bluuebunny/crossref_metadata_2025 huggingface dataset:
Extracellular calcium modulates insulin's action on enzymes controlling cyclic AMP metabolism in intact hepatocytes
Authors: Irvine F , Wallace A V , Sarawak S R , Houslay M D
Published: August 2015

Absence of physiological concentrations of extracellular Ca2+ in the Krebs-Henseleit incubation buffer did not affect the ability of 10 nM glucagon (< 5%) to increase hepatocyte intracellular cyclic AMP concentrations, but severely ablated (by approximately 70%) the ability of 10 nM insulin to decrease these elevated concentrations. Cyclic AMP metabolism is determined by production by adenylate cyclase and degradation by cyclic AMP phosphodiesterase (PDE). In the absence of added extracellular Ca2+ (2.5 mM), insulin's ability to activate PDE activity was selectively compromised, showing a failure of insulin to activate two of the three insulin-stimulated activities, namely the ‘dense-vesicle’ and peripheral plasma-membrane (PPM) PDEs. In the absence of added Ca2+, insulin's ability to inhibit adenylate cyclase activity in intact hepatocytes was decreased dramatically. Vasopressin and adrenaline (+ propranolol) failed to elicit the activation of either the ‘dense-vesicle’ or the PPM-PDEs. The presence of physiological concentrations of extracellular Ca2+ in the incubation medium is shown to be important for the appropriate generation of insulin's actions on cyclic AMP metabolism.
Influence of cellobiose oxidase on peroxidases from Phanerochaete chrysosporium
Authors: Ander P , Sena-Martins G , Duarte J C
Published: August 2015

Reduction of H2O2-oxidized manganese peroxidase (MnP), lignin peroxidase and, to some extent, horseradish peroxidase, was studied in the presence of cellobiose oxidase (CbO) and cellobiose. It was found that the reversion rates for MnP compound II and lignin peroxidase compound II back to native enzymes increased significantly in the presence of CbO and cellobiose. However, the reduction of cytochrome c by CbO plus cellobiose was 40 times faster than the reduction of MnP compound II. Also, the lag phase before reversion to the native states decreased for all three peroxidases in the presence of CbO and cellobiose. Active CbO did not repress formation of compounds I or II of the peroxidases, and Mn2+/veratryl alcohol reduced compound II of the peroxidases much more rapidly than did active CbO. This indicates that, in the presence of Mn2+ or veratryl alcohol, MnP and lignin peroxidase can complete their catalytic cycles and function normally without interference from CbO. Without the presence of peroxidase substrates, active CbO reduced compound II of the above peroxidases.
Characterization of a full-length cDNA encoding human liver S-adenosylmethionine synthetase: tissue-specific gene expression and mRNA levels in hepatopathies
Authors: Alvarez L , Corrales F , Martín-Duce A , Mato J M
Published: August 2015

The sequence of a full-length cDNA coding for human liver S-adenosylmethionine synthetase has been determined. It spans 3217 nucleotides and encodes a protein of 395 amino acid residues, with a calculated molecular mass of 43,647 Da. The structural features deduced from the amino acid sequence show a close similarity to those of the rat liver enzyme. The liver-specific S-adenosylmethionine synthetase gene appears to be present as a single copy in the genome, as revealed by Southern analysis. The occurrence of a single mRNA species for this enzyme has been determined by primer extension and Northern analysis. Among several human tissues examined, this gene is expressed only in the liver. Similar S-adenosylmethionine synthetase mRNA levels have been detected in biopsies from normal human liver and from patients with alcoholic cirrhosis and hepatocellular carcinoma. Based on these results, a possible mechanism of regulation of human liver S-adenosylmethionine synthetase is discussed.
Characterization of a new oriental-mustard (Brassica juncea) allergen, Bra
j IE: detection of an allergenic epitope
Authors: Monsalve R I , Gonzalez de la Peña M A , Menendez-Arias L , Lopez-Otin C , Villalba M , Rodriguez R
Published: August 2015

Bra j IE, a major allergen from oriental-mustard (Brassica juncea) seeds, has been isolated and characterized. Its primary structure has been elucidated. This protein is composed of two chains (37 and 92 amino acids) linked by disulphide bridges. The amino acid sequence obtained is closely related to that previously determined for Sin a I, an allergen isolated from yellow mustard (Sinapis alba). A common epitope has been detected in the large chain of both Bra j IE and Sin a I by means of electroblotting and immunodetection with 2B3, which is a monoclonal antibody raised against the yellow-mustard allergen. A histidine residue of the large chain of both mustard allergens has been found to be essential for the recognition by 2B3 antibody. A synthetic multiantigenic peptide containing this His was recognized by 2B3 as well as by sera of mustard-hypersensitive individuals. Therefore this antigenic determinant must be involved in the allergenicity of these proteins.
Hepatic lipase function and the accumulation of β-very-low-density lipoproteins in the plasma of cholesterol-fed rabbits
Authors: Chang S , Borensztajn J
Published: August 2015

The accumulation of cholesterol-rich beta-very-low-density lipoproteins (beta-VLDL) in the plasma of rabbits fed on a high-fat high-cholesterol diet is due to a defect in the clearance of these lipoprotein remnants from circulation by the liver. In view of the evidence that hepatic lipase participates in the process of rapid removal of remnants from circulation, and considering that rabbits are naturally deficient in hepatic lipase, we examined whether this defect in the clearance of beta-VLDL could be reversed by exogenous hepatic lipase. We report that treatment in vitro of [3H]cholesterol-labelled beta-VLDL, or rat chylomicrons, with hepatic lipase resulted in the formation of particles that were rapidly cleared from circulation by the liver when injected intravenously into hypercholesterolaemic rabbits. These results are consistent with the notion that, in addition to the well-established requirement for lipoprotein lipase activity, the generation of remnants capable of being efficiently taken up by the liver also requires the action of hepatic lipase. Lipoprotein lipase acts on triacylglycerol-rich lipoproteins to transform them into particles (remnants) which bind to the surface of liver cells, where they become accessible to hepatic lipase. Hepatocyte endocytosis of these remnants occurs only after further modification by hepatic lipase. According to this scheme, the results presented suggest that the accumulation of beta-VLDL in the circulation of rabbits fed on a high-fat high-cholesterol diet is the result of the saturation of the available hepatic lipase by abnormally high levels of lipoprotein-lipase-generated chylomicron remnants.
DNA precursors are channelled to nuclear matrix DNA replication sites
Authors: Panzeter P L , Ringer D P
Published: August 2015

Studies of replicative DNA synthesis using DNA precursors have shown that the DNA that was replicated most recently is that associated with the nuclear matrix. Consequently, precursors arising via the salvage and the de novo metabolic pathways are first incorporated into a small percentage of the total nuclear DNA that is termed nuclear matrix-associated DNA. These results have been substantiated in cell culture, as well as in intact mammalian systems. Furthermore, when DNA precursors were injected intravenously into regenerating rat liver, a significant lag in the incorporation of orotic acid-derived nucleotides (de novo pathway precursors) into nuclear DNA was observed, when compared with deoxythymidine-derived nucleotides (salvage pathway precursors). This lag in incorporation kinetics was also evident at the nuclear matrix level, although, once incorporated into nuclear matrix-associated DNA, the distribution patterns of both precursors into extra-matrix nuclear DNA fractions were identical. To determine the basis for this kinetic lag, we compared the incorporation kinetics of orotic acid and of deoxythymidine into dTTP and into nuclear matrix-associated DNA, respectively. Orotic acid-derived nucleotides entered the cytosolic dTTP pool before being incorporated into nuclear matrix-associated DNA, that is, traversing the classical metabolic route of DNA precursors. Conversely, deoxythymidine-derived nucleotides by-passed the soluble dTTP cellular pool and engaged directly in DNA synthesis at the nuclear matrix. Not only is this the first evidence for nucleotide channelling in an intact mammalian system, but it also forms direct evidence that salvage pathway DNA precursors are channelled to nuclear matrix-associated sites of DNA replication.
Signalling through CD28 T-cell activation pathway involves an inositol phospholipid-specific phospholipase C activity
Authors: Nunes J , Klasen S , Franco M D , Lipcey C , Mawas C , Bagnasco M , Olive D
Published: August 2015

Stimulation of the human T-cell line, Jurkat, by a monoclonal antibody (mAb) directed against the CD28 molecule leads to sustained increases in intracellular levels of Ca2+ ([Ca2+]i); the initial rise in Ca2+ comes from internal stores, followed by Ca2+ entry into the cells. The CD28 molecule also appears to activate polyphosphoinositide (InsPL)-specific phospholipase C (PLC) activity in Jurkat cells, as demonstrated by PtdInsP2 breakdown, InsP3 and 1,2-diacylglycerol generation and PtdIns resynthesis. We also observed that interleukin-2 (IL2) production induced via CD28 triggering was sensitive to a selective protein kinase C inhibitor. Of the four other anti-CD28 mAbs (CD28.2, CD28.4, CD28.5, CD28.6) tested, only one (CD28.5) was unable to generate any InsPL-specific PLC or IL2 secretion. However, the cross-linking of cell-bound CD28.5 with anti-mouse Ig antibodies led to an increase in [Ca2+]i. CD28-molecule clustering in itself appears to be a sufficient signal for induction of PLC activity.
Oxygen Michaelis constants for tyrosinase
Authors: Rodríguez-López J N , Ros J R , Varón R , García-Cánovas F
Published: August 2015

The Michaelis constant of tyrosinase for oxygen in the presence of monophenols and o-diphenols, which generate a cyclizable o-quinone, has been studied. This constant depends on the nature of the monophenol and o-diphenol and is always lower in the presence of the former than of the latter. From the mechanism proposed for tyrosinase and from its kinetic analysis [Rodríguez-López, J. N., Tudela, J., Varón, R., García-Carmona, F. and García-Cánovas, F. (1992) J. Biol. Chem. 267, 3801-3810] a quantitative ratio has been established between the Michaelis constants for oxygen in the presence of monophenols and their o-diphenols. This ratio is used for the determination of the Michaelis constant for oxygen with monophenols when its value cannot be calculated experimentally.
Conformational properties of Bacillus subtilis RNA polymerase σA factor during transcription initiation
Authors: Chang B Y , Doi R H
Published: August 2015

By the use of a partial proteolysis method and Western-blot analysis, the conformational properties of Bacillus subtilis sigma A factor in the transcription initiation stage were studied. From a comparison of the trypsin-digestion patterns of free sigma A and of sigma A associated with core enzyme, it was found that the production of 45 kDa sigma A tryptic-derived fragment was enhanced when sigma A was associated with the core enzyme. More importantly, a 40 kDa sigma A tryptic-derived fragment was found exclusively in this associated state. Based on the change of the digestion kinetics when producing the 45 kDa tryptic fragment and the generation of this new 40 kDa tryptic fragment from sigma A, it was apparent that a conformation change of sigma A occurred during the association of sigma A with the core enzyme. Also, similar patterns were found for the sigma A present in the holoenzyme-promoter DNA complex. These findings suggest that no further distinctive conformational change of sigma A occurs at the step of RNA polymerase holoenzyme and promoter DNA complex formation. Trypsin-digestion patterns of sigma A in different RNA polymerase holoenzyme and promoter DNA complexes were also studied. The presence of similar trypsin digestion-patterns of sigma A in those complexes strongly supports the idea that a similar sigma A conformation is used in the recognition of different sigma A-type promoters and the formation of different open complexes.
Disulphide cross-linking of smooth-muscle and non-muscle caldesmon to the C-terminus of actin in reconstituted and native thin filaments
Authors: Graceffa P , Adam L P , Lehman W
Published: August 2015

It was reported that chicken gizzard smooth-muscle caldesmon Cys-580 can be disulphide-cross-linked to the C-terminal pen-ultimate residue (Cys-374) of actin, indicating that these residues are close in the protein complex [Graceffa, P. and Jancso, A. (1991) J. Biol. Chem. 266, 20305-20310]. Since the possibility that the cross-link involves a cysteine residue other than actin Cys-374 was not absolutely excluded, more direct evidence was sought for the identify of the cysteine residues involved in the cross-link. We show here that caldesmon could not be disulphide-cross-linked to actin which had Cys-374 removed by carboxypeptidase A digestion, providing direct support for the participation of actin Cys-374 in the cross-link to caldesmon. In order to assign the caldesmon cysteine residue involved in the cross-link, use was made of caldesmon from porcine stomach muscle, which is shown to contain one cysteine residue close to, or at, position 580, in contrast with chicken gizzard caldesmon, which has an additional cysteine residue at position 153. The porcine stomach caldesmon also formed a disulphide-cross-link to actin, further supporting the original conclusion that Cys-580 of the chicken gizzard caldesmon had been cross-linked to actin. Disulphide-cross-linking with similar yield was also observed in native chicken gizzard muscle thin filaments, indicating that the interaction between actin and the C-terminal domain of caldesmon is the same in native and reconstituted thin filaments. The much smaller non-muscle isoform of caldesmon, from rabbit liver, could be similarly cross-linked to actin, consistent with the sequence similarity between the C-terminal domain of muscle and non-muscle caldesmon. The ability to cross-link caldesmon Cys-580 to actin Cys-374 suggests the possibility that the Cys-580 region of caldesmon and the C-terminus of actin form part of the actin-caldesmon binding interface.
Detection, isolation and characterization of multiple lectins from the haemolymph of the cockroach Blaberus discoidalis
Authors: Chen C , Ratcliffe N A , Rowley A F
Published: August 2015

Three agglutinins (lectins), designated BDL1, BDL2 and BDL3, were identified in the haemolymph of the cockroach Blaberus discoidalis by erythrocyte cross-adsorption and sugar inhibition tests. With the use of (NH4)2SO4 fractionation, anion-exchange and affinity chromatography, BDL1 and BDL2 have been purified to homogeneity, and BDL3 has been partially purified to three bands on SDS/PAGE. BDL1 has a molecular-mass estimate of 390 kDa by gel filtration and approx. 158 kDa by SDS/PAGE under non-reducing conditions, further reduced to subunits of 36 kDa under reducing conditions. BDL2 has a molecular mass of approx. 140 kDa and is composed of subunits of 67 kDa which can be further reduced to identical subunits of 23 kDa. Isoelectric focusing in agarose gels revealed that BDL1 and BDL2 both focused as single bands at pH 6.0 and pH 5.2 respectively. The purified forms of BDL1 and BDL2 were stained by the periodic acid/Schiff's reagent showing that both lectins are glycoproteins. In addition, BDL1 was deglycosylated by endo-beta-N-acetylglucosaminidase H. Immunological tests showed that these three lectins are not structurally related. All three lectins bind galactose but have different specificities for binding other sugars and for a range of vertebrate erythrocytes. BDL1 is specifically inhibited by D-(+)-glucose, D-(+)-mannose and N-acetyl-D-mannosamine, but not by N-acetyl-D-glucosamine, and BDL2 is inhibited by N-acetyl-D-glucosamine, but not by D-(+)-glucose, D-(+)-mannose or N-acetyl-D-mannosamine. BDL3 is strongly inhibited by N-acetyl-D-galactosamine, but not by any of the other above-mentioned sugars. Erythrocyte specificities showed that BDL1 is more specific for rabbit than mouse erythrocytes, whereas BDL2 and BDL3 are more specific for mouse than rabbit erythrocytes. The haemagglutinating activities of both the serum and isolated lectins are Ca(2+)-dependent. Localization of BDL1 and BDL2 with fluorescein isothiocyanate-labelled antibodies showed that both lectins are associated with the granules and other areas of the cytoplasm of all blood cell types.
Ion fluxes through the progesterone-activated channel of the sperm plasma membrane
Authors: Foresta C , Rossato M , Di Virgilio F
Published: August 2015

We have characterized ionic changes triggered by progesterone in human spermatozoa. This steroid, which is a fast-acting stimulator of the acrosome reaction, triggered a rapid increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) which was entirely due to influx across the plasma membrane, as it was obliterated by chelation of extracellular Ca2+. Ca2+ fluxes were insensitive to verapamil and pertussis toxin, thus suggesting that they did not occur via voltage-gated channels and did not involve a pertussis toxin-sensitive G protein, and were potentiated in Na(+)-free, choline-containing or methylglucamine-containing medium. Progesterone also caused a depolarization of the plasma membrane in Na(+)-containing as well as in choline- or methyl-glucamine-containing saline; depolarization was larger in the absence of extracellular Ca2+, suggesting that Na+ and Ca2+ fluxes occurred through the same channel. Progesterone was able to trigger the acrosome reaction in the three media investigated (Na+, choline and methylglucamine), provided that extracellular Ca2+ was also present. We conclude that progesterone activates a membrane ion channel that is permeable to monovalent cations as well as to Ca2+.
Interleukin-2 induces tyrosine phosphorylation of the vav proto-oncogene product in human T cells: lack of requirement for the tyrosine kinase lck
Authors: Evans G A , Howard O M Z , Erwin R , Farrar W L
Published: August 2015

The haematopoietic protein, p95vav, has been shown to be a tyrosine kinase substrate and to have tyrosine kinase-modulated guanine-nucleotide-releasing-factor activity. This implies a function in the control of ras or ras-like proteins. Because ras activation has been shown to be a downstream event following stimulation of the interleukin-2 (IL-2) receptor, we investigated the possibility that vav was involved in IL-2 signal transduction pathways, using human T cells as a model. We found rapid tyrosine phosphorylation of vav in response to IL-2 within 1 min, with maximum increase of phosphorylation of 5-fold occurring by 5 min after treatment in normal human T cells. IL-2 stimulation of the human T-cell line YT and a subclone of the YT cell line (YTlck-) that does not express message for the src-family kinase p56lck also results in a rapid rate of tyrosine phosphorylation of vav of more than 5-fold by 5 min. These results suggest that vav may play an important role in IL-2-stimulated signal transduction and that there is not a strict requirement for the tyrosine kinase p56lck.
A modular esterase from Pseudomonas fluorescens subsp. cellulosa contains a non-catalytic cellulose-binding domain
Authors: Ferreira L M , Wood T M , Williamson G , Faulds C , Hazlewood G P , Black G W , Gilbert H J
Published: August 2015

The 5′ regions of genes xynB and xynC, coding for a xylanase and arabinofuranosidase respectively, are identical and are reiterated four times within the Pseudomonas fluorescens subsp. cellulosa genome. To isolate further copies of the reiterated xynB/C 5′ region, a genomic library of Ps. fluorescens subsp. cellulosa DNA was screened with a probe constructed from the conserved region of xynB. DNA from one phage which hybridized to the probe, but not to sequences upstream or downstream of the reiterated xynB/C locus, was subcloned into pMTL22p to construct pFG1. The recombinant plasmid expressed a protein in Escherichia coli, designated esterase XYLD, of M(r) 58,500 which bound to cellulose but not to xylan. XYLD hydrolysed aryl esters, released acetate groups from acetylxylan and liberated 4-hydroxy-3-methoxycinnamic acid from destarched wheat bran. The nucleotide sequence of the XYLD-encoding gene, xynD, revealed an open reading frame of 1752 bp which directed the synthesis of a protein of M(r) 60,589. The 5′ 817 bp of xynD and the amino acid sequence between residues 37 and 311 of XYLD were almost identical with the corresponding regions of xynB and xynC and their encoded proteins XYLB and XYLC. Truncated derivatives of XYLD lacking the N-terminal conserved sequence retained the capacity to hydrolyse ester linkages, but did not bind cellulose. Expression of truncated derivatives of xynD, comprising the 5′ 817 bp sequence, encoded a non-catalytic polypeptide that bound cellulose. These data indicate that XYLD has a modular structure comprising of a N-terminal cellulose-binding domain and a C-terminal catalytic domain.
Isolation and characterization of the human diacylglycerol kinase gene
Authors: Fujikawa K , Imai S , Sakane F , Kanoh H
Published: August 2015

The 80 kDa diacylglycerol kinase (DGK) is abundantly expressed in oligodendrocytes and lymphocytes but not to a detectable extent in other cells such as neurons and hepatocytes. As an initial attempt to delineate the mechanism of the transcriptional control of the DGK gene, we have cloned from a human genomic library a 22 kb genomic fragment. The genomic clone consists of the 5′-flanking region and 17 exons coding for approx. 53% of the total exons of human DGK, including those encoding EF-hand and zinc-finger regions. The translation initiation site is located in the second exon. S1 nuclease mapping and primer extension analysis of the human DGK mRNA identified a major transcription initiation site (position +1) at 264 bp upstream from the initiator ATG. In the 5′-flanking sequence we detected a single GC box at -35 but no canonical TATA and CAAT sequences. However, the sequence starting from the cap site (AGTTCCTGCCA) is very similar to the initiator element that specifies the transcription initiation site of some housekeeping genes. In addition, the 5′-upstream region contains several putative cis-elements. Jurkat and HepG2 cells were transfected with various 5′-deletion mutants of the upstream region fused to the structural gene of chloramphenicol acetyltransferase (CAT). The CAT assay revealed that among constructs containing up to 3.4 kb of the 5′-flanking region, a fragment of 263 bp from the transcription initiation site contains a basic promoter that is active in both types of cells. Moreover, the region between -263 and -850 contains a negative element that is active in HepG2 but not in Jurkat cells. This negative element may, at least in part, be responsible for the cell type-specific expression of the DGK gene.
Characterization of a B-type esterase involved in insecticide resistance from the mosquito Culex quinquefasciatus
Authors: Karunaratne S H P P , Jayawardena K G , Hemingway J , Ketterman A J
Published: August 2015

The enzyme, esterase B2, involved in insecticide resistance has been purified and characterized from the mosquito Culex quinquefasciatus. The monomeric enzyme has an M(r) of 62000 and a pI of 5.0. This enzyme is compared with the esterase A2 previously characterized [Ketterman, Jayawardena and Hemingway (1992) Biochem. J. 287, 355-360]. The kinetic constants for interaction with several insecticides indicate, as for the esterase A2, that the B2 enzyme has a role in resistance. The rates and affinities of binding observed support the hypothesis that the role mainly is sequestration followed by the slow turnover of the insecticide. Although the B2 esterase appears to have a slightly higher rate of interaction with insecticides, the A2 has a much greater Vmax. with the xenobiotic substrates studied. The B2 esterase also appears to be present in the larvae to a lesser extent than the esterase A2.
Enhanced synthesis and accumulation of proteoglycans in cholesterol-enriched arterial smooth muscle cells
Authors: Vijayagopal P
Published: August 2015

To determine the effects of lipid accumulation on proteoglycan synthesis, we studied proteoglycan biosynthesis in rabbit aortic smooth muscle cells in culture. Cholesterol-enrichment was accomplished by incubating confluent smooth muscle cells with cationized low-density lipoprotein. Control and cholesterol-enriched cells were incubated with [35S]sulphate, [3H]glucosamine, or [3H]serine. Metabolically labelled proteoglycans in the cell layer and medium were quantified. During a 20 h incubation period, proteoglycan synthesis in cholesterol-enriched cells increased by 40-50% above that in control cells. A similar increase in precursor incorporation into proteoglycans was also noted following a short 15 min pulse. The cholesterol-enriched cells also showed a 45-50% increase over control rates in the intralysosomal accumulation of a large chondroitin sulphate proteoglycan and a small dermatan sulphate proteoglycan. The enhanced synthesis of proteoglycans in cholesterol-enriched cultures was inhibited by cycloheximide and actinomycin D, which are inhibitors of protein synthesis and transcription respectively. Proteoglycan turnover was investigated by pulse-chase analysis. Following a 2-h pulse, intracellular proteoglycans in cholesterol-enriched cells disappeared, having a half-life of 26.5 h compared with 2.8 h for those in the control cells. The amount of trypsin-releasable proteoglycan was significantly reduced in cholesterol-enriched cells. In addition, the degradation of proteoglycans was severely retarded in cholesterol-enriched cultures. The activities of three acid hydrolases, N-acetyl-beta-hexosaminidase, beta-glucuronidase and cathepsin C, were significantly reduced in cholesterol-enriched cells compared with activities in control cells. The results indicate that proteoglycan metabolism is altered in cholesterol-enriched smooth muscle cells.
Direct labelling of hormone-sensitive phosphoinositides by a plasma-membrane-associated PtdIns synthase in turkey erythrocytes
Authors: Vaziri C , Downes C P , Macfarlane S C
Published: August 2015

We have previously characterized phosphatidylinositol (PtdIns) synthase and PtdIns/myo-inositol-exchange enzyme activities in ghost membranes prepared by hypotonic lysis of turkey erythrocytes [McPhee, Lowe, Vaziri and Downes (1991) Biochem. J. 275, 187-192]. Here we show that PtdIns synthase activity is relatively enriched in plasma-membrane preparations of turkey erythrocytes and that inositol phospholipids labelled by both PtdIns synthase and PtdIns myo-inositol exchange enzymes are susceptible to hydrolysis by the receptor- and G-protein-regulated phospholipase C (PLC), which is present also in ghost preparations. Specific-radioactivity measurements of [3H]PtdIns from ghosts labelled to equilibrium under conditions favouring [3H]inositol incorporation by PtdIns synthase activity indicate that PtdIns synthase can directly access approx. 14% of the total erythrocyte ghost PtdIns. Approx. 16% of the [3H]PtdIns labelled by the PtdIns synthase reaction can be phosphorylated to polyphosphoinositides, which are then hydrolysed by the receptor- and G-protein-stimulated PLC. Since the mass of PtdIns declines to a similar extent as [3H]PtdIns during stimulation in the presence of guanine nucleotides and ATP, it is evident that both the labelled and unlabelled phosphoinositides are susceptible to hydrolysis by the relevant PLC. Phosphoinositides present in nuclei-free plasma membranes were also labelled by [3H]inositol under conditions favouring PtdIns synthase and PtdIns/myo-inositol-exchange enzyme activities respectively. These membranes lack PLC activity [Vaziri and Downes (1992) J. Biol. Chem. 267, 22973-22981], but the labelled lipids were sensitive to purinergic-receptor-stimulated hydrolysis in reconstitution assays using partially purified turkey erythrocyte PLC. The results strongly suggest that at least a portion of the PtdIns synthase in turkey erythrocytes is located in the plasma membrane and has direct access to an agonist-sensitive pool of inositol phospholipids.
Modulation of insulin secretion from normal rat islets by inhibitors of the post-translational modifications of GTP-binding proteins
Authors: Metz S A , Rabaglia M E , Stock J B , Kowluru A
Published: August 2015

Many GTP-binding proteins (GBPs) are modified by mevalonic acid (MVA)-dependent isoprenylation, carboxyl methylation or palmitoylation. The effects of inhibitors of these processes on insulin release were studied. Intact pancreatic islets were shown to synthesize and metabolize MVA and to prenylate several candidate proteins. Culture with lovastatin (to inhibit synthesis of endogenous MVA) caused the accumulation in the cytosol of low-M(r) GBPs (labelled by the [alpha-32P]GTP overlay technique), suggesting a disturbance of membrane association. Concomitantly, lovastatin pretreatment reduced glucose-induced insulin release by about 50%; co-provision of 100-200 microM MVA totally prevented this effect. Perillic acid, a purported inhibitor of the prenylation of small GBPs, also markedly reduced glucose-induced insulin secretion. Furthermore, both N-acetyl-S-trans, trans-farnesyl-L-cysteine (AFC), which inhibited the base-labile carboxyl methylation of GBPs in islets or in transformed beta-cells, and cerulenic acid, an inhibitor of protein palmitoylation, also reduced nutrient-induced secretion; an inactive analogue of AFC (which did not inhibit carboxyl methylation in islets) had no effect on secretion. In contrast with nutrients, the effects of agonists that induce secretion by directly activating distal components in signal transduction (such as a phorbol ester or mastoparan) were either unaffected or enhanced by lovastatin or AFC. These data are compatible with the hypothesis that post-translational modifications are required for one or more stimulatory GBPs to promote proximal step(s) in fuel-induced insulin secretion, whereas one or more inhibitory GBPs might reduce secretion at a more distal locus.
Thimet oligopeptidase: similarity to ‘soluble angiotensin II-binding protein’ and some corrections to the published amino acid sequence of the rat testis enzyme
Authors: McKie N , Dando P M , Rawlings N D , Barrett A J
Published: August 2015

The deduced amino acid sequence of pig liver soluble angiotensin II-binding protein [Sugiura, Hagiwara and Hirose (1992) J. Biol. Chem. 267, 18067-18072] is similar over most of its length to that reported for rat testis thimet oligopeptidase (EC 3.4.24.15) by Pierotti, Dong, Glucksman, Orlowski and Roberts [(1990) (Biochemistry 29, 10323-10329]. We have found that homogeneous rat testis thimet oligopeptidase binds angiotensin II with the same distinctive characteristics as the pig liver protein. Analysis of the nucleotide sequences reported for the two proteins pointed to the likelihood that sequencing errors had caused two segments of the amino acid sequence of the rat protein to be translated out of frame, and re-sequencing of selected parts of the clone (kindly provided by the previous authors) confirmed this. The revised deduced amino acid sequence of rat thimet oligopeptidase contains 687 residues, representing a protein of 78,308 Da, and is more closely related to those of the pig liver protein and other known homologues of thimet oligopeptidase than that described previously.
Malonyl-CoA metabolism in cardiac myocytes and its relevance to the control of fatty acid oxidation
Authors: Awan M M , Saggerson E D
Published: August 2015

1. Viable myocytes were obtained from rat hearts. Oxidation of [1-14C]palmitate by these cells could be decreased by the addition of glucose (5 mM) or lactate (2 mM). In the presence of glucose, insulin decreased and adrenaline increased palmitate oxidation. 2. The myocytes contained activities of ATP citrate-lyase, acetyl-CoA carboxylase and the condensing enzyme of the fatty acid elongation system. No fatty acid synthase activity was demonstrable in myocytes. 3. In rat hearts perfused with 5 mM glucose, malonyl-CoA content was acutely raised by insulin. In the presence of glucose+insulin, perfusion with palmitate or adrenaline decreased the malonyl-CoA content. 4. It is concluded that malonyl-CoA can be synthesized within cardiac myocytes and that the level of this metabolite can be acutely regulated. This is likely to have consequences for the regulation of carnitine palmitoyltransferase in the heart.
Differential control of the functional cell surface expression and content of hexose transporter GLUT-1 by glucose and glucose metabolism in murine fibroblasts
Authors: Ortiz P A , Haspel H C
Published: August 2015

The present paper evaluates the contributions of glucose and its metabolites to the post-translational regulation of hexose transport and GLUT-1 content in murine fibroblasts. The effects of 3-O-methylglucose, a nearly non-metabolizable glucose analogue, on 2-deoxyglucose-uptake, cell-surface expression and content of GLUT-1, glucose 6-phosphate levels, and phosphoglucose isomerase (PGI) and hexokinase activities of murine fibroblasts were compared with those of glucose and fructose. Glucose (EC50 approximately 6 mM) or 3-O-methylglucose (EC50 approximately 12 mM), which are substrates of GLUT-1, but not fructose, which is not transported by GLUT-1, are able to prevent the glucose-deprivation-induced increases in both hexose transport and cell-surface expression of GLUT-1. In contrast, glucose (EC50 approximately 6 mM), but not 3-O-methylglucose or fructose, prevents the glucose-deprivation-induced accumulation of total GLUT-1 polypeptides. Glucose (> or = 5 mM), but not fructose or 3-O-methylglucose, leads to significant glucose 6-phosphate accumulation. Although 3-O-methylglucose is weakly phosphorylated by fibroblasts, accumulation of phosphorylated product does not correlate with hexose-transport regulation. The activities of hexokinase and PGI are not altered by glucose, fructose or 3-O-methylglucose. We suggest that, in murine fibroblasts: (i) hexose transport and GLUT-1 content are differentially regulated; (ii) substrates of GLUT-1 and/or their immediate metabolites regulate the cell-surface expression of functional GLUT-1; and (iii) glucose metabolism is required for the regulation of GLUT-1 content.
Alterations in the expression of G-proteins and regulation of adenylate cyclase in human neuroblastoma SH-SY5Y cells chronically exposed to low-efficacy μ-opioids
Authors: Ammer H , Schulz R
Published: August 2015

Western-blot analysis of human neuroblastoma SH-SY5Y cells (mu- and delta-receptors) revealed the presence of the following G-protein subunits: Gi alpha 1, Gi alpha 2, Gs alpha, G(o) alpha, Gz alpha, and G beta, a pattern resembling that observed in central nervous tissue. Chronic treatment of differentiated [all-trans-retinoic acid (10 microM; 6 days)] SH-SY5Y cells with D(-)-morphine (10 microM; 3 days) significantly increased the abundance of all G-protein subunits identified. Co-incubation of morphine-exposed cells together with naloxone (10 microM; 3 days) or the mu-selective opioid antagonist CTOP (10 microM; 3 days), but not with the delta-selective antagonist ICI-174,864 (10 microM; 3 days), completely abolished this effect, suggesting that the increase in G-protein abundance is specifically mediated by mu-receptors. Moreover, the biologically inactive enantiomer L(+)-morphine (10 microM; 3 days) failed to produce a similar effect. G-protein up-regulation developed in a time- and dose-dependent manner and is most likely due to enhanced protein synthesis de novo, since concomitant treatment of the cells with cycloheximide (100 micrograms/ml; 3 days) prevented this effect. Chronic treatment with the low-efficacy mu-selective opioid peptide morphiceptin (10 microM; 3 days), but not with the highly potent mu-agonist DAGO (0.1 microM; 3 days) produced a comparable increase in G-protein abundance. Coincident with quantitative effects on G-protein levels in morphine-tolerant/dependent SH-SY5Y cells, we found elevated levels of basal, forskolin (1 microM)- and prostaglandin-E1 (1 microM)-stimulated adenylate cyclase activities. Reconstitution experiments using S49 cyc- lymphoma-cell membranes suggest that this increase is most likely due to elevated levels of functionally intact Gs. Chronic treatment with both morphine and DAGO induces high degrees of tolerance in this cell line. However, the intrinsic activity of G1 was unchanged, as assessed in functional studies with low-nanomolar concentrations of guanosine 5′-[beta gamma- imido]triphosphate. Our data demonstrate that chronic treatment of SH-SY5Y cells with low-efficacy mu-opioids increases G-protein abundance, a phenomenon which might contribute to the biochemical mechanisms underlying opioid tolerance/dependence.
Effect of l-ascorbic acid on the monophenolase activity of tyrosinase
Authors: Ros J R , Rodríguez-López J N , García-Cánovas F
Published: August 2015

The effect of ascorbic acid on the monophenolase activity of tyrosinase, using tyrosine as substrate, has been studied. Over the ranges of ascorbic acid concentration used, no direct effect on the enzyme is found. However, a shortening of the characteristic induction period of the hydroxylation reaction is observed. The evolution of the reaction is dependent on the concentration of ascorbic acid. Low concentrations permit the system to reach the steady state when all ascorbic acid is consumed, whereas high concentrations do not. In the light of these results it is proposed that the influence of ascorbic acid on the reaction is due to its ability to reduce the enzymically generated o-quinones. A relationship between the ascorbic acid concentration, and the induction period generated by it, with the diphenolase activity of tyrosinase is established, which can be used as a basis for the determination of trace amounts of this reducing agent.
Hepoxilin A3 inhibits the rise in free intracellular calcium evoked by formyl-methionyl-leucyl-phenylalanine, platelet-activating factor and leukotriene B4
Authors: Laneuville O , Reynaud D , Grinstein S , Nigam S , Pace-Asciak C R
Published: August 2015

We have previously shown that the hepoxilins are capable of increasing the intracellular free concentration of calcium ([Ca2+]i) in human neutrophils through a pertussis toxin-sensitive, extracellular calcium-independent pathway involving the mobilization of calcium from internal stores. A subsequent hepoxilin-induced and extracellular calcium-dependent influx of calcium is observed. In an effort to investigate further the role of these compounds in the human neutrophil, we investigated their potential effects on the action of known agonists such as formyl-methionine-leucine-phenylalanine (fMLP), platelet-activating factor (PAF) and leukotriene B4 (LTB4) on the mobilization of calcium. Hepoxilis dose-dependently inhibited the increases in [Ca2+]i induced by fMLP, PAF and LTB4. The hepoxilin concentration required for inhibition was around 100 ng/ml (3 x 10(-7) M). This concentration of hepoxilin did not cause any measurable change in [Ca2+]i. The extent of inhibition of the agonist-evoked rise in [Ca2+]i by hepoxilins was proportional to the increase in the calcium response evoked by hepoxilin beyond its threshold concentration. Additional experiments were carried out to investigate the mechanism for the hepoxilin effect. Using calcium-free medium and in the presence of sufficient amounts of thapsigargin (200 ng/ml) to maximally block the calcium pump (thereby achieving a constant rate of calcium leakage from stores), hepoxilin A3 increased further this rate of calcium leakage, indicating that hepoxilin acts by rapidly draining calcium from stores. Its potential (additional) thapsigargin-like action in blocking the pump, however, cannot be ruled out by these experiments. These observations suggest that the hepoxilins may serve an important negative regulatory function in the agonist-induced mobilization of calcium in these cells by depleting calcium stores.
A membrane-bound form of glutamate dehydrogenase possesses an ATP-dependent high-affinity microtubule-binding activity
Authors: Rajas F , Rousset B
Published: August 2015

We previously identified a 50 kDa membrane protein which bound to in vitro assembled microtubules [Mithieux and Rousset (1989) J. Biol. Chem. 264, 4664-4668]. This protein exhibited the expected properties for mediating the ATP-dependent association of vesicles with microtubules [Mithieux, Audebet and Rousset (1988) Biochim. Biophys. Acta 969, 121-130]. The 50 kDa membrane protein (MP50), initially extracted in very low amount from isolated pig thyroid lysosomes/endosomes, has now been purified from membrane preparations of crude vesicle fractions from pig liver and brain. MP50 was isolated from detergent-solubilized membrane protein by affinity chromatography on immobilized ATP; 3-5 mg of MP50 was obtained from 100 g of liver tissue. Phase partitioning in Triton X-114 indicated that MP50 is a peripheral membrane protein. Radioiodinated liver MP50 bound to microtubules assembled in vitro. The binding was inhibited by ATP (Ki = 0.76 mM) and displaced by unlabelled liver or brain MP50. Equilibrium binding studies yielded KD values of 1.8 x 10(-7) M. By N-terminal amino acid sequence analysis, MP50 was identified as glutamate dehydrogenase (GDH), by comparison of V8 protease peptide maps of MP50 with purified liver GDH. Liver MP50 exhibited a low GDH activity; 4-5 units/mg compared with 18 and 34 units/mg for purified bovine and rat liver GDH respectively. Bovine and rat liver GDH yielded six spots from pI 5.7 to 7.2 when analysed by two-dimensional electrophoresis; in contrast, MP50 gave one main spot (corresponding to spot 2 of liver GDH) with a pI of approx. 6.5. Soluble liver GDH from commercial sources exhibited a very low or no microtubule-binding activity. In conclusion, we have found a membrane-bound form of GDH capable of specific and nucleotide-sensitive interaction with microtubules. Our data suggest that GDH isoproteins, the number of which has been undervalued up to now, could have cellular functions other than that of an enzyme.
Changes in molar volume and heat capacity of actin upon polymerization
Authors: Quirion F , Gicquaud C
Published: August 2015

We have used densimetry and microcalorimetry to measure the changes in molar volume and heat capacity of the actin molecule during Mg(2+)-induced polymerization. Molar volume is decreased by 720 ml/mol. This result is in contradiction with previous measurements by Ikkai and Ooi [(1966) Science 152, 1756-1757], and by Swezey and Somero [(1985) Biochemistry 24, 852-860]: both of these groups reported increases in actin volume during polymerization, of 391 ml/mol and 63 ml/mol respectively. We also observed a decrease in heat capacity of about 69.5 kJ.K-1.mol-1 during polymerization. This is in agreement with the concept of conformational fluctuation of proteins proposed by Lumry and Gregory [(1989) J.Mol. Liq. 42, 113-144]whereby either ligand binding by a protein or monomer-monomer interaction decreases the protein's conformational flexibility.
Interaction of thrombospondin with platelet glycoproteins GPIa-IIa and GPIIb-IIIa
Authors: Kowalska M A , Tuszynski G P
Published: August 2015

The interaction of human thrombospondin (TSP) with GPIa-IIa and GPIIb-IIIa was studied. The binding for both proteins became time-independent after 60 min. A 7-fold excess concentration of unlabelled GPIa-IIa added either initially, or after time-dependent binding, resulted in a 50% inhibition of GPIa-IIa bound to TSP. GPIa-IIa and GPIIb-IIIa specifically bound TSP since: (a) the binding of GPIIb-IIIa to TSP was dependent on the presence of 1 mM MgCl2 and 1 mM CaCl2, whereas binding of GPIa-IIa was ion-independent. (b) The binding was saturable, with dissociation constants of 0.69 +/- 0.17 microM and 3.77 +/- 1.02 microM for GPIa-IIa and GPIIb-IIIa respectively. (c) GPIIb-IIIa and GPIa-IIa did not significantly bind to BSA. (d) GPIIb-IIIa bound fibrinogen ion-specifically, whereas little or no binding of GPIa-IIa was detectable. (e) Both GPIIb-IIIa and GPIa-IIa bound collagen in an ion-independent manner. (f) GPIIb-IIIa did not compete with GPIa-IIa for binding to TSP. (g) Binding of GPIa-IIa to TSP was inhibited with anti-(GPIa-IIa) (6F1), whereas mouse IgG and anti-(GPIIb-IIIa) (AP-2) had no effect. (h) The interaction of GPIa-IIa with TSP is 5.5-fold more favourable than that of GPIIb-IIIa suggesting that GPIa-IIa may be a preferred binding protein for TSP-mediated platelet adhesion.
Post-translational processing of progastrin: inhibition of cleavage, phosphorylation and sulphation by brefeldin A
Authors: Varro A , Dockray G J
Published: August 2015

The precursor for the acid-stimulating hormone gastrin provides a useful model for studies of post-translational processing because defined sites of cleavage, amidation, sulphation and phosphorylation occur within a dodecapeptide sequence. The factors determining these post-translational processing events are still poorly understood. We have used brefeldin A, which disrupts transport from rough endoplasmic reticulum to the Golgi complex, to examine the mechanisms of cleavage, phosphorylation and sulphation of rat progastrin-derived peptides. Biosynthetic products were detected after immunoprecipitation using antibodies specific for the extreme C-terminus of progastrin, followed by reversed-phase and ion-exchange h.p.l.c. Gastrin cells incorporated [3H]tyrosine, [32P]phosphate and [35S]sulphate into both progastrin and its extreme C-terminal tryptic (nona-) peptide. Ion-exchange chromatography resolved four forms of the C-terminal tryptic fragment of progastrin which differed in whether they were phosphorylated at Ser96, sulphated at Tyr103, both or neither. The specific activity of [3H]tyrosine in the peak that was both phosphorylated and sulphated was higher than in the others. Brefeldin A inhibited the appearance of [3H]tyrosine-labelled C-terminal tryptic fragment but there was an accumulation of labelled progastrin and a peptide corresponding to the C-terminal 46 residues of progastrin. Brefeldin A also inhibited incorporation of 32P and 35S into both progastrin and its C-terminal fragment. Thus phosphorylation of Ser96, sulphation of Tyr103 and cleavage at Arg94-Arg95 depend on passage of newly synthesized progastrin along the secretory pathway; as brefeldin A is thought to act proximal to the trans-Golgi, these processing steps would appear to occur distal to this point. The data also indicate that the stores of unphosphorylated C-terminal tryptic fragment are not available for phosphorylation, implying that this modification occurs proximal to the secretory granule; cleavage is known to occur in the secretory granule which suggests that it occurs after phosphorylation.