 week in cell culture. To determine if this steady state could be altered by the administration of agents that have been reported to stimulate myosin heavy-chain synthesis, 7-day muscle-cell cultures were treated with 0-1 mM-creatine. Incorporation of [3H]leucine into myosin heavy chain was stimulated by 30-40% at the optimum creatine concentration (0.2 mM), but this stimulation was blocked when actinomycin D (10 micrograms/ml) was also present. However, the quantity of myosin-heavy-chain mRNA as measured by hybridization in vitro was only 15% higher in creatine-treated cultures, and was therefore not entirely responsible for the observed effect. It is important to note that creatine only exerted its action on myosin-heavy-chain synthesis rate in steady-state cultures; creatine was ineffective in altering this rate in rapidly differentiating 3-day muscle cultures. Finally, muscle-cell cultures that had been grown for the entire 7-day culture period in the presence of 0.2 mM-creatine were assayed for quantity of myosin heavy chain. Control and creatine-treated cultures contained 12.7 +/- 1.5 and 20.5 +/- 1.8 micrograms/dish respectively. In conclusion, creatine apparently enhances the quantity of myosin heavy chain in steady-state embryonic muscle-cell cultures, but it probably does not mediate regulation of myosin content in adult skeletal muscle.
Lactose and fatty acid synthesis in lactating-rat mammary gland. Effects of starvation, re-feeding, and administration of insulin, adrenaline, streptozotocin and 2-bromo-α-ergocryptine
Authors: Bussmann L E , Ward S , Kuhn N J
Published: August 2015

Lactose synthesis and fatty acid synthesis in intact lactating-rat mammary gland were measured simultaneously by incorporation of [U-14C]glucose and of both [U-14C]glucose and 3H2O respectively. Both processes were almost abolished by overnight starvation. Self-re-feeding caused recovery of lipogenesis to 100% of normal by 2 h and to 170% by 5 h. Lactose synthesis recovered to 80% of normal by 5 h. Food intubated to starved rats caused partial recovery in 3 h, standard diet favouring lactose synthesis and sugars favouring lipogenesis. Casein and starch were ineffective. Olive oil intubated to fed rats suppressed lipogenesis greatly and lactose synthesis slightly. Paraffin oil or water partly mimicked these effects. Adrenaline (subcutaneous) decreased lipogenesis from glucose, whereas insulin (subcutaneous) caused hypoglycaemia associated with loss of lactose synthesis but unchanged fatty acid synthesis. Streptozotocin and 2-bromo-alpha-ergocryptine (CB-154) impaired lipogenesis but not lactose synthesis. The results are interpreted in terms of competition for intracellular glucose by biosynthetic pathways for lactose and fat, and the possible implications for variations in milk composition are discussed.
Activation of ADP-ribosyltransferase in polyamine-depleted mammalian cells
Authors: Wallace H M , Gordon A M , Keir H M , Pearson C K
Published: August 2015

Mammalian fibroblasts were cultured in the presence of alpha-methylornithine and/or methylglyoxal bis(guanylhydrazone), which inhibit the synthesis of polyamines. This led to a decrease in the cellular content of the polyamines spermine and spermidine by up to 60% when the cells were grown in the presence of both drugs together. The activity of the chromatin-associated enzyme ADP-ribosyltransferase was enhanced 2-3-fold in the drug-treated cells when measured in cells subsequently rendered permeable to exogenous NAD+, the substrate for the transferase. This is a novel and surprising observation, since the transferase is invariably activated by the addition of polyamines to a suitable incubation system such as permeabilized cells, isolated nuclei or the purified enzyme. We found no evidence that the activation was due to the appearance of DNA strand breaks, by using a variety of procedures including both neutral [the ‘nucleoid’ technique of Cook & Brazell [(1975) J. Cell Sci. 19, 261-279; (1976) J. Cell Sci. 22, 287-302]] and alkaline sucrose-gradient centrifugation and gel electrophoresis, suggesting that this therefore may not be the only means of regulating the activity of ADP-ribosyltransferase and that polyamines may have a role to play in this regard in vivo.
The carboxybiotin complex of pyruvate carboxylase. A kinetic analysis of the effects of Mg2+ ions on its stability and on its reaction with pyruvate
Authors: Attwood P V , Wallace J C , Keech D B
Published: August 2015

The enzyme-[14C] carboxybiotin complex of sheep liver pyruvate carboxylase was isolated and the reaction between this and pyruvate was studied by using the quenched-flow rapid-reaction technique. At 0.5 degrees C the reaction was 80% complete within 180 ms. The reaction was monophasic and obeyed pseudo-first-order kinetics. Increasing concentrations of Mg2+ caused a decrease in the magnitude of the observed pseudo-first-order rate constant. Throughout the carboxylation of pyruvate, the rate-limiting step of the reaction occurred after the dissociation of carboxybiotin from the first sub-site, whereas in the slow phase of the reaction with 2-oxobutyrate this dissociation is the rate-limiting step. It is possible, from the reaction scheme proposed, that the inhibition of overall enzymic activity by high concentrations of Mg2+ could be caused by the transfer of the carboxy group from biotin to pyruvate becoming rate-limiting. The efficacy of a substrate as a signal for the movement of carboxybiotin from the first sub-site is reflected by the amount that the effective affinity of the enzyme- carboxybiotin complex for Mg2+ is lowered. In the presence of the substrates tested, the affinities of the carboxybiotin complex can be arranged in order of increasing magnitude, i.e.: (formula; see text). The kinetics of the decay of the enzyme-[14C] carboxybiotin complex at 0 degree C in the absence of substrates are similar to the reaction with pyruvate except that the carboxybiotin is also unstable in the first sub-site, to some degree. This similarity allows for the proposal of a general scheme for the decarboxylation of the enzyme- carboxybiotin complex in the presence or in the absence of substrates.
Investigation of the binding of Ca2+, Mg2+, Mn2+ and K+ to the vitamin D-dependent Ca2+-binding protein from pig duodenum
Authors: Bryant D T W , Andrews P
Published: August 2015

The cation-binding properties of the vitamin D-dependent Ca2+-binding protein from pig duodenum were investigated, mainly by flow dialysis. The protein bound two Ca2+ ions with high affinity, and Mg2+, Mn2+ and K+ were all bound competitively with Ca2+ at both sites. The sites were distinguished by their different affinities for Mn2+, the one with the higher affinity being designated A (Kd 0.61 +/- 0.02 microM) and the other B (Kd 50 +/- 6 microM). Competitive binding studies allied to fluorimetric titration with Mg2+ showed that site A bound Ca2+, Mg2+ and K+ with Kd values of 4.7 +/- 0.8 nM, 94 +/- 18 microM and 1.6 +/- 0.3 mM respectively, and site B bound the same three cations with Kd values of 6.3 +/- 1.8 nM, 127 +/- 38 microM and 2.1 +/- 0.6 mM. For the binding of these cations, therefore, there was no significant difference between the two sites. In the presence of 1 mM-Mg2+ and 150 mM-K+, both sites bound Ca2+ with an apparent Kd of 0.5 microM. The cation-binding properties were discussed relative to those of parvalbumin, troponin C and the vitamin D-dependent Ca2+-binding protein from chick duodenum.