bluuebunny/crossref_metadata_2025 huggingface dataset:
Biosynthesis and metabolism in vivo of intervertebral-disc proteoglycans in the mouse
Authors: Venn G , Mason R M
Published: August 2015

The synthesis and turnover in vivo of 35S-labelled proteoglycans in mouse cervical, thoracic and lumbar intervertebral discs, and in costal cartilage, was investigated after intraperitoneal injection of [35S]sulphate. Intervertebral discs and costal cartilage synthesize similar amounts of 35S-labelled proteoglycans per microgram of DNA. Discs and cartilage all synthesize a major proteoglycan species (approx. 85%) of large hydrodynamic size and a minor species (approx. 15%) of small size. Both proteoglycans carry chondroitin sulphate chains. Keratan sulphate was not found associated with either species. The total 35S-labelled proteoglycan pool had a metabolic half-life (t1/2) of 10-12 days in discs, and 17 days in cartilage. The extractable major and minor species turned over at similar rates. Those proteoglycans left in the tissue after 29 days turn over very slowly. Approx. 50% of the major 35S-labelled proteoglycan species formed mixed aggregates with hyaluronic acid and rat chondrosarcoma proteoglycan. The ability to form aggregates did not decrease up to 45 days after synthesis. Of the heterogeneous population of proteoglycans comprising the major species, those remaining in the tissue 9 days after synthesis were of smaller average hydrodynamic size and had shorter chondroitin sulphate side chains than the average size at the time of synthesis. With increasing time after synthesis, proteoglycans were less readily extracted from the tissue by 4.0 M-guanidinium chloride than at the time of synthesis.
Receptor-mediated endocytosis of proteoglycans by human fibroblasts involves recognition of the protein core
Authors: Glössl J , Schubert-Prinz R , Gregory J D , Damle S P , von Figura K , Kresse H
Published: August 2015

Endocytosis by cultured human skin fibroblasts of 35SO4(2-)-labelled or [3H]leucine-labelled proteoglycans from fibroblast secretions and of 125I-proteodermatan sulphate from pig skin was quantitatively investigated. The following results were obtained. (1) Core proteins prepared by digestion with chondroitin ABC lyase were at least as efficiently endocytosed as native proteoglycans. Pig skin proteodermatan sulphate was a competitive inhibitor of endocytosis of 35SO4(2-)-labelled proteoglycans. (2) Proteoglycans produced in the presence of tunicamycin and native proteoglycans degraded with endoglycosaminidase H were internalized at a normal rate. Several monosaccharides that can be bound by mammalian lectins were unable to influence the internalization of proteoglycans. Treatment of proteoglycans with neuraminidase, however, resulted in an increased clearance rate. (3) Reductive methylation or acetoacetylation of lysine residues was accompanied by a parallel decrease in the rate of proteoglycan endocytosis. Reversal of acetoacetylation normalized the uptake properties. Endocytosis of native proteoglycans was also reduced in the presence of poly-L-lysine, and this reduction in endocytosis was observed as well with proteoglycans synthesized in the presence of the lysine analogue S-2-aminoethylcysteine. These results suggest that the recognition marker required for receptor-mediated endocytosis of proteodermatan sulphate resides in its protein moiety and involves lysine residues.
Endopeptidase-24.11 purified from pig intestine is differently glycosylated from that in kidney
Authors: Fulcher I S , Chaplin M F , Kenny A J
Published: August 2015

Endopeptidase-24.11 (EC 3.4.24.11) was purified from pig intestinal microvilli by immunoadsorbent chromatography, using antibodies raised to kidney endopeptidase-24.11. In many respects, the kidney and intestinal enzymes were indistinguishable, but some structural differences were demonstrated. In particular, the detergent form of the intestinal enzyme had an apparent subunit Mr of 95000, which, on treatment with trypsin, fell to a value of 89000, identical with that of the kidney form. The intestinal enzyme contained 3-4% more carbohydrate and many more fucose residues than that from kidney. Although these results show that post-translational processing was different in the two cell types, the possibility that the primary translation products also differed cannot be excluded.
Bovine lens aldehyde dehydrogenase. Kinetics and mechanism
Authors: Ting H H , Crabbe M J C
Published: August 2015

Bovine lens cytoplasmic aldehyde dehydrogenase exhibits Michaelis-Menten kinetics with acetaldehyde, glyceraldehyde 3-phosphate, p-nitrobenzaldehyde, propionaldehyde, glycolaldehyde, glyceraldehyde, phenylacetylaldehyde and succinic semialdehyde as substrates. The enzyme was also active with malondialdehyde, and exhibited an esterase activity. Steady-state kinetic analyses show that the enzyme exhibits a compulsory-ordered ternary-complex mechanism with NAD+ binding before acetaldehyde. The enzyme was inhibited by disulfiram and by p-chloromercuribenzoate, and studies with with mercaptans indicated the involvement of thiol groups in catalysis.
Transient kinetic studies of Neurospora crassa cytochrome c oxidase
Authors: Brunori M , Silvestrini M C , Wilson M T , Weiss H
Published: August 2015

The reaction of Neurospora crassa cytochrome c oxidase with CO was studied by flash-photolysis and rapid-mixing experiments, leading to the determination of the association and dissociation rate constants (7 X 10(4) M-1 X s-1 and 0.02s-1 respectively). Pre-steady-state kinetic investigations of the catalytic properties of the enzyme showed that under proper conditions Neurospora cytochrome c oxidase can be ‘pulsed’, i.e. activated, like the mammalian enzyme. The ‘pulsed’ species is spectroscopically different from the ‘resting’ one, and the decay into the ‘resting’ state is fast (t1/2 approx. 3 min).
Post-secretional modification of exo-1,3-β-d-glucanases from Saccharomyces cerevisiae
Authors: Sánchez A , Nebreda A R , Villanueva J R , Villa T G
Published: August 2015

Exo-1,3-beta-D-glucanase secreted by Saccharomyces cerevisiae undergoes extracellular modifications in its carbohydrate moiety that change the affinity towards the lectin concanavalin A. The transition of negatively reacting enzyme form into positively reacting one depends on temperature. Results from experiments with glucono-delta-lactone and from treatments in vitro with hydrolases suggest a glycosidase-mediated mechanism.
Kinetic properties of spermine synthase from bovine brain
Authors: Pajula R L
Published: August 2015

A kinetic analysis including initial-velocity and product-inhibition studies were performed with spermine synthase purified from bovine brain. The enzyme activity was assayed in the presence of 5′-methylthioadenosine phosphorylase as an auxiliary enzyme to prevent the accumulation of the inhibitory product, 5′-methylthioadenosine, and thus to obtain linearity of the reaction with time. Initial-velocity studies gave intersecting or converging linear double-reciprocal plots. No substrate inhibition by decarboxylated S-adenosylmethionine was observed at concentrations up to 0.4 mM. Apparent Michaelis constants were 60 microM for spermidine and 0.1 microM for decarboxylated S-adenosylmethionine. Spermine was a competitive product inhibitor with respect to decarboxylated S-adenosylmethionine, but a mixed one with respect to the other substrate, spermidine. 5′-Methylthioadenosine showed a mixed inhibition with both substrates, predominantly competitive with respect to decarboxylated S-adenosylmethionine and predominantly uncompetitive with respect to spermidine. The observed kinetic and inhibition patterns are consistent with a compulsory-order mechanism, where both substrates add to the enzyme before products can be released.
Thermodynamic studies of the activation of rabbit muscle lactate dehydrogenase by phosphate
Authors: Ward L D , Winzor D J
Published: August 2015

In an attempt to trace the source of phosphate activation of the enzyme-catalysed pyruvate-lactate interconversion by rabbit muscle lactate dehydrogenase, equilibrium constants were measured to examine the effects of phosphate on interactions pertinent to the enzymic process. Frontal gel-chromatographic studies of the binding of NADH to the enzyme established that the intrinsic association constant is doubled in the presence of 50 mM-phosphate in the buffer (pH 7.4, I0.15). From kinetic studies of the competition between NAD+ and NADH for the coenzyme-binding sites of the enzyme it is concluded that the binding of oxidized nicotinamide nucleotide is also doubled in the presence of 50 mM-phosphate. Competitive-inhibition studies and fluorescence-quenching measurements indicated the lack of a phosphate effect on ternary-complex formation between enzyme-NADH complex and oxamate, a substrate analogue of pyruvate. The equilibrium constant for the interaction between enzyme-NAD+ complex and oxalate, an analogue of lactate, was also shown, by difference spectroscopy, to be insensitive to phosphate concentration. Provided that the effects observed with the substrate analogues mimic those operative in the kinetic situation, the equilibrium constant governing the isomerization of ternary complex is also independent of phosphate concentration. It is concluded that enhanced coenzyme binding is the source of phosphate activation of the rabbit muscle lactate dehydrogenase system.
Preparation of [B23-d-alanine]des-(B25-B30)-hexapeptide-insulin by a combination of enzymic and non-enzymic synthesis
Authors: Zhang Y S , Cao Q P , Li Z G , Cui D F
Published: August 2015

Des-(B25-B30)-hexapeptide-insulin with B23-glycine replaced by D-alanine was prepared by a combination of enzymic and non-enzymic syntheses. The purified product was homogeneous in polyacrylamide-gel electrophoresis and could be crystallized. The biological activity in vivo of crystalline [B23-D-Ala]des-(B25-B30)-hexapeptide-insulin was determined as 58% of that of standard pig insulin (27 i.u./mg).
Fatty acids reverse the cyclic AMP inhibition of triacylglycerol and phosphatidylcholine synthesis in rat hepatocytes
Authors: Pelech S L , Pritchard P H , Brindley D N , Vance D E
Published: August 2015

The influence of cyclic AMP analogues and fatty acids on glycerolipid biosynthesis in monolayer cultures of rat hepatocytes was investigated. Chlorophenylthio-cyclic AMP and adenosine 3′:5′-cyclic phosphorothioate inhibited the rate of triacylglycerol synthesis from [1(3)-3H]glycerol, and phosphatidylcholine synthesis from [Me-3H]-choline. Supplementation of the hepatocytes with palmitate (1 mM) reversed chlorophenylthio-cyclic AMP inhibition of triacylglycerol synthesis. Similarly, cyclic AMP analogue-inhibition of phosphatidylcholine synthesis was abolished when the cells were simultaneously incubated with oleate (3 mM). Reactivation of phosphatidylcholine synthesis in chlorophenylthio-cyclic AMP-supplemented cells with oleate was accompanied by conversion of CTP: phosphocholine cytidylyltransferase into the membrane-bound form, since these cells released the enzyme more slowly after treatment with digitonin. The opposing actions of cyclic AMP and fatty acids are discussed in relation to the regulation of glycerolipid biosynthesis during starvation, diabetes and stress.
Oxidative phosphorylation by mutant Escherichia coli membranes with impaired proton permeability
Authors: Cox G B , Jans D A , Gibson F , Langman L , Senior A E , Fimmel A L
Published: August 2015

The effect on the function of the Escherichia coli F1F0-ATPase of the substitution of leucine-31 by phenylalanine in the c-subunit of the enzyme was examined. The assembly of the mutant c-subunit requires an increased gene dosage [Jans, Fimmel, Langman, James, Downie, Senior, Ash, Gibson & Cox (1983) Biochem. J. 211, 717-726], and this was achieved by incorporation of the uncE408 or uncE463 alleles on to F-plasmids or multicopy plasmids. Membranes from strains carrying either the uncE463 or uncE408 alleles on F-plasmids or multicopy plasmids were capable of carrying out oxidative phosphorylation. In particular, membranes from strain AN1928 (pAN162, uncE463) gave phosphorylation rates and P/O ratios equal to or greater than those obtained for the control strain AN1460 (pAN45, unc+). However, the mutant membranes, on removal of the F1-ATPase, appeared to be proton-impermeable. The ATPase activity of the mutant membranes was also resistant to the inhibitor dicyclohexylcarbodi-imide.
Degradation of transplanted mitochondrial proteins by hepatocyte monolayers
Authors: Evans P J , Mayer R J
Published: August 2015

Reductively [3H]methylated rat mitochondria and mitochondrial-outer-membrane vesicles and mitochondrial-outer-membrane vesicles where monoamine oxidase is irreversibly labelled by [3H]pargyline have been transplanted into hepatocytes by poly(ethylene glycol)-mediated organelle or organelle-vesicle cell fusion. During subsequent culture of hepatocyte monolayers for 4-5 days, under conditions which mimic endogenous catabolic rates in vivo the transplanted organelle proteins retain their degradation characteristics observed in vivo (e.g. mitochondria: average t 1/2 72.5 h; monoamine oxidase: t1/2 55 h). In all cases protein degradation with first-order kinetics is only observed after an initial lag period (i.e. 24-30 h after fusion). Transplantation of fluorescein-conjugated organelles showed that the fluorescent material is rapidly internalized (average t1/2 1-6 h) and uniformly distributed in the cytoplasm. During a subsequent 18-24 h period (which corresponds to the lag period for intracellular destruction of transplanted mitochondrial material) the transplanted material is translocated to assume a perinuclear distribution. The destruction of transplanted mitochondrial proteins is compared with endogenous mitoribosomally synthesized proteins (average t1/2 52.5 h). Percoll fractionation of cell homogenates containing transplanted mitochondrial outer membranes where the enzyme monoamine oxidase is irreversibly labelled with [3H]pargyline shows a distribution of enzyme similar to lysosomal acid phosphatase. After transplantation of reductively methylated 3H-labelled mitochondrial-outer-membrane vesicles the cells were treated with leupeptin to alter lysosomal density. This treatment leads to the predominant association of acid phosphatase with dense structures, whereas the 3H-labelled transplanted material predominantly does not change density. Therefore transplanted mitochondrial-outer-membrane proteins are found in intracellular vesicular structures from which the proteins are donated for destruction, at least in part, by a lysosomal mechanism.
Inhibition of ureagenesis by valproate in rat hepatocytes. Role of N-acetylglutamate and acetyl-CoA
Authors: Coude F X , Grimber G , Parvy P , Rabier D , Petit F
Published: August 2015

Valproate (0.5-5 mM) strongly inhibited urea synthesis in isolated rat hepatocytes incubated with 10 mM-alanine and 3 mM-ornithine. Valproate at the same concentrations markedly decreased concentrations of N-acetylglutamate, an essential activator of carbamoyl-phosphate synthetase I (EC 6.3.4.16), in parallel with the inhibition of urea synthesis by valproate. This compound also lowered the cellular concentration of acetyl-CoA, a substrate of N-acetylglutamate synthase (EC 2.3.1.1); glutamate, aspartate and citrulline were similarly decreased. Valproate in a dose up to 2 mM did not significantly affect the cellular concentration of ATP and had no direct effect on N-acetylglutamate synthesis, carbamoyl-phosphate synthetase I and ornithine transcarbamoylase (EC 2.1.3.3) activities.
Abnormal secretion of proteins into bile from colchicine-treated isolated perfused rat livers
Authors: Barnwell S G , Coleman R
Published: August 2015

The microtubule poison, colchicine, caused an abnormal output of a variety of proteins into rat bile. After 3 h of exposure to the drug, livers were isolated and perfused with media of defined protein composition. There was no essential change in permeability of the hepatobiliary system to proteins (e.g. bovine serum albumin) entering bile from the perfusion fluid. The rat (serum) albumin and fibrinogen that were secreted into bile from colchicine-treated livers were probably derived from the hepatocytes. Disruption of the microtubular system reduces the secretion of proteins at the sinusoidal face of the hepatocyte and results in an accumulation of secretory vesicles in the cytoplasm. It is suggested that under these conditions some of the vesicles discharge their contents into the bile canaliculus.
The hydrogenation of γ-linolenic acid by pure cultures of two rumen bacteria
Authors: Kemp P , Lander D J
Published: August 2015

Two species of rumen bacteria that have been previously shown to partially hydrogenate alpha-linolenic acid have been examined for their ability to hydrogenate gamma-linolenic acid. Free gamma-linolenic acid is hydrogenated in vitro to stearic acid by a rumen Fusocillus sp. (N.C.I.B. 11026), but only to cis, trans-octadec-6,11-enoic acid by a Butyrivibrio sp. The sequential hydrogenations are preceded by a delta 12-cis-delta 11-trans isomerization identical with that observed in the hydrogenation of alpha-linolenic acid and linoleic acid.
Kinetics of nitrobenzylthioinosine binding to the human erythrocyte nucleoside transporter
Authors: Jarvis S M , Janmohamed S N , Young J D
Published: August 2015

The kinetics of [3H]nitrobenzylthioinosine binding to human erythrocyte membranes was studied. The pseudo-first-order association was linear and consistent with a simple bimolecular reaction mechanism between nitrobenzylthioinosine and the nucleoside-transport mechanism. Dissociation of the [3H]nitrobenzylthioinosine complex at 22 degrees C was also linear (apparent k-1 congruent to 0.20 min-1). Adenosine was a competitive inhibitor of equilibrium high-affinity [3H]nitrobenzylthioinosine-binding activity (apparent Ki 0.1 mM). Dissociation of the [3H]nitrobenzylthioinosine-membrane complex was faster in the presence of adenosine and uridine, and this effect was proportional to the nucleoside concentration. Nucleoside concentrations less than 1 mM had no significant effect on the dissociation rate constant. In contrast, dissociation was slower in the presence of high concentrations (micromolar) of dipyridamole. Low concentrations of dipyridamole (2-200 nM) and nitrobenzylthioinosine concentrations as high as 2.5 microM had no effect on the rate of [3H]nitrobenzylthioinosine dissociation. These results are discussed in terms of possible distinct inhibitor and permeation sites, and are suggested to be consistent with both a single-site model for the binding of nitrobenzylthioinosine and permeant to the same site, or an allosteric-site model in which permeant and inhibitor bind to different sites.
Determination of the desensitization of β-adrenergic receptors by [3H]CGP-12177
Authors: Hertel C , Müller P , Portenier M , Staehelin M
Published: August 2015

Isoprenaline treatment of C6-glioma cells induced a fast decrease in the number of beta-adrenergic receptors as determined by binding of [3H]CGP-12177, which paralleled the decrease in the hormonally stimulated adenylate cyclase activity. The total number of receptors, as determined by binding of (-)-[3H]dihydroalprenolol, did not decrease. Separation of the beta-adrenergic receptors on a sucrose density gradient showed that the decrease in the number of receptors detectable with CGP-12177 was due to a movement of the receptors from the plasma membrane to a vesicular cell compartment. By using both (-)-[3H]dihydroalprenolol and [3H]CGP-12177 it is thus possible to differentiate between the total number of receptors and those present at the plasma membrane in an unfractionated cell lysate.
Evidence concerning the mechanism of insulin-receptor interaction and the structure of the insulin receptor from biological properties of covalently linked insulin dimers
Authors: Tatnell M A , Jones R H , Willey K P , Schüttler A , Brandenburg D
Published: August 2015

Covalently linked insulin dimers have been prepared by cross-linking two insulin monomers with a flexible suberoyl chain at either the B1 phenylalanine or the B29 lysine residue. Binding potencies of dimers determined by inhibition of binding of 125I-insulin to isolated rat liver plasma membranes or adipocytes were 2.5-7-fold greater than their abilities to stimulate lipogenesis in adipocytes. Rates of liver plasma-membrane-associated degradation of labelled insulin and dimers, measured by gel filtration, were similar at 37 degrees C. Binding and lipogenesis potencies of dimers prepared by substitution of each monomeric half of an asymmetrical dimer with desoctapeptide insulin, an almost inactive derivative, implicated the B1-cross-linked monomeric half as predominantly interacting with the insulin receptor. These results suggest that (1) dimers bind univalently to a bivalent insulin-receptor complex, in which the two individual binding subunits are arranged with anti-parallel symmetry and (2) the mechanism by which insulin binds and initiates its biological responses requires a conformational change within the insulin-receptor complex and/or in the insulin molecule for full biological expression.
Catecholamine-induced insulin resistance of glucose transport in isolated rat adipocytes
Authors: Kirsch D M , Baumgarten M , Deufel T , Rinninger F , Kemmler W , Häring H U
Published: August 2015

The effects of pre-incubation with isoprenaline and noradrenaline on insulin binding and insulin stimulation of D-glucose transport in isolated rat adipocytes are reported. (1) Pre-incubation of the cells with isoprenaline (0.1-10 microM) in Krebs-Ringer-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid] buffer (30 min, 37 degrees C) at D-glucose concentrations of 16 mM, in which normal ATP levels were maintained, caused a rightward-shift in sensitivity of D-glucose transport to insulin stimulation by 50% and a decrease in maximal responsiveness by 30% (2) [A14-125I]insulin binding was reduced significantly by 35% at insulin concentrations less than 100 mu-units/ml and Scatchard analysis showed that this consisted mainly of a decrease in high-affinity binding. (3) Pre-incubation with catecholamines under the same conditions but at low glucose concentrations (0-5 mM) caused a fall in intracellular ATP levels of 65 and 45% respectively. (4) The fall in ATP additionally lowered insulin binding by 50% at all insulin concentrations and a parallel shift of the binding curves in the Scatchard plot showed that this was due to a decrease in the number of receptors. (5) At low and high ATP concentrations the insulin stimulation of D-glucose transport was inhibited to a similar extent. (6) Pre-incubation with catecholamines thus inhibited insulin stimulation of D-glucose transport in rat adipocytes mainly by a decrease in high-affinity binding of insulin, which was not mediated by low ATP levels. This mechanism may play a role in the pathogenesis of catecholamine-induced insulin resistance in vivo.
Ca2+-binding protein from human kidney. Purification and properties
Authors: Staun M , Norén O , Sjöström H
Published: August 2015

A Ca2+-binding protein (CaBP) from human kidney was purified by two different procedures. The first involved heat-precipitation of a kidney cytosol fraction followed by gel filtration and chromatofocusing. This resulted in a 200-fold increase in the specific Ca2+-binding activity with a yield of 10%. A specific antibody was raised against the purified CaBP, as demonstrated by one precipitate in crossed immunoelectrophoresis of a kidney cytosol fraction. The antibody was coupled to Sepharose 4B and CaBP was then purified by immunoadsorbent chromatography. Applying this technique, a 500-fold purification of CaBP with a yield of 50% was obtained. Both preparations appeared homogeneous in crossed immunoelectrophoresis against a polyvalent antiserum and migrated as a single band corresponding to a mol.wt. of 26000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In gel filtration under non-denaturing conditions CaBP was eluted corresponding to a mol.wt. of 28000. The association constant for the high-affinity Ca2+-binding sites of CaBP was estimated by gel filtration to be 0.1 × 10(6)M-1, and the protein displayed Ca2+-dependent electrophoretic mobility, with more rapid anodic migration in the presence of EDTA. The protein eluted at a position corresponding to a pI of 4.5 in chromatofocusing. Immunochemical experiments with the specific antibody showed no cross-reaction between renal and intestinal CaBP.
Effects of carbachol and pancreozymin (cholecystokinin-octapeptide) on polyphosphoinositide metabolism in the rat pancreas in vitro
Authors: Orchard J L , Davis J S , Larson R E , Farese R V
Published: August 2015

We studied the possibility that hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] may be the initiating event for the increase in [32P]Pi incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) during carbachol and pancreozymin (cholecystokinin-octapeptide) action in the rat pancreas. After prelabelling acini for 2h, [32P]Pi incorporation into PtdA, PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate (PtdIns4P) had reached equilibrium. Subsequent addition of carbachol or pancreozymin caused 32P in PtdIns(4,5)P2 to decrease by 30-50% within 10-15 s, and this was followed by sequential increases in [32P]Pi incorporation into PtdA and PtdIns. Similar changes in 32P-labelling of PtdIns4P were not consistently observed. Confirmation that the decrease in 32P in chromatographically-purified PtdIns(4,5)P2 reflected an actual decrease in this substance was provided by the fact that similar results were obtained (a) when PtdIns(4,5)P2 was prelabelled with [2-3H]inositol, and (b) when PtdIns(4,5)P2 was measured as its specific product (glycerophosphoinositol bisphosphate) after methanolic alkaline hydrolysis and ion-exchange chromatography. The secretogogue-induced breakdown of PtdIns(4,5)P2 was not inhibited by Ca2+ deficiency (severe enough to inhibit amylase secretion and Ca2+-dependent hydrolysis of PtdIns), and ionophore A23187 treatment did not provoke PtdIns(4,5)P2 hydrolysis. The increase in the hydrolysis of PtdIns(4,5)P2 and the increase in [32P]Pi incorporation into PtdA commenced at the same concentration of carbachol in dose-response studies. Our findings suggest that the hydrolysis of PtdIns(4,5)P2 is an early event in the action of pancreatic secretogogues that mobilize Ca2+, and it is possible that this hydrolysis may initiate the Ca2+-independent labelling of PtdA and PtdIns. Ca2+ mobilization may follow these responses, and subsequently cause Ca2+-dependent hydrolysis of PtdIns and exocytosis.
Stoichiometry of reactions of α2-macroglobulin with trypsin and chymotrypsin
Authors: Björk I , Larsson L J , Lindblom T , Raub E
Published: August 2015

The stoichiometry of the individual steps, i.e. polypeptide chain cleavage, hydrolysis of the putative thioester bond and conformational change, of the reaction between alpha 2-macroglobulin and trypsin or chymotrypsin was analysed. The chain cleavage was monitored by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the thioester hydrolysis by both a spectroscopic and a fluorimetric technique and the conformational change by tryptophan fluorescence. A stoichiometry of close to 2:1 was obtained for all reactions. This finding indicates that the alpha 2-macroglobulin half-molecule is an independent functional unit of the inhibitor, within which co-operativity between the two subunits may occur.
Rat liver Golgi galactosyltransferases. Distinct enzymes for glycolipid and glycoprotein acceptor substrates
Authors: Kaplan F , Hechtman P
Published: August 2015

Two enzymes that catalyse the transfer of galactose from UDP-galactose to GM2 ganglioside were partially purified from rat liver Golgi membranes. These preparations, designated enzyme I (basic) and enzyme II (acidic), utilized as acceptors GM2 ganglioside and asialo GM2 ganglioside as well as ovalbumin, desialodegalactofetuin, desialodegalacto-orosomucoid, desialo bovine submaxillary mucin and GM2 oligosaccharide. Enzyme II catalysed disaccharide synthesis in the presence of the monosaccharide acceptors N-acetylglucosamine and N-acetylgalactosamine. The affinity adsorbent alpha-lactalbumin-agarose, which did not retard GM2 ganglioside galactosyltransferase, was used to remove most or all of galactosyltransferase activity towards glycoprotein and monosaccharide acceptors from the extracted Golgi preparation. After treatment of the extracted Golgi preparation with alpha-lactalbumin-agarose, enzyme I and enzyme II GM2 ganglioside galactosyltransferase activities, prepared by using DEAE-Sepharose chromatography, were distinguishable from transferase activity towards GM2 oligosaccharide and glycoproteins by the criterion of thermolability. This residual galactosyltransferase activity towards glycoprotein substrates was also shown to be distinct from GM2 ganglioside galactosyltransferase in both enzyme preparations I and II by the absence of competition between the two acceptor substrates. The two types of transferase activities could be further distinguished by their response to the presence of the protein effector alpha-lactalbumin. GM2 ganglioside galactosyltransferase was stimulated in the presence of alpha-lactalbumin, whereas the transferase activity towards desialodegalactofetuin was inhibited in the presence of this protein. The results of purification studies, comparison of thermolability properties and competition analysis suggested the presence of a minimum of five galactosyltransferase species in the Golgi extract. Five peaks of galactosyltransferase activity were resolved by isoelectric focusing. Two of these peaks (pI 8.6 and 6.3) catalysed transfer of galactose to GM2 ganglioside, and three peaks (pI 8.1, 6.8 and 6.3) catalysed transfer to glycoprotein acceptors.
16S acetylcholinesterase of the extracellular matrix is assembled within mouse muscle cells in culture
Authors: Inestrosa N C
Published: August 2015

The present paper examines where the extracellular-matrix (ECM) 16S acetylcholinesterase (AChE, EC 3.1.1.7) is assembled in muscle cells in culture. The existence of an internal pool of 16S AChE was detected by using AChE inhibitors of differing membrane permeability. After irreversible inhibition of all cellular esterase, the newly synthesized 16S form appears in an intracellular compartment and is only later detected on the cell surface. Results show that the ECM 16S AChE is assembled within muscle cells.
The hepatic defect in glycogen synthesis in chronic diabetes involves the G-component of synthase phosphatase
Authors: Bollen M , Stalmans W
Published: August 2015

Hepatocytes from normal fed rats and from chronically (90 h) alloxan-diabetic rats were compared. The rate and the extent of activation of glycogen synthase in response to 60 mM-glucose were greatly decreased in diabetes. During incubation of gel-filtered extracts from broken hepatocytes, diabetes only decreased the rate of the activation, which became ultimately complete in either preparation. Synthase phosphatase activity, as measured by the activation of purified hepatic synthase b, was decreased in chronic diabetes. The decrease was proportional to the severity of the diabetes, and reached 90% when the plasma glucose concentration was greater than or equal to 55 mM. In contrast, phosphorylase phosphatase activity was not decreased. Synthase phosphatase activity was progressively restored by treatment with insulin for 20-68 h. During the induction of diabetes and during insulin treatment there was a good correlation between the activity of synthase phosphatase and the maximal activation of synthase in glucose-stimulated hepatocytes from the same livers. The decreased activity of synthase phosphatase in diabetes cannot be explained by an inhibitor. The decrease was much less marked when synthase phosphatase was assayed by the dephosphorylation of 32P-labelled synthase from muscle. This observation suggested a loss of only one component of synthase phosphatase. Cross-combination of subcellular fractions from control rats and from diabetic rats showed a preferential loss of G-component, with little or no loss of S-component. No G-component could be detected in severe diabetes. The concentration of G-component is therefore of critical importance in the glucose-induced activation of glycogen synthase in the liver.
Membrane potential of mitochondria in intact lymphocytes during early mitogenic stimulation
Authors: Brand M D , Felber S M
Published: August 2015

The mitochondrial membrane potential (delta psi m) in intact lymphocytes was calculated by measuring the distribution of radiolabelled methyltriphenylphosphonium cation. The value obtained was 120 mV. The pH gradient across the mitochondrial membrane in situ (delta pH m) was estimated to be 73 mV (1.2 pH units). Thus the electrochemical gradient of protons was about 190 mV. Addition of the mitogen concanavalin A did not alter delta psi m, showing that, if movement of Ca2+ across the inner membrane of lymphocyte mitochondria occurs when concanavalin A is added, it is accompanied by charge-compensating ion movements.
Protein turnover and growth in the whole body, liver and kidney of the rat from the foetus to senility
Authors: Goldspink D F , Kelly F J
Published: August 2015

Changes in the growth and protein turnover (measured in vivo) of the rat liver, kidney and whole body were studied between 16 days of life in utero and 105 weeks post partum. Tissue and whole-body growth were related to changes in both cellular hyperplasia (i.e. changes in DNA) and hypertrophy (protein/DNA values) and to the protein composition within the enlarging tissue mass. The suitability of using a single large dose of phenylalanine for measuring the rates of protein synthesis during both pre- and post-natal life was established. The declining growth rates in the whole animal and the two visceral tissues were then explained by developmental changes in the fractional rates of protein synthesis and breakdown, turnover rates being age-for-age higher in the liver than in the kidney, which in turn were higher than those measured in the whole animal. The declining fractional rates of synthesis in both tissues and the whole body with increasing age were related to changes in the tissues' ribosomal capacity and activity. The fall in the hepatic rate between 18 and 20 days of foetal life (from 134 to 98% per day) corresponded to a decrease in both the ribosomal capacity and the rate of synthesis per ribosome. No significant changes in any of these parameters were, however, found in the liver between weaning (3 weeks) and senility (105 weeks). In contrast, the fractional synthetic (and degradative) rates progressively declined in the kidney (from 95 to 24% per day) and whole body (from 70 to 11% per day) throughout both pre- and post-natal life, mainly as a consequence of a progressive decline in the ribosomal capacity, but with some fall in the ribosomal activity also during foetal life. The age-related contributions of these visceral tissues to the total amount of protein synthesized per day by the whole animal were determined. The renal contribution remained fairly constant at 1.6-2.9%, whereas the hepatic contribution declined from 56 to 11%, with increasing age. Approximate-steady-state conditions were reached at, and between, 44 and 105 weeks post partum, the half-life values of mixed whole-body, kidney and liver proteins being 6.4, 3.0 and 1.5 days, respectively, at 105 weeks.
Cysteine-dependent inactivation of hepatic ornithine decarboxylase
Authors: Murakami Y , Kameji T , Hayashi S
Published: August 2015

When rat liver homogenate or its postmitochondrial supernatant was incubated with L-cysteine, but not D-cysteine, ornithine decarboxylase (ODC) lost more than half of its catalytic activity within 30 min and, at a slower rate, its immunoreactivity. The inactivation correlated with production of H2S during the incubation. These changes did not occur in liver homogenates from vitamin B6-deficient rats. A heat-stable inactivating factor was found in both dialysed cytosol and washed microsomes obtained from the postmitochondrial supernatant incubated with cysteine. The microsomal inactivating factor was solubilized into Tris/HCl buffer, pH 7.4, containing dithiothreitol. Its absorption spectrum in the visible region resembled that of Fe2+ X dithiothreitol in Tris/HCl buffer. On the other hand FeSO4 inactivated partially purified ODC in a similar manner to the present inactivating factor. During the incubation of postmitochondrial supernatant with cysteine, there was a marked increase in the contents of Fe2+ loosely bound to cytosolic and microsomal macromolecules. Furthermore, the content of such reactive iron in the inactivating factor preparations was enough to account for their inactivating activity. These data suggested that H2S produced from cysteine by some vitamin B6-dependent enzyme(s) converted cytosolic and microsomal iron into a reactive loosely bound form that inactivated ODC.
The effect of complete or specific partial acetimidylation on the biological properties of cytochrome c and cytochrome c-T
Authors: Wallace C J A
Published: August 2015

The biological consequences of acetimidylation of all 19 epsilon-amino groups of horse cytochrome c are a slight decrease in both the redox potential of the protein and its ability to stimulate oxygen uptake in the cytochrome c-depleted-mitochondria assay. Examination of a number of specific partially acetimidylated analogues and acetimidylated cytochromes c of other species has shown that the changes in biological properties, which are associated with a slight structural change as monitored by n.m.r. spectroscopy [Boswell, Moore, Williams, Harris, Wallace, Bocieck & Welti (1983) Biochem. J. 213, 679-686], appear to stem from modification of residues in a restricted region of the sequence. The failure of the redox potential of Saccharomyces cerevisae cytochrome c to be affected by acetimidylation suggests that it is lysine-53, absent from that species, that is the sensitive residue.
19F-n.m.r. studies of 3′,5′-difluoromethotrexate binding to Lactobacillus casei dihydrofolate reductase. Molecular motion and coenzyme-induced conformational changes
Authors: Clore G M , Gronenborn A M , Birdsall B , Feeney J , Roberts G C K
Published: August 2015

19F-n.m.r. spectroscopy was used to study the binding of 3′,5′-difluoromethotrexate to dihydrofolate reductase (tetrahydrofolate dehydrogenase) from Lactobacillus casei. The benzoyl ring of the bound difluoromethotrexate was found to ‘flip’ about its symmetry axis, and the rate (7.3 × 10(3) s-1 at 298 K) and activation parameters for this process were determined by lineshape analysis of the 19F-n.m.r. spectrum at a series of temperatures in the range 273-308 K. The contributions to the barrier for this process are discussed. Addition of NADP+ or NADPH to form the enzyme-difluoromethotrexate-coenzyme ternary complex led to an increase in the rate of benzoyl ring flipping by a factor of 2.6-2.8-fold, and to substantial changes in the 19F-n.m.r. chemical shifts. The possible nature of the coenzyme-induced conformational changes responsible for these effects is discussed.
An investigation of the iron-sulphur proteins of benzene dioxygenase from Pseudomonas putida by electron-spin-resonance spectroscopy
Authors: Geary P J , Saboowalla F , Patil D , Cammack R
Published: August 2015

Benzene dioxygenase from Pseudomonas putida comprises three components, namely a flavoprotein (NADH:ferredoxin oxidoreductase; Mr 81000), an intermediate electron-transfer protein, or ferredoxin (Mr 12000) with a [2Fe-2S] cluster, and a terminal dioxygenase containing two [2Fe-2S] iron-sulphur clusters (Mr 215000), which requires two additional Fe2+ atoms/molecule for oxygenase activity. The ferredoxin and the dioxygenase give e.s.r. signals in the reduced state with rhombic symmetry and average g values of 1.92 and 1.896 respectively. The mid-point redox potentials were determined by e.s.r. titration at pH 7.0 to be −155 mV and −112 mV respectively. The signal from the dioxygenase shows pronounced g anisotropy and most closely resembles those of 4-methoxybenzoate mono-oxygenase from Pseudomonas putida and the [2Fe-2S] ‘Rieske’ proteins of the quinone-cytochrome c region of electron-transport chains of respiration and photosynthesis.
Inhibition by heparin-modulated antithrombin III of amidolysis catalysed by m
β-acrosin
Authors: Long W F , Williamson F B
Published: August 2015

Purified m beta-acrosin catalysed amidolysis of several p-nitroanilides with C-terminal arginine residues. Antithrombin III inhibited amidolysis catalysed by the enzyme. This effect of antithrombin III was potentiated by heparin, and to a modest extent by heparan sulphate, cellulose sulphate, dextran sulphate and xylan sulphate. De-N-sulphated heparin, de-N-sulphated N-acetylated heparin, heparin of low relative molecular mass, chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate and hyaluronic acid were ineffective.
Changes in the DNA content of amoebae of Dictyostelium discoideum during growth and development
Authors: Sharpe P T , Knight G M , Watts D J
Published: August 2015

A fluorimetric assay has been used to determine the DNA content of amoebae of Dictyostelium discoideum during growth and development. Amoebae grown in axenic culture tended to be multinucleate and had a greater DNA content than amoebae grown with a bacterial substrate, which were mononucleate. During the first 10 h of development there was little change in the DNA content of amoebae grown with a bacterial substrate, but the average DNA content per cell in amoebae grown axenically decreased as the amoebae became virtually mononucleate. Amoebae at 10 h development that had been harvested during exponential axenic growth were divided into two populations by countercurrent distribution in a polymer two-phase system. DNA content indicated that one population was largely in the G2-phase of the cell cycle, whereas the other population was largely in the G1-phase. Similar results were obtained at 10 h development with amoebae harvested during the stationary phase of axenic growth, although these amoebae start development all in the G2-phase of the cell cycle. Spores had a low DNA content, indicating that they were in G1-phase. It is proposed that all amoebae in G2-phase after early development differentiate, after mitosis, into spores and that stalk cells are formed from amoebae that remain in G1-phase after 10 h development.
Concentration of free oxaloacetate in the mitochondrial compartment of isolated liver cells
Authors: Siess E A , Kientsch-Engel R I , Wieland O H
Published: August 2015

The concentration of metabolically active (i.e. ‘free’) oxaloacetate in the mitochondrial compartment of isolated liver cells was investigated by two independent approaches. On the basis of mitochondrial aspartate aminotransferase maintaining equilibrium and the direct measurements of mitochondrial aspartate, 2-oxoglutarate and glutamate, the concentration of free oxaloacetate was calculated to be 5 microM after incubation of hepatocytes in the presence of 1.5 mM-lactate and 0.05 mM-oleate. Gradually increasing oleate up to 0.5 mM decreased the free oxaloacetate to 2 microM. Very similar results were obtained when free oxaloacetate concentration was derived from the CO2 production of hepatocytes as a measure of citrate flux through the tricarboxylic acid cycle, and the kinetic data on citrate synthase in situ. The decrease in free oxaloacetate on increasing oleate concentration was associated with lowered rates of cycle-dependent CO2 output and O2 uptake, indicating a decrease in the disposal of acetyl-CoA into the tricarboxylic acid cycle. This decrease could explain 25-30% of the increase in ketone-body production occurring at elevated fatty acid supply. This work documents on a quantitative basis the role of free oxaloacetate in the regulation of ketogenesis.
Transport and processing of β-hexosaminidase in normal and mucolipidosis-II cultured fibroblasts. Effect of monensin and nigericin
Authors: Vladutiu G D
Published: August 2015

The univalent-cation ionophores monensin (4.0 microM) and nigericin (0.5 microM) inhibited the abnormal excretion of beta-hexosaminidase from mucolipidosis-II cultured fibroblasts by 62 and 76% respectively, with a corresponding intracellular accumulation of the enzyme. As shown by lectin binding, the enzyme which accumulated in monensin-treated cells did not contain galactose residues, whereas the corresponding enzyme from nigericin-treated cells was galactosylated. The results suggest that monensin acts at an early point in the process of hydrolase glycosylation, and nigericin acts later, both presumably within the Golgi region, allowing the accumulation of different glycosylated forms of the enzyme. The intra- and extra-cellular distribution of beta-hexosaminidase in ionophore-treated normal cells was essentially unchanged, whereas concanavalin A precipitability of excreted enzyme was increased and its ability to be taken up by deficient fibroblasts was decreased. The bivalent-cation ionophore A23187 (1 microM) reduced beta-hexosaminidase excretion from mucolipidosis-II cells by 82% and by 96% when used with EGTA (1 mM). However, there was no intracellular accumulation of enzyme, suggesting that the effect of this ionophore was restricted to the inhibition of synthesis. It therefore appears that the actual transport of beta-hexosaminidase in mucolipidosis-II cells is affected by univalent-cation ionophores in a selective manner. These findings suggest that individual ionophores could be used to identify the sites of hydrolase oligosaccharide processing in the Golgi region by causing intermediate glycosylated forms of the transported hydrolase to accumulate in a specific Golgi compartment preceding the blocking site of the ionophore.
Conformation, structure and activation of bovine cathepsin D. Unfolding and refolding studies
Authors: Lah T , Drobniĉ-Koŝorok M , Turk V , Pain R H
Published: August 2015

Cathepsin D is found in the cell in two forms, one a single polypeptide chain (Mr 44 000) and the other a non-covalent complex of two peptides of Mr 14 000 and 30 000. These correspond to the N-terminal and C-terminal regions of the single chain from which they originate. It has been shown that the two forms of the enzyme are closely similar in secondary-structure content, in aromatic amino acid environment and in denaturation behaviour. The two-chain enzyme has half the specific activity of the single-chain form. The denaturation and renaturation of the single-chain cathepsin D has now been studied by c.d., fluorescence and enzyme activity. Activity is lost irreversibly on unfolding, but the loss of backbone ellipticity and of folded aromatic environment is 75% reversible. The enzyme unfolds in two main stages, and the kinetics of these transitions indicate the existence of at least two intermediate forms between the native and the fully unfolded states. A further form of the enzyme exists in 0.5 M-guanidinium chloride. It is characterized by having an activity 40% greater than that of the native state. This increase is not reversed on removing the denaturant. The similarities between cathepsin D and pepsin are discussed.
Specificity studies on the oligosaccharide neuraminidase of human fibroblasts
Authors: Mendla K , Cantz M
Published: August 2015

Competition and thermal inactivation experiments with different potential natural substrates indicated that in homogenates of human fibroblasts one single enzyme is acting on both (alpha 2-3) and (alpha 2-6) sialosyl linkages of oligosaccharides and glycoproteins, but not of the ganglioside GM3. N-Acetylneuraminic and 2-deoxy-2,3-dehydro-N-acetylneuraminic acids are competitive inhibitors, whereas chondroitin 4-sulphate and the drug Suramin are potent inhibitors of undefined type.