bluuebunny/crossref_metadata_2025 huggingface dataset:
A general framework of cysteine-proteinase mechanism deduced from studies on enzymes with structurally different analogous catalytic-site residues Asp-158 and −161 (papain and actinidin), Gly-196 (cathepsin B) and Asn-165 (cathepsin H). Kinetic studies up to pH 8 of the hydrolysis of N-α-benzyloxycarbonyl-l-arginyl-l-arginine 2-naphthylamide catalysed by cathepsin B and of l-arginine 2-naphthylamide catalysed by cathepsin H.
Authors: Willenbrock F , Brocklehurst K
Published: August 2015

The pH-dependences of kcat, Km and kcat./Km for the hydrolysis at 25 degrees C at I 0.1 of L-arginine 2-naphthylamide catalysed by cathepsin H from bovine spleen were determined in the pH range approx. 4-8. The pH-dependences of these kinetic parameters were determined also for the hydrolysis at 25 degrees C at I 0.1 of N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide catalysed by cathepsin B (EC 3.4.22.1) from bovine spleen in the pH range 7-8, which extends the studies in acidic media reported by Willenbrock & Brocklehurst [(1984) Biochem. J. 222, 805-814]. These results are discussed and related to those from the reactivity-probe kinetics reported in the preceding paper [Willenbrock & Brocklehurst (1985) Biochem. J. 227, 511-519] and to known structural features present in rat liver cathepsins B and H and in papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14). Consideration of the kinetic data leads to the suggestion that in the cysteine proteinases rearrangement of intimate S-/ImH+ ion-pairs in catalytic sites is brought about by a combination of field effects in the immediate vicinity of the ion-pair and consequences of protonic dissociation of a group with pKa 5-6 remote from the catalytic site. The contributions of the two types of effect seem to differ from enzyme to enzyme. Of the four cysteine proteinases considered, only cathepsin B exerts an absolute requirement for the proton-deficient form of a group with pKa 5-6 for catalytic activity. Protonic dissociation with pKa 5-6 enhances catalytic activity in cathepsin H and in actinidin and appears to have little or no effect in papain. Only cathepsin B lacks a polar or negatively charged side chain in the residue analogous to Asp-158 in papain, and this is suggested to account for its total dependence on a protonic dissociation remote from the catalytic site.
Characterization of plasma-membrane glycoproteins from functional domains of the rat hepatocyte
Authors: Enrich C , Gahmberg C G
Published: August 2015

Plasma-membrane glycoproteins from the three different functional domains of the rat hepatocyte were radioactively labelled by oxidation with NaIO4, followed by reduction with NaB3H4. Analysis of the radioactively labelled glycoproteins by polyacrylamide-gel electrophoresis revealed the presence of at least 12 major sialoglycoproteins in each different region of the hepatocyte surface. The Mr-110 000 component was homogeneously distributed over the plasma membrane, whereas the Mr-90 000 polypeptide was only located at the sinusoidal face. These radiolabelled glycoproteins were solubilized in 1% Triton X-100, and the soluble fraction was subjected to affinity chromatography on Sepharose-conjugated wheat-germ agglutinin (WGA). The labelled glycoproteins were poorly bound to WGA. Membrane glycoproteins were also labelled by the galactose oxidase/NaB3H4 method. The results show that the polypeptides with apparent Mr 170 000 from the sinusoidal, 230 000 from the canalicular and 170 000 from the lateral membranes were specifically labelled. When the membranes were treated with neuraminidase and galactose oxidase/NaB3H4, the electrophoretic patterns showed changes in the apparent Mr values of the glycoproteins, owing to loss of sialic acid, and a clear increase in labelling in the sinusoidal and canalicular membranes compared with the lateral membranes. When these labelled membranes were solubilized in 1% Triton X-100 and subjected to affinity chromatography on Sepharose-conjugated Ricinus communis agglutinin and/or Lens culinaris agglutinin, the results showed that the former columns efficiently bound the radiolabelled glycoproteins, whereas the latter columns bound poorly. The results show that there is a differential distribution of glycoproteins along the hepatocyte's surface.
3-Hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver. Properties of its acetyl derivative
Authors: Lowe D M , Tubbs P K
Published: August 2015

Ox liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) reacts with acetyl-CoA to form a complex in which the acetyl group is covalently bound to the enzyme. This acetyl group can be removed by addition of acetoacetyl-CoA or CoA. The extent of acetylation and release of CoA were found to be highly temperature-dependent. At temperatures above 20 degrees C, a maximum value of 0.85 mol of acetyl group bound/mol of enzyme dimer was observed. Below this temperature the extent of rapid acetylation was significantly lowered. Binding stoichiometries close to 1 mol/mol of enzyme dimer were also observed when the 3-hydroxy-3-methylglutaryl-CoA synthase activity was titrated with methyl methanethiosulphonate or bromoacetyl-CoA. This is taken as evidence for a ‘half-of-the-sites’ reaction mechanism for the formation of 3-hydroxy-3-methylglutaryl-CoA by 3-hydroxy-3-methylglutaryl-CoA synthase. The Keq. for the acetylation was about 10. Isolated acetyl-enzyme is stable for many hours at 0 degrees C and pH 7, but is hydrolysed at 30 degrees C with a half-life of 7 min. This hydrolysis is stimulated by acetyl-CoA and slightly by succinyl-CoA, but not by desulpho-CoA. The site of acetylation has been identified as the thiol group of a reactive cysteine residue by affinity-labelling with the substrate analogue bromo[1-14C]acetyl-CoA.
Kinetic studies with the low-Km aldehyde reductase from ox brain
Authors: Ryle C M , Tipton K F
Published: August 2015

Initial-rate studies of the low-Km aldehyde reductase-catalysed reduction of pyridine-3-aldehyde by NADPH gave families of parallel double-reciprocal plots, consistent with a double-displacement mechanism being obeyed. Studies on the variation of the initial velocity with the concentration of a mixture of the two substrates were also consistent with a double-displacement mechanism. In contrast, the initial-rate data indicated that a sequential mechanism was followed when NADH was used as the coenzyme. Product-inhibition studies, however, indicated that a compulsory-order mechanism was followed in which NADPH bound before pyridine-3-aldehyde with a ternary complex being formed and the release of pyrid-3-ylcarbinol before NADP+. The apparently parallel double-reciprocal plots obtained in the initial-rate studies with NADPH and pyridine-3-aldehyde were thus attributed to the apparent dissociation constant for the binary complex between the enzyme and coenzyme being finite but very low.
Two mechanisms produce tissue-specific inhibition of fatty acid oxidation by oxfenicine
Authors: Stephens T W , Higgins A J , Cook G A , Harris R A
Published: August 2015

Oxfenicine [S-2-(4-hydroxyphenyl)glycine] is transaminated in heart and liver to 4-hydroxyphenylglyoxylate, an inhibitor of fatty acid oxidation shown in this study to act at the level of carnitine palmitoyltransferase I (EC 2.3.1.21). Oxfenicine was an effective inhibitor of fatty acid oxidation in heart, but not in liver. Tissue specificity of oxfenicine inhibition of fatty acid oxidation was due to greater oxfenicine transaminase activity in heart and to greater sensitivity of heart carnitine palmitoyltransferase I to inhibition by 4-hydroxyphenylglyoxylate [I50 (concentration giving 50% inhibition) of 11 and 510 microM for the enzymes of heart and liver mitochondria, respectively]. Branched-chain-amino-acid aminotransferase (isoenzyme I, EC 2.6.1.42) was responsible for the transamination of oxfenicine in heart. A positive correlation was found between the capacity of various tissues to transaminate oxfenicine and the known content of branched-chain-amino-acid aminotransferase in these tissues. Out of three observed liver oxfenicine aminotransferase activities, one may correspond to asparagine aminotransferase, but the major activity could not be identified by partial purification and characterization. As reported previously for malonyl-CoA inhibition of carnitine palmitoyltransferase I, 4-hydroxyphenylglyoxylate inhibition of this enzyme was found to be very pH-dependent. In striking contrast with the kinetics of malonyl-CoA inhibition, 4-hydroxyphenylglyoxylate inhibition was not affected by oleoyl-CoA concentration, but was partially reversed by increasing carnitine concentrations.
Effect of dexamethasone on the synthesis of dolichol-linked saccharides and glycoproteins in hepatocytes prepared from control and inflamed rats
Authors: Sarkar M , Mookerjea S
Published: August 2015

Hepatocytes were prepared from control and inflamed rats. The incorporation of [14C]mannose into protein was increased in inflamed compared with control hepatocytes. The incorporation of [14C]mannose into protein was also increased when the hepatocytes were cultured in presence of dexamethasone (1 microM), either from control or inflamed rats. At the same time the incorporation of [14C]mannose into dolichol phosphate mannose and dolichol-linked oligosaccharide was increased due to inflammation. The presence of dexamethasone in the hepatocyte culture caused an increased formation of these two products; in particular its effect on oligosaccharide lipid formation was very pronounced. The ratios of activities of formation of [14C]mannose-labelled oligosaccharide lipid in inflamed over control hepatocytes gradually decrease when increasing amounts of exogenous dolichol phosphate was added in cell homogenate assay mixture. These results suggest that the increase of oligosaccharide lipid formation in inflammation could be due to a higher concentration of endogenous dolichol phosphate, as was shown for dolichol phosphate mannose formation in inflammation [Sarkar & Mookerjea (1984) Biochem. J. 219, 429-436]. In contrast, the ratio of activities of [14C]mannose-labelled oligosaccharide lipid between dexamethasone-treated and untreated hepatocytes shows only a slight increase when increasing concentrations of exogenous dolichol phosphate were added to the assays. This suggests that the stimulation of dolichol pyrophosphate oligosaccharide synthesis observed in dexamethasone treatment is probably due to the higher level of enzymes involved in oligosaccharide synthesis rather than higher level of endogenous dolichol phosphate in these cells.
Induction of selective phosphorylation of a fat body protein of Sarcophaga peregrina larvae by 20-hydroxyecdysone
Authors: Itoh K , Ueno K , Natori S
Published: August 2015

20-Hydroxyecdysone was shown to induce selective phosphorylation of a fat body protein of Sarcophaga peregrina larvae with a molecular mass of 30 kDa. This phosphorylation was not associated with synthesis of new protein. Fractionation of 32P-labelled fat body by differential centrifugation showed that this protein was mainly present in the membrane-rich fraction, although we could not specify the membrane. Thus, 20-hydroxyecdysone may modify the function of the fat body by inducing phosphorylation of a specific membrane protein.
Synthesis of retinyl phosphate mannose in vitro. Non-enzymic breakdown and reversibility
Authors: Creek K E , Rimoldi D , Silverman-Jones C S , De Luca L M
Published: August 2015

Hamster liver microsomal membranes catalyse the synthesis of retinyl phosphate mannose (Ret-P-Man) from GDP-mannose and exogenous retinyl phosphate (Ret-P). We have previously shown that maximal Ret-P-Man synthesis occurs in vitro at 20-30 min, followed by a subsequent loss of mannose from Ret-P-Man, suggestive of an intermediary function of Ret-P-Man and/or Ret-P-Man breakdown [Shidoji, Silverman-Jones & De Luca (1982) Biochem. J. 208, 865-868; Creek, Morre, Silverman-Jones, Shidoji & De Luca (1983) Biochem. J. 210, 541-547). To monitor Ret-P-Man synthesis and breakdown carefully, we developed a chromatographic system in which mannose, Ret-P-Man, mannose phosphate and GDP-mannose are separated in a single analysis on a Mono Q column eluted with a gradient of NaCl. Using this chromatographic system, we have determined that 80-90% of the Ret-P-Man made in vitro by hamster liver membranes in 30 min is recovered with the membranes upon centrifugation. Subsequent incubation of Ret-P-Man-loaded membranes at 37 degrees C results in a non-enzymic breakdown of Ret-P-Man to beta-mannopyranosyl phosphate and anhydroretinol. However, incubation of the Ret-P-Man-loaded hamster liver membranes with GDP, but not GMP, ADP, CDP or UDP, results in a loss of mannose from Ret-P-Man and the formation of GDP-mannose and Ret-P. These results demonstrate that Ret-P-Man synthesized in vitro is subject to non-enzymic breakdown to beta-mannopyranosyl phosphate and anhydroretinol and that the GDP-mannose:retinyl phosphate mannosyltransferase reaction is reversible.
Distribution, metabolism and excretion of [1-14C]dolichol injected intravenously into rats
Authors: Rip J W , Carroll K K
Published: August 2015

[1-14C]Dolichol mixed in vitro with rat serum and injected intravenously into rats was rapidly cleared from the circulation in a manner consistent with a two-compartment model. About 80% of the radioactivity recovered from animals killed after 1 day was in the liver, with smaller amounts being found in lung, carcass (internal organs removed), gastrointestinal tract and contents, and spleen. The kidneys, testes and heart contained little radioactivity, and the brain did not appear to take up any [1-14C]dolichol. The half-life for the turnover of radioactivity from [1-14C]dolichol in tissues varied considerably, being 2 days for the lung, 17 for liver and about 50 days for the carcass. After 1 day, and also after 4 and 21 days, most of the radioactivity in all tissues was as [1-14C]dolichol and as [1-14C]dolichyl fatty acyl ester, although a small amount of incorporation of [1-14C]dolichol radioactivity into phospholipids was also observed. Faeces collected over the first 4 days after injection contained 13% of the [1-14C]dolichol dose, but urine and expired air contained only small amounts of radioactivity. Radioactivity in faeces was nearly all as unchanged [1-14C]dolichol and as [1-14C]dolichyl fatty acyl ester. The [1-14C]dolichol remaining in liver after 21 days appeared to be in a pool (possibly lysosomes) where most of it was not subject to excretion.
Metal-ion binding to parvalbumin. A 113Cd-n.m.r. study of the binding of different lanthanide ions
Authors: Drakenberg T , Swärd M , Cavé A , Parello J
Published: August 2015

113Cd-n.m.r. studies were used to investigate the binding of the lanthanide ions La3+, Gd3+, Tb3+, Yb3+ and Lu3+ to parvalbumins. It was shown that lanthanide ions with a smaller ionic radius bind sequentially to Cd2+-saturated parvalbumin, whereas those with a larger ionic radius bind with similar affinity to both the CD site and the EF site. The smallest ion, Lu3+, does in fact not compete significantly with Cd2+ for the CD site in carp parvalbumin, but appears to bind only to the EF site. This preference of the smaller lanthanide ions for the EF site was used to assign the n.m.r. signals for protein-bound 113Cd. By using Cd n.m.r. and Tb3+ fluorescence it was also shown for alpha-lineage parvalbumin from pike that these proteins possess a third site that can bind lanthanide ions. This site is, however, much weaker than in the beta-lineage parvalbumins. It was used to assign the 113Cd resonances from protein-bound Cd2+ ions in the spectrum of pike pI5.0 parvalbumin.
Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Location and regulation of expression
Authors: Allison N , O'Donnell M J , Hoey M E , Fewson C A
Published: August 2015

Acinetobacter calcoaceticus possesses an L(+)-lactate dehydrogenase and a D(-)-lactate dehydrogenase. Results of experiments in which enzyme activities were measured after growth of bacteria in different media indicated that the two enzymes were co-ordinately induced by either enantiomer of lactate but not by pyruvate, and repressed by succinate or L-glutamate. The two lactate dehydrogenases have very similar properties to L(+)-mandelate dehydrogenase and D(-)-mandelate dehydrogenase. All four enzymes are NAD(P)-independent and were found to be integral components of the cytoplasmic membrane. The enzymes could be solubilized in active form by detergents; Triton X-100 or Lubrol PX were particularly effective D(-)-Lactate dehydrogenase and D(-)-mandelate dehydrogenase could be selectively solubilized by the ionic detergents cholate, deoxycholate and sodium dodecyl sulphate.
The glutathione S-transferases in selenium and vitamin E deficiency
Authors: Mehlert A , Diplock A T
Published: August 2015

Preliminary experiments confirmed the work of others showing that the total glutathione peroxidase (GSH-px) activity of rat liver supernatant fraction may be resolved into two peaks of activity (peaks I and II) by gel filtration, and that peak I is the selenium-containing enzyme and peak II is another peroxidase indistinguishable from glutathione S-transferase (GST). In selenium and vitamin E deficiency, the total activity of the GSH-px became very low, and the total activity of GST with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate was enhanced. Study of the time course of these changes as deficiency progressed indicated that the stimulus for the rise in GST (CDNB) activity was the fall in GSH-px activity which preceded it. The peroxidase activity of GST was found to reside only in the GST AA, B and B2 forms of the enzyme, which were shown to be respectively a homodimer of the Yc subunit, a homodimer of the Ya subunit and a heterodimer of the YaYc subunit. As vitamin E and selenium deficiency progressed, the B2 and AA forms of the enzyme showed enhanced activity, which was interpreted as implying that the Yc subunit of the enzyme becomes enriched as a consequence of the withdrawal of selenium from the animal's diet. Densitometric measurements of the Yc and Ya subunits confirmed that the amount of the Yc subunit was nearly doubled in selenium deficiency, relative to the Ya subunit.
Assimilation of α-glutamyl-peptides by human erythrocytes. A possible means of glutamate supply for glutathione synthesis
Authors: King G F , Kuchel P W
Published: August 2015

Human erythrocytes are essentially impermeable to glutamate and yet there is a continual requirement for the amino acid for glutathione synthesis. In addition, the intracellular glutamate concentration is approximately five times that of plasma. We present evidence that glutamate enters the red cell as small peptides which are rapidly hydrolysed by cytoplasmic peptidase(s) and that with the estimated physiological levels of plasma glutamyl-peptides the rate of inward flux would be adequate to maintain the glutamate pool at its observed level. Experimentally, we used 1H spin-echo n.m.r. spectroscopy to follow peptide hydrolysis, since peptide spectra are different from those of the free amino acids and the spin-echo sequence enables the monitoring of reactions in concentrated lysates and whole cell suspensions. Thus, the system was studied under near-physiological conditions. Weighted non-linear regression analysis of progress curves using the integrated Michaelis-Menten equation was used to obtain estimates of Km and Vmax. for the hydrolysis of alpha-L-glutamyl-L-alanine and L-alanyl-alpha-L-glutamate in lysates and whole cell suspensions; the values for lysates were Km = 3.60 +/- 0.29 and 5.4 +/- 0.4 mmol/l and Vmax. = 120 +/- 4 and 46.7 +/- 1.7 mmol/h per 1 of packed cells respectively. In whole cell suspensions the rate of peptide hydrolysis was much slower and dominated by the transmembrane flux-rate. The estimates of the steady-state kinetic parameters for the transport were Kt = 2.35 +/- 0.41 and 11.2 +/- 1.0 mmol/l and Vmax. = 3.26 +/- 0.13 and 19.7 +/- 0.7 mmol/h per 1 of packed cells respectively for the previously mentioned peptides. Using the n.m.r. procedure we failed to detect any glutaminase activity in whole cells or lysates; thus, we exclude the possibility that glutamate gains entry to the cell as glutamine which is subsequently hydrolysed by glutaminase.
Gas chromatographic-mass spectrometric study of metabolites of C21 and C19 steroids in neonatal porcine testicular microsomes
Authors: Kwan T K , Taylor N F , Watson D , Gower D B
Published: August 2015

Microsomal fractions obtained from testes of 3-week-old piglets have been incubated, separately, with progesterone, 17-hydroxyprogesterone, 5-pregnene-3 beta,20 beta-diol, 16 alpha-hydroxypregnenolone, 5-androstene-3 beta,17 alpha-diol and dehydro-epiandrosterone. The metabolites, after derivatization, have been separated by capillary gas chromatography and identified by mass spectrometry. Quantification was by selected ion monitoring. Progesterone was shown to be 17-hydroxylated and also converted into 4,16-androstadien-3-one (androstadienone). The major metabolite of 17-hydroxyprogesterone was 4-androstene-3,17-dione (4-androstenedione), but little, if any, androstadienone was formed, indicating that this particular biosynthesis did not require 17-hydroxylation. The metabolites of 5-pregnene-3 beta, 20 beta-diol were found to be 17-hydroxypregnenolone, 3 beta-hydroxy-5,16-pregnadien-20-one (16-dehydropregnenolone) and 5,16-androstadien-3 beta-ol. Dehydroepiandrosterone and 5-androstene-3 beta,17 alpha-diol were interconvertible but neither steroid acted as a substrate for 16-androstene formation. However, dehydroepiandrosterone was metabolized to a small quantity of 4-androstenedione. Under the conditions used, no metabolites of 16 alpha-hydroxypregnenolone could be detected. The present results, together with those obtained earlier, indicate that the neonatal porcine testis has the capacity to synthesize weak androgens, mainly by the 4-en-3-oxo steroid pathway. Although 16-androstenes cannot be formed from C19 steroids, progesterone served as a substrate and may be converted directly to androstadienone, without being 17-hydroxylated first. The pathway to 5,16-androstadien-3 beta-ol, however, involves 17-hydroxypregnenolone and 16-dehydropregnenolone as intermediates.
The structure of Artemia sp. (brine shrimp) haemoglobins. Purification of a structural unit to homogeneity
Authors: Moens L , Van Hauwaert M L , Wolf G
Published: August 2015

The extracellular haemoglobins (Mr 260 000) of the brine shrimp Artemia sp. were cleaved by limited digestion with subtilisin. Structural units of Mr 16 000, which can bind dioxygen reversibly, were isolated. Analysis of the 16 000-Mr fraction (E) reveals the presence of a limited number of structural units. A single type of structural unit, E1 (Mr 15 800; pI4.8), was purified to homogeneity and characterized.
Pertussis toxin does not inhibit muscarinic-receptor-mediated phosphoinositide hydrolysis or calcium mobilization
Authors: Masters S B , Martin M W , Harden T K , Brown J H
Published: August 2015

Pertussis toxin was used to examine the role of the inhibitory guanine nucleotide regulatory protein, Ni, in muscarinic-receptor-mediated stimulation of phosphoinositide turnover and calcium mobilization. In cultured chick heart cells, pertussis-toxin treatment inhibited muscarinic-receptor-mediated attenuation of isoprenaline-stimulated cyclic AMP accumulation. This finding is consistent with the proposal that pertussis toxin blocks the capacity of Ni to couple muscarinic receptors to adenylate cyclase. In contrast, treatment of chick heart cells or 1321N1 human astrocytoma cells with pertussis toxin did not block muscarinic-receptor-mediated stimulation of phosphoinositide hydrolysis, as measured by [3H]inositol phosphate accumulation in the presence of Li+. Pertussis-toxin treatment also had little effect on basal and muscarinic-receptor-stimulated phosphatidylinositol synthesis, as measured by the incorporation of [3H]inositol into phosphatidylinositol. Activation of muscarinic receptors also enhances the rate of unidirectional 45Ca2+ efflux in 1321N1 cells; this response, like phosphoinositide hydrolysis, was not prevented by pertussis-toxin treatment. Our data suggest that muscarinic receptors are not coupled to phosphoinositide hydrolysis or calcium mobilization through Ni.
Participation of endogenous fatty acids in the secretory activity of the pancreatic B-cell
Authors: Malaisse W J , Malaisse-Lagae F , Sener A , Hellerström C
Published: August 2015

The pancreatic B-cell may represent a fuel-sensor organ, the release of insulin evoked by nutrient secretagogues being attributable to an increased oxidation of exogenous and/or endogenous substrates. The participation of endogenous fatty acids in the secretory response of isolated rat pancreatic islets was investigated. Methyl palmoxirate (McN-3716, 0.1 mM), an inhibitor of long-chain-fatty-acid oxidation, suppressed the oxidation of exogenous [U-14C]palmitate and inhibited 14CO2 output from islets prelabelled with [U-14C]palmitate. Methyl palmoxirate failed to affect the oxidation of exogenous D-[U-14C]glucose or L-[U-14C]glutamine, the production of NH4+ and the output of 14CO2 from islets prelabelled with L-[U-14C]glutamine. In the absence of exogenous nutrient and after a lag period of about 60 min, methyl palmoxirate decreased O2 uptake to 69% of the control value. Methyl palmoxirate inhibited insulin release evoked by D-glucose, D-glyceraldehyde, 2-oxoisohexanoate, L-leucine, 2-aminobicyclo[2.2.1]heptane-2-carboxylate or 3-phenylpyruvate. However, methyl palmoxirate failed to affect insulin release when the oxidation of endogenous fatty acids was already suppressed, e.g. in the presence of pyruvate or L-glutamine. These findings support the view that insulin release evoked by nutrient secretagogues tightly depends on the overall rate of nutrient oxidation, including that of endogenous fatty acids.
The amino acid sequence of the cytochrome c-554(547) from the chemolithotrophic bacterium Thiobacillus neapolitanus
Authors: Ambler R P , Meyer T E , Trudinger P A , Kamen M D
Published: August 2015

An amino acid sequence is proposed for the cytochrome c-554(547) from the bacterium Thiobacillus neapolitanus N.C.I.B. 8539). It consists of a polypeptide chain of 91 residues, with a pair of haem-attachment cysteine residues at positions 15 and 18. There is similarity in sequence with each of the halves of the sequence of the dihaem cytochromes c4 and with a cytochrome c-554(548) from a halophilic strain of Paracoccus. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50127 (11 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1985) 225, 5.
Δ5-desaturase activity in liver and brain microsomes during development of the pig
Authors: Clandinin M T , Wong K , Hacker R R
Published: August 2015

Microsomes isolated from liver and brain tissue were assayed to examine transitions in metabolic capability to synthesize tetraenes and pentaenes by chain elongation-desaturation of C20:3(8,11,14) during the perinatal development of the pig. Rates of synthesis of tetraenes and pentaenes by chain elongation-desaturation of C20:3(8,11,14) were greatest in liver. During the latter half of gestation, the capability to synthesize tetraenes increased 7- or 23-fold on a per mg of microsomal protein basis for brain and liver respectively. Increase in the capacity to synthesize tetraenes from C20:3(8,11,14) suggests a significant transition in the activity of the delta 5-desaturase during the last half of gestation. These observations indicate that in liver and brain the capability to chain elongate-desaturate C18:2(9,12) to longer chain homologues increases significantly during early development as a function of transitions in the activity of the delta 5-desaturase.
GTP-binding proteins may stimulate insulin biosynthesis in rat pancreatic islets by enhancing the signal-recognition-particle-dependent translocation of the insulin mRNA poly-/mono-some complex to the endoplasmic reticulum
Authors: Welsh N , Oberg C , Welsh M
Published: August 2015

We aimed to elucidate the putative role of GTP-binding proteins in the regulation of insulin biosynthesis. For this purpose, freshly isolated rat islets were incubated in the presence of liposomes containing GDP, guanosine 5′-[beta-thio]diphosphate (GDP[S]), GTP, guanosine 5′-[gamma-thio]triphosphate (GTP[S]), guanosine 5′-[beta gamma-methylene]triphosphate (p[CH2]ppG), guanosine 5′[beta gamma-imido]triphosphate (p[NH]ppG) and ATP, and the effects of the liposomal delivery of these substances on rates of biosynthesis of insulin and total protein were determined. Insulin biosynthesis during a 1 h incubation at 1.67 mM-glucose was stimulated by ATP- and GTP[S]-containing liposomes as compared with control liposomes. At 16.7 mM-glucose, only the GTP[S]-containing liposomes stimulated insulin biosynthesis. No inhibition of islet protein and insulin synthesis was observed with GDP-, GDP[S]-, p[CH2]ppG- and p[NH]ppG-containing liposomes. By determining the subcellular distribution of insulin mRNA, it was found that the mRNA content associated with microsomes was increased and that associated with the cytosolic mono-/poly-somes decreased when the islets were incubated with GTP[S]-containing liposomes, resulting in an approximate doubling of the ratio of microsomal to polysomal-associated insulin mRNA. ATP-containing liposomes produced no effects on the association of insulin mRNA with microsomes. By using photoaffinity labelling and immunoprecipitation techniques, specific binding of GTP[35S] to the alpha-subunit of the signal-recognition particle (SRP) receptor in islet homogenates containing physiological concentrations of GTP and GDP was demonstrated. These findings suggest that the GTP-binding subunit(s) of the SRP receptor, and possibly also of other GTP-binding proteins involved in this process, may regulate insulin biosynthesis by stimulating the translocation of insulin mRNA to the endoplasmic reticulum and by increasing preproinsulin-peptide translocation into the lumen of the reticulum.
Phorbol ester only partially mimics the effects of insulin on glucose transport and glucose-transporter distribution in 3T3-L1 adipocytes
Authors: Gibbs E M , Calderhead D M , Holman G D , Gould G W
Published: August 2015

We examined the effects of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on the rate of hexose transport into 3T3-L1 adipocytes. Exposure of adipocytes to PMA (1 microM) for 60 min results in a 1.7-2.5-fold increase in the rate of hexose transport. This effect was mediated by translocation of two isoforms of glucose transporters to the plasma membrane, as determined by labelling in situ, photoaffinity labelling with a membrane-impermeant glucose analogue, and by immunoblotting of subcellular fractions. The PMA-induced stimulation of both transport and transporter translocation was substantially less than that induced by insulin in this cell line; the PMA-induced increase in plasma-membrane GLUT 1 and GLUT 4 transporter isoforms was only about 40% and 10% respectively of that induced by insulin. We suggest that the stimulation of transport by insulin and PMA occurs via different mechanisms, which is manifested by the ability of insulin to induce a much greater increase in the plasma-membrane content of GLUT 4 compared with the phorbol ester.
The contribution of the C-terminal sequence to the catalytic activity of GST2, a human Alpha-class glutathione transferase
Authors: Board P G , Mannervik B
Published: August 2015

A plasmid vector was constructed that encodes the expression in Escherichia coli of a truncated form of GST2, a human Alpha-class glutathione transferase. The truncated enzyme, GST2del210, has 12 residues deleted from the C-terminus and has the last two residues of the new C-terminal mutated from aspartic acid and glutamic acid to histidine and glycine respectively. GST2del210 has substantially diminished specific activity with either 1-chloro-2,4-dinitrobenzene or cumene hydroperoxide as substrate. The affinity of the truncated enzyme for a GSH-agarose matrix was also diminished, but sufficient interaction remained to enable affinity purification. Inhibition of GST2del210 by bromosulphophthalein was not altered. In contrast, this truncated form was not inhibited by S-pentylglutathione, a competitive inhibitor of the wild-type GST2 isoenzyme. The results show that the C-terminal segment of the Alpha-class glutathione transferases may form a component of the hydrophobic substrate-binding site. In contrast, this region appears not to be directly involved in GSH binding and is not absolutely essential for catalytic activity.
Uptake and degradation of several pyrenesphingomyelins by skin fibroblasts from control subjects and patients with Niemann-Pick disease. Effect of the structure of the fluorescent fatty acyl residue
Authors: Levade T , Gatt S , Salvayre R
Published: August 2015

Three fluorescent analogues of sphingomyelin (SPM), each containing pyrene in the fatty acyl residue, were synthesized and employed for the study of their mode of uptake by, and degradation within, intact cultured human skin fibroblasts. These were prepared by condensing sphingosylphosphocholine and the following fatty acids: pyrenedodecanoic acid (P12), pyrenesulphonylaminoundecanoic acid (PSA11) and pyrenepropenoic acid (P3:1). The cell association and catabolism of these SPM analogues by normal, Niemann-Pick-disease-Type-A and low-density-lipoprotein (LDL)-receptor-negative familial hypercholesterolaemia fibroblasts were investigated and compared with the metabolism of [cholinemethyl-14C]sphingomyelin. The catabolism of the fluorescent derivatives was monitored by measuring the appearance of the corresponding fluorescent ceramides. Two modes of uptake and degradation patterns were observed. Thus P12-SPM and radiolabelled SPM were taken up by LDL-receptor-mediated endocytosis when incubated with serum-containing medium, this conclusion being supported by the very low uptake by familial-hypercholesterolaemia fibroblasts, which lack the apolipoprotein-B/E receptor. After uptake, these compounds were metabolically degraded solely by the lysosomal sphingomyelinase, as evidenced by the fact that more than 98% of the SPM remained undegraded in Niemann-Pick-disease cells. By contrast, PSA11- and P3:1-SPMs were taken up by a receptor-independent endocytic pathway, as indicated by the similar rates of uptake in control and familial-hypercholesterolaemia cells in the absence or presence of fetal-calf serum in the culture medium. The degradation of PSA11-SPM and P3:1-SPM was brought about, in the main, by the lysosomal sphingomyelinase, but also by a yet uncharacterized process. The latter catabolic pathway, active in Niemann-Pick-disease-Type-A fibroblasts, seems to differ from the neutral Mg2(+)-dependent sphingomyelinase whose activity was undetectable in homogenates of skin fibroblasts. The present study emphasizes the influence of the structure of the fatty acyl moiety of SPM on its association with lipoproteins and/or cell membranes and on its intracellular routing and metabolic degradation.
Expression of human recombinant lipocortin I in a wheat-germ cell-free system and Xenopus oocytes. Lipocortin is not secreted
Authors: Frey B M , Frey F J , Lingappa V R , Trachsel H
Published: August 2015

Lipocortin I has been presumed to be synthesized and secreted in response to glucocorticoids yet the amino acid sequence of lipocortin I reveals no signal sequence typically necessary for proteins to enter the secretory pathway. The translocation of lipocortin I across membranes was analyzed in a cell-free system and in Xenopus oocytes. Based on the published sequence, the cDNA of human lipocortin I was cloned and expressed in Escherichia coli. Lipocortin I was purified and used to raise monoclonal antibodies. To test whether lipocortin I is secreted in vitro, transcribed lipocortin mRNA was translated in a wheat germ cell-free system in the absence and presence of microsomal membranes. Prolactin mRNA was used as a control for translocation of newly synthesized protein into membrane vesicles. Prolactin, but not lipocortin I, was translocated into the membranes. To test for secretion of lipocortin I in vivo, Xenopus oocytes were co-injected with transcripts encoding lipocortin I and prolactin, with and without the signal sequence. Prolactin with the signal sequence was released into the medium. However, neither prolactin without a signal sequence nor lipocortin I was released. Carbonate extraction, using an integral transmembrane protein as control, revealed no evidence for membrane integration of lipocortin I. Thus lipocortin I is not a secreted protein.
Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phosphatase activity of protein phosphatases
Authors: Takai A , Mieskes G
Published: August 2015

The phosphatase activities of type 2A, type 1 and type 2C protein phosphatase preparations were measured against p-nitrophenyl phosphate (pNPP), a commonly used substrate for alkaline phosphatases. Of the three types of phosphatase examined, the type 2A phosphatase exhibited an especially high pNPP phosphatase activity (119 +/- 8 mumol/min per mg of protein; n = 4). This activity was strongly inhibited by pico- to nano-molar concentrations of okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases that has been shown to have no effect on alkaline phosphatases. The dose-inhibition relationship was markedly shifted to the right and became steeper by increasing the concentration of the enzyme, as predicted by the kinetic theory for tightly binding inhibitors. The enzyme concentration estimated by titration with okadaic acid agreed well with that calculated from the protein content and the molecular mass for type 2A phosphatase. These results strongly support the idea that the pNPP phosphatase activity is intrinsic to type 2A protein phosphatase and is not due to contamination by alkaline phosphatases. pNPP was also dephosphorylated, but at much lower rates, by type 1 phosphatase (6.4 +/- 8 nmol/min per mg of protein; n = 4) and type 2C phosphatase (1.2 +/- 3 nmol/min per mg of protein; n = 4). The pNPP phosphatase activity of the type 1 phosphatase preparation shows a susceptibility to okadaic acid similar to that of its protein phosphatase activity, whereas it was interestingly very resistant to inhibitor 2, an endogenous inhibitory factor of type 1 protein phosphatase. The pNPP phosphatase activity of type 2C phosphatase preparation was not affected by up to 10 microM-okadaic acid.
Models of human platelet thrombospondin in solution. A dynamic light-scattering study
Authors: Vuillard L , Clezardin P , Miller A
Published: August 2015

The translational diffusion coefficient (D20,w) of human platelet thrombospondin was measured by dynamic light-scattering. D20,w, measured in 20 mM-Hepes buffer, pH 7.4, containing 350 mM-NaCl and 2 mM-CaCl2, was 1.73(+/- 0.02) x 10(-7) cm2.s-1. After removal of bound Ca2+ by addition of EDTA, D20,w decreased to 1.56(+/- 0.04) x 10(-7) cm2.s-1; this was not a consequence of aggregation. D20,w showed little sensitivity to NaCl concentration between 130 and 550 mM. Through hydrodynamic analysis combining D20,w and other parameters taken from the literature, two major types of models for thrombospondin can be proposed: either classic compact models (i.e. low degree of hydration) such as prolate or oblate ellipsoids with a high axial ratio (greater than 20) or models of low axial ratio made of multiple subunits with significant cavities (i.e. high degree of hydration).
Staurosporine both activates and inhibits serine/threonine kinases in human platelets
Authors: Kocher M , Clemetson K J
Published: August 2015

The effects of staurosporine on a selection of protein kinases were investigated with thrombin-stimulated and control human blood platelets. The results demonstrate that staurosporine (1 microM) can lead to activation of certain protein kinases in intact platelets and has a general inhibitory effect on the renaturable protein kinases in vitro. New candidates for protein kinases involved in signal transduction are identified.
The reactivities and ionization properties of the active-site dithiol groups of mammalian protein disulphide-isomerase
Authors: Hawkins H C , Freedman R B
Published: August 2015

1. The number of reactive thiol groups in mammalian liver protein disulphide-isomerase (PDI) in various conditions was investigated by alkylation with iodo[14C]acetate. 2. Both the native enzyme, as isolated, and the urea-denatured enzyme contained negligible reactive thiol groups; the enzyme reduced with dithiothreitol contained two groups reactive towards iodoacetic acid at pH 7.5, and up to five reactive groups were detectable in the reduced denatured enzyme. 3. Modification of the two reactive groups in the reduced native enzyme led to complete inactivation, and the relationship between the loss of activity and the extent of modification was approximately linear. 4. Inactivation of PDI by alkylation of the reduced enzyme followed pseudo-first-order kinetics; a plot of the pH-dependence of the second-order rate constant for inactivation indicated that the essential reactive groups had a pK of 6.7 and a limiting second-order rate constant at high pH of 11 M-1.s-1. 5. Since sequence data on PDI show the presence within the polypeptide of two regions closely similar to thioredoxin, the data strongly indicate that these regions are chemically and functionally equivalent to thioredoxin. 6. The activity of PDI in thiol/disulphide interchange derives from the presence of vicinal dithiol groups in which one thiol group of each pair has an unusually low pK and high nucleophilic reactivity at physiological pH.
Identification and characterization of a hepatic microsomal glucose transport protein. T3 of the glucose-6-phosphatase system?
Authors: Waddell I D , Scott H , Grant A , Burchell A
Published: August 2015

A 52 kDa polypeptide in rat liver microsomes was identified as a glucose-binding protein by its ability to weakly bind cytochalasin B and by its cross-reactivity to an antibody raised against the human erythrocyte glucose transport protein. The microsomal glucose binding polypeptide was purified by affinity chromatography and an antibody was raised against it. The inhibitory effect of this antibody on rat microsomal glucose-6-phosphatase activity and on glucose transport out of microsomal vesicles indicates that this protein is a microsomal glucose transport protein.
The herpes simplex virus virion protein Vmw65 transcriptionally activates the gene encoding the U4 snRNA but not that encoding the U2 snRNA during lytic infection
Authors: Latchman D S
Published: August 2015

Although lytic infection with herpes simplex virus (HSV) causes the repression of most host cell biosynthesis, it results in increased transcription of the cellular gene encoding the U4 snRNA, leading to accumulation of this snRNA. In contrast, no increased transcription of the gene encoding the U2 snRNA or accumulation of this RNA is observed in infected cells. These effects are mediated by the HSV virion protein Vmw65, which activates the U4 gene but does not affect the U2 gene. The significance of this difference between the U2 and U4 genes is discussed with regard to the presence in both of these genes of an identical octamer-binding site for the cellular transcription factor Oct-1 which complexes with Vmw65.
Determination of mass changes in phosphatidylinositol 4,5-bisphosphate and evidence for agonist-stimulated metabolism of inositol 1,4,5-trisphosphate in airway smooth muscle
Authors: Chilvers E R , Batty I H , Challiss R A , Barnes P J , Nahorski S R
Published: August 2015

Stimulation of muscarinic receptors in bovine tracheal smooth muscle (BTSM) causes a sustained increase in muscle tone, but a transient increase in the second messenger Ins(1,4,5)P3. To examine whether this brief increase in Ins(1,4,5)P3 mass results from transient formation or is due to agonist-stimulation of Ins(1,4,5)P3 metabolism, we have studied the relationship between mass changes in PtdIns(4,5)P2 and Ins(1,4,5)P3 accumulation, and changes in [3H]InsP3, [3H]PtdIns, [3H]PtdInsP1 and [3H]PtdInsP2 in carbachol-stimulated myo-[3H]inositol-prelabelled BTSM slices. Carbachol (0.1 mM) caused a rapid transient increase in Ins(1,4,5)P3 concentration (basal, 12.9 +/- 0.8 pmol/mg of protein; 5 s carbachol treatment, 27.1 +/- 1.5 pmol/mg of protein), with values returning to basal levels by 30 s, but a sustained accumulation of total [3H]InsP3s, with [3H]Ins(1,3,4)P3 being the predominant isomer present at later time points. In contrast, PtdIns(4,5)P2 mass, determined by radioreceptor assay of Ins(1,4,5)P3 in desalted alkaline hydrolysates of acidified chloroform/methanol tissue extracts, declined rapidly (basal, 941 +/- 22 pmol/mg of protein; 120 s carbachol, 365 +/- 22 pmol/mg of protein; t1/2 14 s) and remained at this new steady-state level for at least 20 min in the continued presence of carbachol. Addition of 10 microM-atropine 2 min after carbachol caused a prompt return of PtdIns(4,5)P2 concentration to prestimulated values (t1/2 210 s). Ongoing resynthesis of PtdIns(4,5)P2 after carbachol stimulation was demonstrated in [3H]inositol-labelled tissue by observing a persistent increase in the specific radioactivity of [3H]PtdInsP2, shown to be exclusively [3H]PtdIns(4,5)P2, over a 10 min period. These findings strongly suggest the occurrence of persistent receptor-mediated increases in PtdIns(4,5)P2 hydrolysis and Ins(1,4,5)P3 formation which, in conjunction with the transient accumulation of Ins(1,4,5)P3 observed, provide evidence that regulation of the metabolism of Ins(1,4,5)P3 is a major determinant of Ins(1,4,5)P3 concentration in this tissue under agonist-stimulated conditions.
2-acyl-sn-glycero-3-phosphoethanolamine lysophospholipase A2 activity in guinea-pig heart microsomes
Authors: Badiani K , Arthur G
Published: August 2015

We have recently described a lysophospholipase A2 activity in guinea-pig heart microsomes that hydrolyses 2-acyl-sn-glycero-3-phosphocholine (2-acyl-GPC). The presence of a similar activity that hydrolyses 2-acyl-sn-glycero-3-phosphoethanolamine (2-acyl-GPE) was not known. In this study, a lysophospholipase A2 activity in guinea-pig heart microsomes that hydrolyses 2-acyl-GPE has been characterized. The enzyme did not require Ca2+ for activity and exhibited a high specificity for 2-arachidonoyl-GPE and 2-linoleoyl-GPE over 2-oleoyl-GPE and 2-palmitoyl-GPE. The specificity for these unsaturated substrates was observed in the presence and absence of detergents. Selective hydrolysis of 2-arachidonoyl-GPE over 2-palmitoyl-GPE was observed when equimolar quantities of the two substrates were incubated with the enzyme. There was no preferential hydrolysis of either 2-linoleoyl- or 2-arachidonoyl-GPE when presented individually or as a mixture. Significant differences in the characteristics of 2-acyl-GPE-hydrolysing and 2-acyl-GPC-hydrolysing activities included differences in their optimum pH, the effect of Ca2+ and their acyl specificities. Taken together, these results suggest that the two activities are catalysed by different enzymes. 2-Acyl-GPE lysophospholipase activity with a preference for 2-arachidonoyl-GPE over 2-oleoyl-GPE was observed in guinea-pig brain, liver, kidney and lung microsomes. Lysophospholipase A1 activity that catalyses the hydrolysis of 1-acyl-GPE was also present in guinea-pig heart microsomes and had different characteristics from the 2-acyl-GPE-hydrolysing activity, including a preference for saturated over unsaturated substrates. The 2-acyl-GPE lysophospholipase A2 activity appeared to be distinct from Ca(2+)-independent phospholipase A2. The characteristics of the 2-acyl-GPE lysophospholipase A2 suggest it could play a role in the selective release of arachidonic and linoleic acids for further metabolism in cells.