bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of 3-phenylpyruvate-induced insulin release from isolated pancreatic islets
Authors: Panten U , Langer J
Published: August 2015

3-Phenylpyruvate evoked a monophasic insulin release from perifused mouse islets. L-Phenylalanine was not an insulin secretagogue and was oxidized by islets at a very low rate, suggesting that 3-phenylpyruvate does not trigger insulin release by enhancing production of reducing equivalents. Moreover, allosteric activation of glutamate dehydrogenase does not play a role in 3-phenylpyruvate-induced insulin secretion.
Renal glycerol metabolism and the distribution of glycerol kinase in rabbit nephron
Authors: Wirthensohn G , Vandewalle A , Guder W G
Published: August 2015

Glycerol and dihydroxyacetone are metabolized by rabbit kidney-cortex tubules, isolated by collagenase treatment. Half-maximal concentrations of both substrates were determined with regard to uptake rates and product formations. Maximal uptake rates were 643 and 329 mumol/h per g of protein for dihydroxyacetone and glycerol respectively. Glucose and lactate were found as major metabolic products. Glycerol kinase, the enzyme catalysing the first step in renal glycerol and dihydroxyacetone metabolism, was measured radiochemically as described by Newsholme, Robinson & Taylor [(1967) Biochim, Biophys. Acta 132, 338-346] and adapted for studies of the localization of this enzyme along the different structures of rabbit nephron. The results show that glycerol kinase is located exclusively in the proximal segments, i.e. the proximal convoluted tubules and the pars recta, but is negligible in the other structures studied. The activities were close to the maximal dihydroxyacetone uptake rates measured in tubule suspensions.
Developmental changes in rat blood choline concentration
Authors: Zeisel S H , Wurtman R J
Published: August 2015

1. Serum choline concentration in the newborn rat is extremely high and declines as the rat matures until adult values are attained at 20 days of age. 2. Rat milk is a rich source of choline, and rat pups denied access to milk had significantly lower serum choline concentrations than did fed littermates. We conclude that dietary intake of choline contributes to the maintenance of high serum choline concentrations in the neonatal rat. 3. In vivo, choline disappears with a half-life of 70 min. It is converted into betaine, phosphocholine and phosphatidylcholine. The rate of phosphocholine formation is identical in 3- and 10-day-old rats (3.3 mumol/h), whereas the rate of betaine formation is slower in younger animals (0.15 mumol/h at 3 days versus 0.69 mumol/h at 10 days). In vitro, choline oxidase activity [choline dehydrogenase (EC 1.1.99.1) and betaine aldehyde dehydrogenase (EC 1.2.1.8)] increased between birth and 40 days of age. The age-related acceleration in choline's conversion into betaine probably tends to diminish unesterified choline concentration in the rat.
Assembly of proteoglycan aggregates in cultures of chondrocytes from bovine tracheal cartilage
Authors: Björnsson S , Heinegård D
Published: August 2015

The assembly of proteoglycan aggregates in chondrocyte cell cultures was examined in pulse-chase experiments with the use of [35S]sulphate for labelling. Rate-zonal centrifugation in linear sucrose density gradients (10-50%, w/v) was used to separate the aggregated proteoglycans from monomers and to assess the size of the newly formed aggregates. The proportion of aggregates stabilized by link protein was assessed by competition with added exogenous aggregate components. The capacity of the proteoglycans synthesized in culture to compete with exogenous nasal-cartilage proteoglycans for binding was studied in dissociation-reassociation experiments. The results were as follows. (a) The proteoglycan monomers and the hyaluronic acid are exported separately and combined extracellularly. (b) The size of the aggregates increases gradually with time as the proportion of monomers bound to hyaluronic acid increases. (c) All of the aggregates present at a particular time appear to be link-stabilized and therefore not dissociated by added excess of nasal-cartilage proteoglycan monomer or hyaluronic acid oligomers. (d) The free monomer is apparently present as a complex with link protein. The monomer-link complexes are then aggregated to the hyaluronic acid. (e) The aggregates synthesized in vitro and the nasal-cartilage aggregates differ when tested for link-stabilization by incubation at low pH. The aggregates synthesized in vitro were completely dissociated whereas the cartilage proteoglycans remained aggregated. The results obtained from dissociation-reassociation experiments performed at low pH indicate that the proteoglycan monomer synthesized in vitro does not bind the hyaluronic acid or the link protein as strongly as does the nasal-cartilage monomer.
Affinity chromatography and immunosorption with acetylcholine receptor attached to nylon tubes
Authors: Yang B H , Sundaram P V , Maelicke A
Published: August 2015

Nylon-linked proteins were used for affinity trapping and chromatography. As representative examples purified acetylcholine receptor, alpha-cobratoxin and bovine serum albumin were coupled to the activated matrix to serve as biospecific ligands. In particular, acetylcholine receptor was coupled without significant loss of biochemical properties. The resulting affinity tubes bind receptor-specific ligands including immunoglobulins and thus can be used for affinity-chromatographic purposes and immunoassays.
Extractable proteoglycan from human femoral-head articular cartilage
Authors: Bullimore D W , Phelps C F , Jayson M I V
Published: August 2015

Proteoglycans were prepared from human femoral-head articular cartilage by using either guanidinium hydrochloride or MgCl2 as extractant, followed by density-gradient centrifugation. The proteoglycan subunit had a particle weight of 2.6 x 10(6), with a radius of gyration, RG, of 68.5 nm in 150 mM-NaCl/20 mM-sodium phosphate buffer, pH 7.0. The proteoglycan aggregate had a particle weight of 3.7 x 10(6) (RG 84 nm) for guanidinium hydrochloride extracts and 8.7 x 10(6) (RG 118 nm) for MgCl2 extracts in the same buffer. The addition of excess of high-molecular-weight hyaluronate did not significantly alter the particle size of the aggregate. The small increase in size probably reflects a rapid equilibrium between hyaluronate and proteoglycan monomers, and is not due to proteolytic cleavage producing non-aggregating units. Experiments that support the rapid-interaction hypothesis include analytical ultracentrifugation and column chromatography. This interaction does not appear to be pressure-sensitive at 20 degrees C, but is sensitive to temperature variation near the physiological range.
Detection of a glycosylated subunit in human serum ferritin
Authors: Cragg S J , Wagstaff M , Worwood M
Published: August 2015

Ferritin was purified from the serum of two patients with idiopathic haemochromatosis. The protein contained three types of subunit--the H and L subunits of tissue ferritins (although only a trace of H could be detected) and a third subunit, ‘G’, with the highest apparent molecular weight. Only the ‘G’ subunit band stained for carbohydrate, indicating that a proportion of the subunits of human serum ferritin are glycosylated. Although serum was obtained from patients with idiopathic haemochromatosis, it is probable that the ‘G’ subunit is a component of normal serum ferritin.
Identification of an arginine residue important for catalytic activity in the primary structure of d-glyceraldehyde 3-phosphate dehydrogenase. Studies with the rat skeletal-muscle enzyme
Authors: Vospelnikova N D , Safronova M I , Shuvalova E R , Baratova L A , Kniazev S P , Nagradova N K
Published: August 2015

The reaction of holo-(D-glyceraldehyde 3-phosphate dehydrogenase) (EC 1.2.1.12) from rat skeletal muscle with [14C]butanedione in 0.05 M-NH4HCO3, pH 8.0, resulted in modification (*) of two arginine residues per subunit with a concomitant loss of catalytic activity. From a tryptic digest of the modified protein two radiolabelled peptides were isolated, with the following sequences: (1)Val-Ile-Ile-Asn-Ala-Pro-Thr-Ala-Asp-Ala(Glx,Met,Leu,Phe,Met)Gly-Val-Asx-Arg- Glx(His,Tyr)Ser-Lys and (2) Asp-Ala-Gly-Ala-Thr-Ile-Ala-Leu(Asx,Glx,Arg,Phe,Val)Lys. By comparison of the data with the known sequences of homologous enzymes, the localization of the modified residues was established. The first peptide was identified as corresponding to residues 116--139, the second to residues 293--306. Experimental evidence from this and previous studies suggests that arginine-134 is important for the catalytic activity of the rat muscle enzyme, being involved in structural rearrangements accompanying the organization of the active centre on the binding of coenzyme and substrate.
Plasma clearance and endocytosis of mitochondrial malate dehydrogenase in the rat
Authors: Bijsterbosch M K , Duursma A M , Bouma J M W , Gruber M , Nieuwenhuis P
Published: August 2015

1. Pig mitochondrial malate dehydrogenase was labelled with 125I and intravenously injected into rats. Enzyme activity and radioactivity were cleared from plasma identically, with first-order kinetics, with a half-life of only 7 min. 2. Radioactivity accumulated in liver, spleen, bone (marrow) and kidneys, reaching maxima of 3 1, 4, 6 and 9% of the injected dose respectively, at 10 min after injection. 3. Our data allow us to calculate that in the long run 59, 5, 11 and 13% of the injected dose is taken up and subsequently broken down by liver, spleen, bone and kidneys respectively. 4. Differential fractionation of liver showed that the acid-precipitable radioactivity was mainly present in the lysosomal and microsomal fractions, suggesting that the endocytosed protein is transported via endosomes to lysosomes, where it is degraded. 5. Radioautography of liver and spleen suggested that the labelled protein was taken up by macrophages of the reticuloendothelial system. 6. Mitochondrial malate dehydrogenase is probably internalized in liver, spleen and bone marrow by adsorptive endocytosis, since uptake of the enzyme of these tissues is saturable.
Light activation of fructose bisphosphatase in photosynthetically competent pea chloroplasts
Authors: Charles S A , Halliwell B
Published: August 2015

The fructose bisphosphatase (EC 3.1.3.11) activity of type A chloroplasts isolated from young (9-day-old) pea (Pisum sativum var. Progress no. 9) plants, assayed at physiological pH, substrate and Mg2+ concentrations, increased rapidly on illumination. The enzyme activity detected was more than sufficient to account for observed rates of Co2 fixation both during the induction period and during steady-state CO2 fixation, whether or not dihydroxyacetone phosphate had been added to the preparation. Omission of catalase from the suspension medium had no effect. On switching off the light, CO2 fixation by the chloroplasts ceased at once, yet fructose bisphosphatase activity decreased much more slowly. Changes in enzyme activity were much less marked if assays were conducted at 3 mM substrate and 10 mM-Mg2+. Chloroplasts from older (13--20-day-old) peas only fixed CO2 rapidly if catalase was present in the assay medium. The fructose bisphosphatase activity detected under physiological assay conditions was again more than sufficient to account for observed rates of Co2 fixation. In the presence of added dihydroxyacetone phosphate, however, the rate of Co2 fixation appeared to be determined by the rate of light activation of fructose bisphosphatase. In general, the rates of Co2 fixation and enzyme activation, and the final enzyme activity achieved, decreased markedly with increasing age of the plants. The role of light activation of fructose bisphosphatase as a means of controlling the rate of CO2 fixation in pea chloroplasts is discussed.
The incorporation in vitro of sulphate ions into synaptic-membrane glycoproteins
Authors: White C J B
Published: August 2015

Synaptosomes from sheep brain cortex were incubated with carrier-free Na235SO4 and the synaptic plasma membranes were isolated. The membranes were free of contamination from cytosol, mitochondria and microsomal material and accounted for 30% of the radioactivity present in the synaptosomal particulate fraction. Control experiments demonstrated that the radioactivity present in the preparation was not due to non-specific binding of sulphate ions. The synaptic membranes contained at least six 35S-containing protein bands with molecular weights between 160 000 and 16 000. Analysis showed that the radioactivity was located in the carbohydrate moiety of a glycopeptide.
Adenosine production inside rat polymorphonuclear leucocytes
Authors: Newby A C , Holmquist C A
Published: August 2015

Adenosine synthesis was studied during 2-deoxyglucose-induced ATP catabolism in intact rat polymorphonuclear leucocytes. When both adenosine kinase (EC 2.7.1.20) and adenosine deaminase (EC 3.5.4.4) were selectively inhibited, adenosine accumulated. Adenosine formation took place inside the intact cells by a metabolic pathway independent of the ecto-5′-nucleotidase (EC 3.1.3.5). Distinct metabolic pathways are proposed for adenosine production from intracellular or extracellular nucleotides.
Hormonal regulation of retinoic acid-binding proteins in the mammary gland
Authors: Mehta R G , Moon R C
Published: August 2015

A cytosolic retinoic acid-binding (RAB) protein that sediments specifically as a 2S component on sucrose density gradients was detected in the mammary glands of virgin, pregnant and lactating rats. Mammary cytosol from pregnant rats contained significantly higher concentrations of cytosolic RAB protein than did cytosol from either virgin or lactating rats. The glands of pregnant animals exhibited increased concentration of cytosolic RAB protein during the first 5 days of pregnancy, and a steady decline was observed thereafter. The concentration of cytosolic RAB protein dropped to the value observed during lactation on the day 20 of pregnancy. Moreover, throughout lactation, low concentrations of cytosolic RAB protein were maintained. Daily treatment of virgin and lactating animals with 5 micrograms of oestradiol-17 beta for 1 week increased cytosolic RAB protein to concentrations comparable with those seen in pregnant rats. Progesterone, however, did not affect the mammary cytosolic RAB protein content of virgin rats. These results suggest hormonal involvement in the regulation of cytosolic RAB protein concentration of mammary gland during differentiation.
Composition, stability and electrolyte permeability of Golgi membranes from lactating-rat mammary gland
Authors: White M D , Ward S , Kuhn N J
Published: August 2015

1. Golgi membrane vesicles, isolated from lactating-rat mammary gland and greatly enriched in galactosyltransferase (EC 2.4.1.22), contained over 40 separate bands of protein, including some periodic acid)(Schiff-staining material and free thiol groups, when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 2. The membrane lipids were enriched in phosphatidylcholine, phosphatidylethanolamine and unesterified cholesterol. 3. Membrane fluidity, as monitored by the fluorescence polarization of 1,6-diphenylhexa-1,3,5-triene, increased linearly over 5-37 degrees C. 4. The vesicle membranes were impermeable to lactose over a wide pH range, but admitted electrolytes of molecular weight below about 300. 5. These properties are discussed with respect to other cellular membranes and the secretion of milk products.
Evaluation of the role of peroxisome-proliferator-activated receptor α in the regulation of cardiac pyruvate dehydrogenase kinase 4 protein expression in response to starvation, high-fat feeding and hyperthyroidism
Authors: HOLNESS Mark J. , SMITH Nicholas D. , BULMER Karen , HOPKINS Teresa , GIBBONS Geoffrey F. , SUGDEN Mary C.
Published: August 2015

Inactivation of cardiac pyruvate dehydrogenase complex (PDC) after prolonged starvation and in response to hyperthyroidism is associated with enhanced protein expression of pyruvate dehydrogenase kinase (PDK) isoform 4. The present study examined the potential role of peroxisome-proliferator-activated receptor α (PPARα) in adaptive modification of cardiac PDK4 protein expression after starvation and in hyperthyroidism. PDK4 protein expression was analysed by immunoblotting in homogenates of hearts from fed or 48h-starved rats, rats rendered hyperthyroid by subcutaneous injection of tri-iodothyronine and a subgroup of euthyroid rats maintained on a high-fat/low-carbohydrate diet, with or without treatment with the PPARα agonist WY14,643. In addition, PDK4 protein expression was analysed in hearts from fed, 24h-starved or 6h-refed wild-type or PPARα-null mice. PPARα activation by WY14,643 in vivo over the timescale of the response to starvation failed to up-regulate cardiac PDK4 protein expression in rats maintained on standard diet (WY14,643, 1.1-fold increase; starvation, 1.8-fold increase) or influence the cardiac PDK4 response to starvation. By contrast, PPARα activation by WY14,643 in vivo significantly enhanced cardiac PDK4 protein expression in rats maintained on a high-fat diet, which itself increased cardiac PDK4 protein expression. PPARα deficiency did not abolish up-regulation of cardiac PDK4 protein expression in response to starvation (2.9-fold increases in both wild-type and PPARα-null mice). Starvation and hyperthyroidism exerted additive effects on cardiac PDK4 protein expression, but PPARα activation by WY14,643 did not influence the response of cardiac PDK4 protein expression to hyperthyroidism in either the fed or starved state. Our data support the hypothesis that cardiac PDK4 protein expression is regulated, at least in part, by a fatty acid-dependent, PPARα-independent mechanism and strongly implicate a fall in insulin in either initiating or facilitating the response of cardiac PDK4 protein expression to starvation.
Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA
Authors: de Jonge Jørgen , Leenhouts Johanna M. , Holtrop Marijke , Schoen Pieter , Scherrer Peter , Cullis Pieter R. , Wilschut Jan , Huckriede Anke
Published: August 2015

Reconstituted influenza virosomes (virus membrane envelopes) have been used previously to deliver pDNA (plasmid DNA) bound to their external surface to a variety of target cells. Although high transfection efficiencies can be obtained with these complexes in vitro, the virosome-associated DNA is readily accessible to nucleases and could therefore be prone to rapid degradation under in vivo conditions. In the present study, we show a new method for the production of DNA–virosomes resulting in complete protection of the DNA from nucleases. This method relies on the use of the short-chain phospholipid DCPC (dicaproylphosphatidylcholine) for solubilization of the viral membrane. The solubilized viral membrane components are mixed with pDNA and cationic lipid. Reconstitution of the viral envelopes and simultaneous encapsulation of pDNA is achieved by removal of the DCPC from the mixture through dialysis. Analysis by linear sucrose density-gradient centrifugation revealed that protein, phospholipid and pDNA physically associated to particles, which appeared as vesicles with spike proteins inserted in their membranes when analysed by electron microscopy. The DNA–virosomes retained the membrane fusion properties of the native influenza virus. The virosome-associated pDNA was completely protected from degradation by nucleases, providing evidence for the DNA being highly condensed and encapsulated in the lumen of the virosomes. DNA–virosomes, containing reporter gene constructs, transfected a variety of cell lines, with efficiencies approaching 90%. Transfection was completely dependent on the fusogenic properties of the viral spike protein haemagglutinin. Thus, DNA–virosomes prepared by the new procedure are highly efficient vehicles for DNA delivery, offering the advantage of complete DNA protection, which is especially important for future in vivo applications.
Regulation of the renal-specific Na+–K+–2Cl− co-transporter NKCC2 by AMP-activated protein kinase (AMPK)
Authors: Fraser Scott A. , Gimenez Ignacio , Cook Natasha , Jennings Ian , Katerelos Marina , Katsis Frosa , Levidiotis Vicki , Kemp Bruce E. , Power David A.
Published: August 2015

The renal-specific NKCC2 (Na+–K+–2Cl− co-transporter 2) is regulated by changes in phosphorylation state, however, the phosphorylation sites and kinases responsible have not been fully elucidated. In the present study, we demonstrate that the metabolic sensing kinase AMPK (AMP-activated protein kinase) phosphorylates NKCC2 on Ser126in vitro. Co-precipitation experiments indicated that there is a physical association between AMPK and the N-terminal cytoplasmic domain of NKCC2. Activation of AMPK in the MMDD1 (mouse macula densa-derived 1) cell line resulted in an increase in Ser126 phosphorylation in situ, suggesting that AMPK may phosphorylate NKCC2 in vivo. The functional significance of Ser126 phosphorylation was examined by mutating the serine residue to an alanine residue resulting in a marked reduction in co-transporter activity when exogenously expressed in Xenopus laevis oocytes under isotonic conditions. Under hypertonic conditions no significant change of activity was observed. Therefore the present study identifies a novel phosphorylation site that maintains NKCC2-mediated transport under isotonic or basal conditions. Moreover, the metabolic-sensing kinase, AMPK, is able to phosphorylate this site, potentially linking the cellular energy state with changes in co-transporter activity.
Activation mechanisms for the cystic fibrosis transmembrane conductance regulator protein involve direct binding of cAMP
Authors: Pereira Malcolm M. C. , Parker Jody , Stratford Fiona L. L. , McPherson Margaret , Dormer Robert L.
Published: August 2015

The CFTR [CF (cystic fibrosis) transmembrane conductance regulator] chloride channel is activated by cyclic nucleotide-dependent phosphorylation and ATP binding, but also by non-phosphorylation-dependent mechanisms. Other CFTR functions such as regulation of exocytotic protein secretion are also activated by cyclic nucleotide elevating agents. A soluble protein comprising the first NBD (nucleotide-binding domain) and R-domain of CFTR (NBD1–R) was synthesized to determine directly whether CFTR binds cAMP. An equilibrium radioligand-binding assay was developed, firstly to show that, as for full-length CFTR, the NBD1–R protein bound ATP. Half-maximal displacement of [3H]ATP by non-radioactive ATP at 3.5 μM and 3.1 mM was demonstrated. [3H]cAMP bound to the protein with different affinities from ATP (half-maximal displacement by cAMP at 2.6 and 167 μM). Introduction of a mutation (T421A) in a motif predicted to be important for cyclic nucleotide binding decreased the higher affinity binding of cAMP to 9.2 μM. The anti-CFTR antibody (MPNB) that inhibits CFTR-mediated protein secretion also inhibited cAMP binding. Thus binding of cAMP to CFTR is consistent with a role in activation of protein secretion, a process defective in CF gland cells. Furthermore, the binding site may be important in the mechanism by which drugs activate mutant CFTR and correct defective ΔF508-CFTR trafficking.
Oxygen-dependent regulation of mitochondrial respiration by hypoxia-inducible factor 1
Authors: Semenza Gregg L.
Published: August 2015

The survival of metazoan organisms is dependent upon the utilization of O2 as a substrate for COX (cytochrome c oxidase), which constitutes Complex IV of the mitochondrial respiratory chain. Premature transfer of electrons, either at Complex I or at Complex III, results in the increased generation of ROS (reactive oxygen species). Recent studies have identified two critical adaptations that may function to prevent excessive ROS production in hypoxic cells. First, expression of PDK1 [PDH (pyruvate dehydrogenase) kinase 1] is induced. PDK1 phosphorylates and inactivates PDH, the mitochondrial enzyme that converts pyruvate into acetyl-CoA. In combination with the hypoxia-induced expression of LDHA (lactate dehydrogenase A), which converts pyruvate into lactate, PDK1 reduces the delivery of acetyl-CoA to the tricarboxylic acid cycle, thus reducing the levels of NADH and FADH2 delivered to the electron-transport chain. Secondly, the subunit composition of COX is altered in hypoxic cells by increased expression of the COX4-2 subunit, which optimizes COX activity under hypoxic conditions, and increased degradation of the COX4-1 subunit, which optimizes COX activity under aerobic conditions. Hypoxia-inducible factor 1 controls the metabolic adaptation of mammalian cells to hypoxia by activating transcription of the genes encoding PDK1, LDHA, COX4-2 and LON, a mitochondrial protease that is required for the degradation of COX4-1. COX subunit switching occurs in yeast, but by a completely different regulatory mechanism, suggesting that selection for O2-dependent homoeostatic regulation of mitochondrial respiration is ancient and likely to be shared by all eukaryotic organisms.
The control by Ca2+ of the polyphosphoinositide phosphodiesterase and the Ca2+-pump ATPase in human erythrocytes
Authors: Downes C. Peter , Michell Robert H.
Published: August 2015

1. Both the Ca2+-pump ATPase and the polyphosphoinositide phosphodiesterase of the erythrocyte membrane can, when assayed under appropriate conditions, be activated by Ca2+ in the micromolar range. We have therefore compared the mechanisms and affinities for Ca2+ activation of the two enzymes in human erythrocyte membranes, to see whether the polyphosphoinositide phosphodiesterase would be active in normal healthy erythrocytes. 2. At physiological ionic strength and in the presence of calmodulin, the Ca2+-pump ATPase was activated by Ca2+ in a highly co-operative manner, with half-maximal activation occurring at about 0.3μm-Ca2+. At an optimal Ca2+concentration, calmodulin stimulated the Ca2+-sensitive ATPase activity about 10-fold. 3. Ca2+ activated the polyphosphoinositide phosphodiesterase in a non-co-operative manner. The Ca2+ requirements for breakdown of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were identical, which supports our previous conclusion that Ca2+ activates a single polyphosphoinositide phosphodiesterase that degrades both lipids with equal facility. Added calmodulin did not affect the activity of the polyphosphoinositide phosphodiesterase. 4. At low ionic strength in the absence of Mg2+, half-maximal activation of the phosphodiesterase was at about 3μm-Ca2+. The presence of 1mm-Mg2+ shifted the Ca2+ activation curve to the right, as did elevation of the ionic strength. When the Ca2+-pump ATPase and the polyphosphoinositide phosphodiesterase were assayed in the same incubations and under conditions of intracellular ionic strength and Mg2+concentration, the ATPase was fully activated at 3μm-Ca2+, whereas no polyphosphoinositide phosphodiesterase activity was detected below 100μm-Ca2+. 5. The Ca2+-pump ATPase of the erythrocyte membrane normally maintains the Ca2+ concentration of healthy erythrocytes below approx. 0.1μm. It therefore seems unlikely that the polyphosphoinositide phosphodiesterase of the erythrocyte membrane ever expresses its activity in a healthy erythrocyte.
Studies on the interactions of Ca2+ and pyruvate in the regulation of rat heart pyruvate dehydrogenase activity. Effects of starvation and diabetes
Authors: McCormack J G , Edgell N J , Denton R M
Published: August 2015

1. Previous studies showed that the activation of pyruvate dehydrogenase within intact rat heart mitochondria of pyruvate is much diminished in mitochondria from starved or diabetic animals [see Kerbey, Randle, Cooper, Whitehouse, Pask & Denton (1976) Biochem. J. 154, 327-348]. In the present study, diminished responses to added Ca2+ and ADP were also found in these mitochondria. 2. Starvation or diabetes did not affect the mitochondrial respiratory control ratio of the ATP content. Moreover, starvation and diabetes did not alter the response of the intramitochondrial Ca2+-sensitive enzyme, 2-oxoglutarate dehydrogenase, to changes in the extramitochondrial concentration of Ca2+ and 2-oxoglutarate, thus indicating that there were no appreciable changes in the distribution of Ca2+ and H+ across the mitochondrial inner membrane. 3. Pyruvate, Ca2+ and ADP were found to have synergistic effects on pyruvate dehydrogenase activity, particularly in mitochondria from starved and diabetic rats. 4. The results suggest that the effects of diabetes and starvation on pyruvate dehydrogenase are not brought about by changes in the distribution of these effectors across the mitochondrial inner membrane or by changes in the intrinsic sensitivity of the kinase or phosphatase of the pyruvate dehydrogenase system to pyruvate, Ca2+ or ADP; rather it is probably that there is an increase in the maximum activity of kinase relative to that of the phosphatase. 6. The results also lend further support to the hypothesis that adrenaline may bring about the activation of pyruvate dehydrogenase in the rat heart by an increase in the intramitochondrial concentration of Ca2+.
Evidence for the existence of regulatory sites for Ca2+ on the Na+/Ca2+ carrier of cardiac mitochondria
Authors: Hayat L H , Crompton M
Published: August 2015

The Na+-induced efflux of Ca2+ catalysed by the Na+/Ca2+ carrier of cardiac mitochondria is strongly inhibited by extramitochondrial Ca2+. The nature of this inhibition was investigated as follows. (a) The apparent association of external Na+ and the Ca2+ analogue Sr2+ with substrate-binding sites (i.e. those sites involved in cation translocation) is promoted markedly by K+. The inhibition of Na+/Ca2+ exchange by external Ca2+ is affected little by K+. (b) There is a competitive relationship between the binding of external Na+ and external Ca2+ to substrate-binding sites, whereas at low concentrations (less than 4 microM) extramitochondrial Ca2+ is a partial non-competitive inhibitor with respect to external Na+. (c) This inhibiton by external Ca2+ is characterized by a maximal decrease of about 70% in the Vmax of Na+/Ca2+ exchange and by cooperative binding of external Ca2+ to sites that are half saturated by 0.7-0.8 microM free Ca2+. The binding of Ca2+ and Sr2+ to substrate-binding sites shows no co-operativity. These criteria suggest that the Na+/Ca2+ carrier may contain regulatory sites that render the carrier sensitive to changes in extramitochondrial [Ca2+] within the physiological range.
Effect of inhibition of polyamine synthesis on the content of decarboxylated S-adenosylmethionine
Authors: Pegg Anthony E. , Pösö Hannu , Shuttleworth Kay , Bennett Richard A.
Published: August 2015

1. The content of decarboxylated S-adenosylmethionine (AdoMet) in transformed mouse fibroblasts (SV-3T3 cells) was increased 500-fold to about 0.4fmol/cell when ornithine decarboxylase was inhibited by α-difluoromethylornithine. This increase was due to the absence of putrescine and spermidine, which serve as substrates for aminopropyltransferases with decarboxylated AdoMet as an aminopropyl donor, and to the enhanced activity of AdoMet decarboxylase brought about by depletion of spermidine. The increase in decarboxylated AdoMet content was abolished by addition of putrescine, but not by 1,3-diaminopropane. 2. 5′-Methylthiotubercidin also increased decarboxylated AdoMet content, presumably by direct inhibition of aminopropyl-transferase activities, but the increase in its content and the decline in spermidine content were much less than those produced by α-difluoromethylornithine. 3. Decarboxylated AdoMet content of regenerating rat liver was measured in rats treated with inhibitors of ornithine decarboxylase. The content was increased by 60% 32h after partial hepatectomy in control rats, by 90% when α-difluoromethylornithine was given to the partially hepatectomized rats, and by 330% when 1,3-diaminopropane was used to inhibit putrescine and spermidine synthesis. After 48h of exposure to 1,3-diaminopropane, which completely prevented the increase in spermidine after partial hepatectomy, there was a 5-fold rise in hepatic decarboxylated AdoMet concentration. These increases were prevented by treatment with putrescine or with methylglyoxal bis(guanylhydrazone), an inhibitor of AdoMet decarboxylase. 4. These results show that changes in AdoMet metabolism result from the administration of specific inhibitors of polyamine synthesis. The possible consequences of the accumulation of decarboxylated AdoMet, which could, for example, interfere with normal cellular methylation or lead to depletion of cellular adenine nucleotides, should be considered in the interpretation of results obtained with such inhibitors.
Effects of cross-linked dimers of ribonuclease A or of lysozyme on the processing of endocytosed peroxidase by hepatoma cells
Authors: Bartholeyns J , Baudhuin P
Published: August 2015

Cross-linked dimers of ribonuclease, added at a concentration of 0.05 mg/ml to the culture medium of hepatoma (HTC) cells, were previously shown to inhibit intracellular degradation of peroxidase taken up by endocytosis. Intracellular localization showed that endocytosed peroxidase does not reach lysosomes in dimer-treated cells. The present study shows that preloading of lysosomes with fluorescent anti-peroxidase IgG, obtained by exposing HTC cells for 48 h to 0.1 mg of antibody/ml, restores intracellular degradation of endocytosed peroxidase. Moreover, accumulation of peroxidase into lysosomes, which no longer occurs in dimer-treated cells, occurs again under these conditions. We conclude that inhibition of transfer of peroxidase from phagosomes to lysosomes is most likely to be the alteration resulting from the exposure of the cells to ribonuclease dimer, rather than inhibition of fusion between phagosomes and lysosomes. The dimer of another basic protein, lysozyme added at a concentration of 0.2 mg/ml to the culture medium, is shown to induce the same type of effects as does the dimer of ribonuclease; the half-life of endocytosed peroxidase increased from 5 to 15 h after 2 h exposure of HTC cells to dimerized lysozyme. The effect of both dimers on intracellular protein processing can be reversed by addition of 100 mm-galactose to the culture medium, up to 5 h after pretreatment of the cells. The dimers of ribonuclease A or of lysozyme have thus probably the same mechanism of action. Evidence that the two dimers share the same binding sites on the cells is presented.
Glucagon inhibits triacylglycerol synthesis in isolated hepatocytes by lowering their glycerol 3-phosphate content
Authors: Declercq P E , Debeer L J , Mannaerts G P
Published: August 2015

Triacylglycerol synthesis by glycogen-depleted hepatocytes from fed rats that have low glycerol 3-phosphate contents was stimulated by the addition of glycerol 3-phosphate precursors. Glucagon decreased triacylglycerol synthesis only when it also lowered glycerol 3-phosphate content. The hyperbolic-like relationship between glycerol 3-phosphate content and rates of triacylglycerol synthesis was identical in the absence or presence of glucagon, indicating that the glucagon effect on triacylglycerol synthesis was not mediated through changes in enzyme activities of the esterification pathway but through changes in cellular glycerol 3-phosphate content.
Reactions of Adriamycin with haemoglobin. Superoxide dismutase indirectly inhibits reactions of the Adriamycin semiquinone
Authors: Bates D A , Winterbourn C C
Published: August 2015

The Adriamycin semiquinone produced by the reaction of xanthine oxidase and xanthine with Adriamycin has been shown to reduce both methaemoglobin and cytochrome c. In air, but not N2, both reactions were inhibited by superoxide dismutase. With cytochrome c, superoxide formed by the rapid reaction of the semiquinone with O2, was responsible for the reduction. However, even in air, methaemoglobin was reduced directly by the Adriamycin semiquinone. Superoxide dismutase inhibited this reaction by removing superoxide and hence the semiquinone by displacing the equilibrium: Semiquinone + O2 in equilibrium or formed from quinone + O2-. to the right. This ability to inhibit indirectly reactions of the semiquinone could have wider implications for the protection given by superoxide dismutase against the cytotoxicity of Adriamycin. Oxidation of haemoglobin by Adriamycin has been shown to be initiated by a reversible reaction between the drug and oxyhaemoglobin, producing methaemoglobin and the Adriamycin semiquinone. Reaction of the semiquinone with O2 gives superoxide and H2O2, which can also react with haemoglobin. Catalase, by preventing this reaction of H2O2, inhibits oxidation of oxyhaemoglobin. Superoxide dismutase, however, accelerates oxidation, by inhibiting the reaction of the semiquinone with methaemoglobin by the mechanism described above. Although superoxide dismutase has a detrimental effect on haemoglobin oxidation, it may protect the red cell against more damaging reactions of the Adriamycin semiquinone.
Isolation and partial characterization of wheat-germ-agglutinin-like lectins from rye (Secale cereale) and barley (Hordeum vulgare) embryos
Authors: Peumans Willy J. , Stinissen Hetty M. , Carlier Albert R.
Published: August 2015

Lectins have been isolated from embryos of Secale cereale (rye) and Hordeum vulgare (barley) by affinity chromatography on immobilized N-acetylglucosamine. Both lectins are dimeric proteins of two identical subunits of mol.wt. 18000. They resemble strongly wheat-germ agglutinin with respect to their chemical, physical, biological and immunological properties.
Localization of the membrane-associated thiol oxidase of rat kidney to the basal-lateral plasma membrane
Authors: Lash L H , Jones D P
Published: August 2015

The localization of the membrane-associated thiol oxidase in rat kidney was investigated. Fractionation of the kidney cortex by differential centrifugation demonstrated that the enzyme is found in the plasma membrane. The crude plasma membrane was fractionated by density-gradient centrifugation on Percoll to obtain purified brush-border and basal-lateral membranes. Gamma-Glutamyltransferase, alkaline phosphatase and aminopeptidase M were assayed as brush-border marker enzymes, and (Na+ + K+)-stimulated ATPase was assayed as a basal-lateral-membrane marker enzyme. Thiol oxidase activity and distribution were determined and compared with those of the marker enzymes. Its specific activity was enriched 18-fold in the basal-lateral membrane fraction relative to its activity in the cortical homogenate, and its distribution paralleled that of (Na+ + K+)-stimulated ATPase. This association indicates that thiol oxidase is localized in the same fraction as (Na+ + K+)-stimulated ATPase, i.e. the basal-lateral region of the plasma membrane of the kidney tubular epithelium.
Relation of internal thioesters to conformational change and receptor-recognition site in α2-macroglobulin complexes
Authors: Van Leuven F , Marynen P , Cassiman J J , Van den Berghe H
Published: August 2015

The unique steric type of inhibition of endopeptidases by human alpha 2-macroglobulin (alpha 2-M) and the inactivation of the latter by methylamine were examined in relation to the internal thioesters in alpha 2M. The present results confirm our previous findings that disruption of the internal thioesters, is not in itself sufficient to cause the conformational change of alpha 2M typical of alpha 2-M-proteinase complexes; the electrophoretically slow form of alpha 2M with [14C]methylamine incorporated was isolated. Moreover, this group is stabilized by derivatization of the exposed cysteine thiol groups. Cyanylation with 2,4-dinitrophenyl thiocyanate during the methylamine reaction was the most effective procedure, yielding essentially only slow-form alpha 2M. Other thiol-specific reagents were less effective. When allowed to react with trypsin the cyanylated derivative (slow-form alpha 2M with thioesters broken) produced anomalous complexes; only half the expected amount of trypsin was bound, whereas the complexes were fully inhibited by soya-bean trypsin inhibitor and were proteolytically active. Despite this, the anomalous complexes were recognized by two highly specific probes: the fibroblast alpha 2M-complex receptor and the monoclonal antibody (F2B2) directed against the receptor-recognition site on alpha 2M complexes. The results show that the internal thioesters in alpha 2M are necessary for the conformational change producing sterically inhibited endoproteinase complexes, but do not participate as such in receptor-mediated endocytosis of these complexes.
Precise elimination of the N-terminal domain of histone H1
Authors: Böhm L , Sautière P , Cary P D , Crane-Robinson C
Published: August 2015

The proteinase from mouse submaxillary gland was used to cleave total calf thymus histone H1 between residues 32 and 33. The C-terminal peptide, comprising residues 33 to the C-terminus, was purified and identified by amino acids analysis and Edman degradation. Spectroscopic characterization by n.m.r. for tertiary structure and by c.d. for secondary structure shows the globular domain of the parent histone H1 to be preserved intact in the peptide. It has therefore lost only the N-terminal domain and is a fragment of histone H1 comprising the globular plus C-terminal domains only. Precise elimination of only the N-terminal domain makes the fragment suitable for testing domain function in histone H1.
Tubulin carbamoylation. Functional amino groups in microtubule assembly
Authors: Mellado W , Slebe J C , Maccioni R B
Published: August 2015

The characteristics of the carbamoylation of pig brain tubulin were examined by using the modification conditions with cyanate described previously [Mellado, Slebe + Maccioni (1980) Biochem. Int. I, 584-590]. The carbamoylation reaction resulted in an inhibition of microtubule assembly, which was dependent on the concentration of the modifying agent. This tubulin modification appears to inhibit the growth of microtubules. The presence of GTP did not protect tubulin against this inhibition. Electron microscopy showed a marked decrease in the number of tubules after carbamoylation, but no alterations were observed in the microtubule morphology. The incorporation of KN14CO into alpha- and beta-subunits with similar kinetics was also shown, and the carbamoylated residues were identified as epsilon-N-carbamoyl-lysine residues.
Immunodiffusion studies of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density
Authors: Keiser Harold D.
Published: August 2015

Tryptic fragments of bovine nasal-cartilage proteoglycan, fractionated by dissociative density-gradient ultracentrifugation, were made to react by immunodiffusion against antiserum to a hyaluronidase-digest subfraction of cartilage proteoglycan monomer. This reaction produced two families of partly superimposed precipitin lines. One family was restricted to gradient fractions of medium or low buoyant density and included the immunoprecipitation reaction attributed to the hyaluronic acid-binding region of the cartilage proteoglycan monomer. The second family of precipitin lines was present alone in gradient fractions of high buoyant density. Immunodiffusion studies with antisera to relatively homogeneous keratan sulphate-rich and chondroitin sulphate-bearing fragment subfractions isolated from the gradient fraction of highest density indicated that both subfractions contained the antigenic determinants responsible for the second family of precipitin lines. Additional immunodiffusion studies, with the use of multispecific antisera to chondroitinase ABC digest and hyaluronidase digest of proteoglycan monomer, confirmed that the two subfractions shared antigenic determinants, and, in addition, indicated that these determinants were on one molecular species in the keratan sulphate-rich fragment subfraction and divided among at least three in the chondroitin sulphate-bearing fragment subfraction. Although an unprecedentedly large number of cartilage proteoglycan antigens could be recognized with the antisera employed in this cartilage proteoglycan antigens could be recognized with the antisera employed in this study, it was not possible to identify antigenic determinants unambiguously specific for the three structurally and functionally distinct regions of the cartilage proteoglycan monomer.
A circular-dichroism study of epidermolytic toxins A and B from Staphylococcus aureus
Authors: Bailey C J , Martin S R , Bayley P M
Published: August 2015

The far-u.v. circular-dichroism spectra of the two epidermolytic toxins was analysed into fractional contributions of 0.09 helix and 0.46 beta-sheet to each toxin structure. Trifluoroethanol perturbation caused an initial increase in dichroic absorption at 205 nm and then a change characterized as a beta-sheet-to-alpha-helix transition. The intense near-u.v. spectra suggested that the toxins have unusually rigid, though different, aromatic-side-chain arrangements.
Isolation and partial characterization of rat duodenal-gland (Brunner's-gland) mucus glycoprotein
Authors: Smits H L , van Kerkhof P J M , Kramer M F
Published: August 2015

A mucus glycoprotein was isolated from the duodenal glands of the rat and purified by repeated density-gradient centrifugation. The characterized glycoprotein is unique to the mucous cells of the duodenal glands and is not present in parts of the small intestine devoid of these glands. The chemical composition of the purified glycoprotein is characteristic for glycoproteins of the mucin-type. Its protein content is relatively high and amount to 35% by weight. No neuraminic acid and little sulphate (2%) is present. Evidence is presented that the native glycoprotein is built up from subunits held together via disulphide bridges in a non-glycosylated region of the protein core.
The purification and some properties of a stereospecific d-asparaginase from an extremely thermophilic bacterium, Thermus aquaticus
Authors: Guy G R , Daniel R M
Published: August 2015

A specific D-asparaginase was isolated and crystallized from Thermus aquaticus strain T351. It is present in larger amounts than the L-asparaginase. The enzyme has a molecular weight of 60 000, an isoelectric point of 4.8 and a Km of 2 mM. It has 6 disulphide bonds/molecule, and a histidine residue at the active site. It is inhibited by keto acids and by high salt concentrations.
Assay of free and glycine- and taurine-conjugated bile acids in serum by high-pressure liquid chromatography by using post-column reaction after group separation
Authors: Onishi S , Itoh S , Ishida Y
Published: August 2015

An accurate and sensitive method that involves the group separations of serum bile acids (i.e. free and glycine- and taurine-conjugated bile acid fractions) by ion-exchange chromatography on piperidinohydroxypropyl-Sephadex LH-20 is described. Each group was then analysed by high-pressure liquid chromatography by using the post-column reaction technique with immobilized 3 alpha-hydroxy steroid dehydrogenase. The bile acid patterns in the umbilical venous serum samples were analysed by this method. Taurochenodeoxycholate predominated in the umbilical cord.