bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucose regulates its transport in L8 myocytes by modulating cellular trafficking of the transporter GLUT-1
Authors: Greco-Perotto R , Wertheimer E , Jeanrenaud B , Cerasi E , Sasson S
Published: August 2015

The effect of culture conditions simulating hypo- and hyper-glycaemia on glucose transport and on the subcellular localization of the glucose transporter GLUT-1 was studied in L8 myocytes. Incubation of the cells with 20 mM-glucose for 25 h decreased the rate of 2-deoxy-D-[3H]glucose (dGlc) uptake to 0.106 +/- 0.016 nmol/min per 10(6) cells compared with 0.212 +/- 0.025 in cells maintained at 2 mM-glucose (final glucose concentrations at the end of the incubation period were 16-17 mM and 0.7-1.0 mM respectively). An additional 5 h incubation of these cells with medium containing the opposite glucose concentration (i.e. change from 17 mM to 1 mM and from 1 mM to 17 mM) increased the transport rate to 0.172 +/- 0.033 nmol/min per 10(6) cells in cultures initially conditioned at high glucose, and decreased the transport to 0.125 +/- 0.029 in those conditioned at low glucose. Plasma-membrane- and microsomal-membrane-enriched fractions were prepared from these cells for [3H]cytochalasin B (CB) binding and Western-blot analysis with antibodies against GLUT-1 and GLUT-4. A decrease in glucose concentration increased the number of D-glucose-displaceable CB-binding sites and GLUT-1 protein in the plasma-membrane fraction to the same extent as the increase in dGlc transport. Under downregulatory conditions, the lower dGlc-transport capacity could be accounted for by a decreased number of transporters in the plasma membrane of the cells. No apparent modification of the intrinsic activity of the glucose transporters was observed in up- or down-regulated cells. Under downregulatory conditions, the CB-binding data indicated a large increase in the number of transporters in the intracellular membranes of the myocytes. Western blots of the same membranes also indicated an increase in GLUT-1 content. However, the interaction of the intracellular GLUT-1 protein with the polyclonal antibodies was much weaker than that of the plasma-membrane-associated GLUT-1. The GLUT-4 concentration was too low to permit quantification in membrane fractions. Our findings suggest that autoregulation of glucose transport in L8 myocytes is accompanied by parallel changes in the number of GLUT-1 transporters in the plasma membrane, and that the rate of transporter degradation may be augmented in the upregulated myocytes. These glucose-induced changes are fully reversible.
An easy method for deriving steady-state rate equations
Authors: Waley S G
Published: August 2015

The scope and limitations of a simple and satisfactory method of deducing steady-state rate equations is described. This method (called the Flux Method) consists in writing down the flux in successive steps of the reaction, and calculating the relative concentration of enzyme forms and thence the turnover time. Kinetic mechanisms for linear and branched pathways are used as examples of this method.
Properties of protein kinase C associated with nuclear membranes
Authors: Buchner K , Otto H , Hilbert R , Lindschau C , Haller H , Hucho F
Published: August 2015

To study signal transduction directed towards the cell nucleus and at the nuclear membranes, we investigated the association of protein kinase C (PKC) with nuclear membranes obtained from nuclei isolated from bovine brain. By use of phorbol-ester-binding assays, significant amounts of PKC could be demonstrated in nuclei and nuclear membranes. Nuclear membranes are shown to be able to activate purified PKC. The PKC endogenously present in nuclear membranes appears to be a so-called ‘membrane-inserted’ form: it is permanently active, still binds phorbol ester, but its activity is no longer dependent on Ca2+ and cannot be activated by phorbol ester. On the other hand, this form of PKC can be inhibited by specific PKC inhibitors. By using histone HIIIS and a specific peptide substrate, it could be shown that after extraction with Triton X-100 the PKC can be stimulated by phospholipid again. Immunoblot analysis with isoenzyme-specific antibodies revealed that the alpha- and gamma-isoenzymes, but not the beta-isoenzyme, are associated with membranes derived from brain nuclei.
Involvement of tyrosine kinase and protein kinase C in platelet-activating-factor-induced c-fos gene expression in A-431 cells
Authors: Tripathi Y B , Lim R W , Fernandez-Gallardo S , Kandala J C , Guntaka R V , Shukla S D
Published: August 2015

In A-431 cells, platelet-activating factor (PAF) induces the expression of c-fos and TIS-1 genes in both the absence and the presence of cycloheximide in a structurally specific and receptor-coupled manner. We have now investigated the molecular mechanisms of this response, particularly in relation to the role of protein kinases. Pretreatment of cells with genistein or methyl-2,5-dihydroxycinnamate (tyrosine kinase inhibitors) or staurosporine (a protein kinase C inhibitor) for 20 min abolished the c-fos expression induced by PAF. Interestingly, when genistein was added 90 s after addition of PAF, no inhibition was observed. Similarly, staurosporine did not inhibit c-fos expression when added 8 min after PAF addition to the cells. These inhibitions were dose-dependent (IC50 for staurosporine was 180 nM, and for genistein 50 microM). Simultaneous addition of PAF and phorbol 12-myristate 13-acetate (PMA) did not give a synergistic effect on c-fos expression. Pretreatment of cells with PMA had no effect on [3H]PAF binding, but abolished the PAF-induced gene expression. PAF-stimulated gene expression was desensitized if cells were pretreated with PAF. Interestingly, epidermal growth factor was able to stimulate c-fos expression in PAF-desensitized cells, and thus indicated involvement of distinct mechanisms for the two stimuli. Forskolin, an activator of adenylate cyclase, did not induce c-fos expression and had no effect on the PAF response. Exposure of cells to PAF for as little as 1 min, followed by its removal, was sufficient to activate the gene expression and demonstrated the rapidity and the exquisite nature of the signalling involved in this process. It is concluded that activation of PAF receptor (a proposed G-protein-coupled receptor) causes rapid production of signals which induce the expression of c-fos gene and that this is mediated via tyrosine kinase and protein kinase C.
A mammalian epididymal protein with remarkable sequence similarity to snake venom haemorrhagic peptides
Authors: Perry A C F , Jones R , Barker P J , Hall L
Published: August 2015

Following spermatogenesis in the testis, mammalian spermatozoa pass into the epididymis, where they undergo changes which confer on them forward motility and the ability to recognize and penetrate the egg. Many of these maturation events involve androgen-regulated epididymal proteins which become associated with the sperm membrane, and/or effect changes to integral sperm membrane proteins. Here we report the sequence of an 89 kDa androgen-regulated protein from rat (Rattus norvegicus) and monkey (Macaca fascicularis) epididymis that is synthesized exclusively in the caput region and is localized on the apical surface of its principal epithelial cells. This protein shows remarkable similarity to a variety of proteases and disintegrins found in snake venoms and is similar to, but distinct from, the guinea-pig sperm surface PH-30 alpha/beta complex recently implicated in sperm-egg recognition and fusion.
Okadaic acid produces changes in phosphorylation and translocation of proteins and in intracellular calcium in human neutrophils. Relationship with the activation of the NADPH oxidase by different stimuli
Authors: Garcia R C , Whitaker M , Heyworth P G , Segal A W
Published: August 2015

Okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, profoundly influenced the activity of the NADPH oxidase of human neutrophils. It strongly inhibited stimulation of superoxide generation by phorbol 12-myristate 13-acetate (PMA) and impaired translocation of protein kinase activity and of the two cytosolic components p47-phox and p67-phox to the plasma membrane. The increase in the phosphorylation of the cytochrome b-245 subunits p22-phox and gp91-phox after stimulation was also blocked. Inhibition of activity was associated with a decrease in cytosolic free Ca2+ and was reversed by the Ca2+ ionophore A23187, which also restored protein translocation and phosphorylation of the cytochrome. This effect of A23187 was itself blocked by preincubation with cyclosporin A, suggesting that calcineurin might be involved in the re-activation process. In contrast with PMA, the response to the bacterial peptide fMet-Leu-Phe was greatly prolonged after an initial decrease in the rate of onset of NADPH oxidase activity.
Isolation and characterization of the promoter for the gene coding for the 68 kDa carnitine palmitoyltransferase from the rat
Authors: Brady P S , Park E A , Liu J S , Hanson R W , Brady L J
Published: August 2015

Carnitine palmitoyltransferase (CPT) regulates the flux of long-chain fatty acids into the mitochondria for subsequent beta-oxidation. A 485 bp segment of the promoter for the gene encoding the 68 kDa CPT was isolated from a rat lambda DASH genomic library using the polymerase chain reaction. The promoter contained a consensus binding sequence for CREB (cyclic AMP response element binding protein) at -153 to -166, and for C/EBP alpha (CCAAT/enhancer binding protein) at -115 to -128. DNAase I footprinting using proteins isolated from rat liver nuclei indicated the presence of several regions of nuclear protein binding, most notably at -95 to -130, at -273 to -295, and at a wide region encompassing -395 to -465. DNAase I footprinting studies with purified CREB and C/EBP alpha confirmed that protein binding to DNA occurred at the sites predicted by the consensus sequences. The segment containing 481 bp of 5′ flanking sequence plus 181 bp of untranslated mRNA was ligated to the structural gene for chloramphenicol acetyltransferase (CAT). When this plasmid was transfected into Hep G2 cells, CAT activity was stimulated 7-fold by addition of 1 mM-8-bromo-cyclic AMP (8-Br-cAMP) or co-transfection of the expression vector coding for the catalytic subunit of protein kinase A (PKA). The ability of several known second messengers and transcription factors to stimulate transcription of 68 kDa CPT promoter-CAT reporter was tested in co-transfection experiments. 68 kDa CPT promoter-CAT reporter transcription activity was stimulated 7-fold by addition of 8-Br-cAMP, and this induction was depressed 50% by the addition of phorbol esters. When the 68 kDa CPT promoter-CAT reporter was co-transfected with an expression vector for CREB or C/EBP alpha, transcription was increased 3- and 10-fold respectively. 8-Br-cAMP caused an additional 8-fold induction in the presence of each factor to yield 25- and 80-fold induction respectively. Co-transfection of the expression vector for c-jun also increased the CAT activity driven by the 68 kDa CPT promoter, while co-transfection with the expression vector for c-fos had no effect. When expression vectors for both c-jun and c-fos were co-transfected with the 68 kDa CPT promoter, c-fos depressed the induction seen with c-jun alone.
Stimulation of phosphatidate synthesis in endothelial cells in response to P2-receptor activation. Evidence for phospholipase C and phospholipase D involvement, phosphatidate and diacylglycerol interconversion and the role of protein kinase C
Authors: Purkiss J R , Boarder M R
Published: August 2015

To investigate the stimulation of phosphatidic acid formation in bovine aortic endothelial cells by P2-purinergic agonists, we labelled AG4762 cells with [32P]P1 and stimulated in the presence of butanol. Under these conditions phospholipase D generated [32P]phosphatidylbutanol, whereas the [32P]phosphatidic acid from phospholipase C and diacylglycerol kinase was unchanged. The action of various purinergic agonists on both [32P]phosphatidic acid and [32P]phosphatidylbutanol was consistent with the presence of a P2Y receptor. The stimulation of phospholipase D was dependent on extracellular Ca2+ and was mostly transient (completed within 3 min), whereas the initial stimulation of phospholipase C was independent of extracellular Ca2+, followed by a Ca(2+)-dependent phase. The agonist stimulation of phospholipase D was dependent on protein kinase C, as judged by its sensitivity to the relatively selective protein kinase C inhibitor Ro 31-8220. These results show that purinergic-receptor-mediated stimulation of phosphatidic acid has three phases: an initial Ca(2+)-independent stimulation of phospholipase C, an early but transient Ca(2+)- and protein kinase C-dependent stimulation of phospholipase D, and a sustained Ca(2+)-dependent stimulation of phospholipase C. Using propranolol to inhibit phosphatidate phosphohydrolase, we provide evidence that phosphatidic acid derived from purinergic-receptor-mediated stimulation of the phospholipase C/diacylglycerol kinase route can itself be converted back into diacylglycerol.
Evidence that β-hydroxyacyl-CoA dehydrase purified from rat liver microsomes is of peroxisomal origin
Authors: Cook L , Nagi M N , Suneja S K , Hand A R , Cinti D L
Published: August 2015

The present study provides strong evidence that the previously isolated hepatic microsomal beta-hydroxyacyl-CoA dehydrase (EC 4.2.1.17), believed to be a component of the fatty acid chain-elongation system, is derived, not from the endoplasmic reticulum, but rather from the peroxisomes. The isolated dehydrase was purified over 3000-fold and showed optimal enzymic activity toward beta-hydroxyacyl-CoAs or trans-2-enoyl-CoAs with carbon chain lengths of 8-10. The purified preparation (VDH) displayed a pH optimum at 7.5 with beta-hydroxydecanoyl-CoA, and at 6.0 with beta-hydroxystearoyl-CoA. Competitive-inhibition studies suggested that VDH contained dehydrase isoforms, and SDS/PAGE showed three major bands at 47, 71 and 78 kDa, all of which reacted to antibody raised to the purified preparation. Immunocytochemical studies with anti-rabbit IgG to VDH unequivocally demonstrated gold particles randomly distributed throughout the peroxisomal matrix of liver sections from both untreated and di-(2-ethylhexyl) phthalate-treated rats. No labelling was associated with endoplasmic reticulum or with the microsomal fraction. Substrate-specificity studies and the use of antibodies to VDH and to the peroxisomal trifunctional protein indicated that VDH and the latter are separate enzymes. On the other hand, the VDH possesses biochemical characteristics similar to those of the D-beta-hydroxyacyl-CoA dehydrase recently isolated from rat liver peroxisomes [Li, Smeland & Schulz (1990) J. Biol. Chem. 265, 13629-13634; Hiltunen, Palosaari & Kunau (1989) J. Biol. Chem. 264, 13536-13540]. Neither enzyme utilizes crotonoyl-CoA or cis-2-enoyl-CoA as substrates, but both enzymes convert trans-2-enoyl substrates into the D-isomer only. In addition, the VDH also contained beta-oxoacyl-CoA reductase (beta-hydroxyacyl-CoA dehydrogenase) activity, which co-purified with the dehydrase.
Characterization of an ATP-driven H+ pump in human placental brush-border membrane vesicles
Authors: Simon B J , Kulanthaivel P , Burckhardt G , Ramamoorthy S , Leibach F H , Ganapathy V
Published: August 2015

The presence of an ATP-driven H+ pump as measured by H+ uptake upon addition of ATP was not demonstrable in human placental brush-border membrane vesicles when used in their native form, owing to their right-side-out orientation. However, the presence of the H+ pump in these membranes became evident when the membrane vesicles were transiently exposed to 1% cholate, with subsequent removal of the detergent to re-form the vesicles. Apparently, cholate pretreatment reoriented the H+ pump from an inward-facing configuration to outward-facing. Consequently, H+ uptake in response to externally added ATP was easily demonstrable in these cholate-pretreated vesicles by using the delta pH indicator Acridine Orange. In addition, bafilomycin A1-sensitive ATPase activity was measurable in cholate-pretreated vesicles, but not in native intact vesicles, indicating reorientation of the H+ pump. The reoriented H+ pump was electrogenic because H+ uptake was stimulated by an inside-negative anion-diffusion potential or when the vesicles were voltage-clamped. ATP supported H+ uptake with an apparent Km of 260 microM. ITP and GTP supported the pump activity partially, whereas CTP and UTP did not. Mg2+ and Mn2+ were the most preferred bivalent cations. Co2+ and Zn2+ showed partial activity, whereas Ca2+ and Ba2+ showed little or no activity. The pump was inhibited by nanomolar concentrations of bafilomycin A1 and micromolar concentrations of N-ethylmaleimide, p-chloromercuribenzenesulphonate, NN-dicyclohexylcarbodi-imide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but was relatively insensitive to oligomycin, vanadate and NaN3. The inhibition by N-ethylmaleimide was protectable by ATP. It is concluded that human placental brush-border membranes possess an ATP-driven H+ pump and that, on the basis of its characteristics, it belongs to the class of vacuolar (V-type) H+ pumps.
Structural features of the low-molecular-mass human salivary mucin
Authors: Reddy M S , Bobek L A , Haraszthy G G , Biesbrock A R , Levine M J
Published: August 2015

The low-molecular-mass human salivary mucin has at least two isoforms, MG2a and MG2b, that differ primarily in their sialic acid and fucose content. In this study, we characterize further these isoforms, particularly their peptide moieties. Trypsin digests of MG2a and MG2b yielded high- and low-molecular-mass glycopeptides following gel filtration on Sephacryl S-300. The larger glycopeptides from MG2a and MG2b had similar amino acid compositions and identical N-terminal sequences, suggesting common structural features between their peptides. An oligonucleotide probe generated from the amino acid sequence of the smaller glycopeptide from MG2a was employed in Northern-blot analysis. This probe specifically hybridized to two mRNA species from human submandibular and sublingual glands. A cDNA clone selected from a human submandibular gland cDNA expression library with antibody generated against deglycosylated MG2a also hybridized to these two mRNA species. In both cases, the larger mRNA was polydisperse, and the hybridization signal was more intense in the sublingual gland. In addition, the N-terminal amino acid sequence of the larger glycopeptide was found to be part of one of the selected MG2 cDNA clones.
Investigation of the first step of biotin biosynthesis in Bacillus sphaericus. Purification and characterization of the pimeloyl-CoA synthase, and uptake of pimelate
Authors: Ploux O , Soularue P , Marquet A , Gloeckler R , Lemoine Y
Published: August 2015

The pimeloyl-CoA synthase from Bacillus sphaericus has been purified to homogeneity from an overproducing strain of Escherichia coli. The purification yielded milligram quantities of the synthase with a specific activity of 1 unit/mg of protein. Analysis of the products showed that this enzyme catalysed the transformation of pimelate into pimeloyl-CoA with concomitant hydrolysis of ATP to AMP. Using a continuous spectrophotometric assay, we have examined the catalytic properties of the pure enzyme. The pH profile under Vmax. conditions showed a maximum around 8.5. Apparent Km values for pimelate, CoASH, ATP. Mg2- and Mg2+ were respectively 145 microM, 33 microM, 170 microM and 2.3 mM. The enzyme was inhibited by Mg2+ above 10 mM. This acid-CoA ligase exhibited a very sharp substrate specificity, e.g. neither GTP nor pimelate analogues (di- or mono-carboxylic acids) were processed. The bivalent metal ion requirement was also investigated: Mn2+ (73%) and Co2+ (32%) but not Ca2+ could replace Mg2+. The enzyme was inhibited by metal chelators such as 1,10-phenanthroline and EDTA. The synthase was a homodimer with a 28,000-M(r) subunit. N-Terminal sequencing definitely proved that this enzyme was encoded by the bioW gene. A careful study of pimelate uptake by B. sphaericus, E. coli and Pseudomonas dentrificans showed that this metabolite crossed the membrane of these microorganisms by passive diffusion, ruling out the involvement of the bioX gene product as pimelate carrier.
Bicarbonate-dependent ATP cleavage catalysed by pyruvate carboxylase in the absence of pyruvate
Authors: Attwood P V , Graneri B D L A
Published: August 2015

Preparations of pyruvate carboxylase catalyse the cleavage of MgATP in the absence of pyruvate and acetyl-CoA. The rate of this cleavage is higher in the presence of HCO3- than in its absence. Incubation of the enzyme preparations with an excess of the pyruvate carboxylase inhibitor, avidin, completely abolishes the pyruvate carboxylating activity of the enzyme preparations but only abolishes the HCO3(-)-dependent MgATP cleaving activity, with no effect on the HCO3(-)-independent ATPase activity. The HCO3(-)-dependent MgATP cleavage is also sensitive to inhibition by a pyruvate carboxylase inhibitor, oxamate, and the dependence of the reaction on the free Mg2+ concentration is similar to that of the pyruvate-carboxylation reaction, whereas the HCO3(-)-independent MgATP cleavage is not dependent on the concentration of free Mg2+ in the range tested. This indicates that MgATP cleavage by pyruvate carboxylase is entirely dependent on the presence of HCO3- and that there may be a low level of ATPase contamination in the enzyme preparations. In addition, inhibition of the HCO3(-)-dependent MgATP cleavage by both avidin and oxamate indicate that although biotin does not directly participate in the reaction, its presence is required in that part of the active site of the enzyme. The rate of HCO3(-)-dependent MgATP cleavage is about 0.07% of that of the full pyruvate carboxylation reaction under similar conditions with saturating substrates. The reaction mechanism is sequential with respect to MgATP and HCO3- addition and Mg2+ adds at equilibrium before MgATP. Acetyl-CoA stimulates the HCO3(-)-dependent MgATP cleavage at low MgATP concentrations, with the stimulation being greater at low Mg2+ concentrations. At high levels of MgATP in the presence of acetyl-CoA, substrate inhibition is evident and is more pronounced at increasing concentrations of Mg2+. This inhibition appears to be, at least in part, caused by inhibition of decarboxylation of the enzyme-carboxybiotin complex by the binding to this complex of Mg2+ and MgATP, which probably act to reduce the rate of movement of carboxybiotin from the site of the MgATP cleavage reaction to that of the pyruvate carboxylation reaction where it is unstable and decarboxylates.
Inositol 1,3,4,5-tetrakisphosphate binding sites in neuronal and non-neuronal tissues. Properties, comparisons and potential physiological significance
Authors: Cullen P J , Irvine R F
Published: August 2015

1. Ins(1,3,4,5)P4 binding sites were studied in cerebellar and hepatic microsomes from rat, and in bovine adrenal-cortical microsomes. 2. At pH 7.0, all three tissues showed specific binding, with Ins(1,3,4,5)P4 being the most potent competing ligand of those tested [which included Ins(1,4,5)P3, Ins(1,3,4,5,6)P5 and InsP6] and Scatchard analysis suggested two sites; a site with high affinity and high specificity [Kd (1-6) x 10(-9) M] and a site with low affinity and low specificity [Kd (2-6) x 10(-7) M]. 3. At pH 5.5, cerebellar and bovine adrenal microsomes showed similar binding properties: two affinities with a similar specificity for Ins(1,3,4,5)P4 as at pH 7.0. 4. However, when assayed in a low-ionic strength acetate-based buffer at pH 5.0, cerebellar microsomes retain specific Ins(1,3,4,5)P4 binding sites, whereas bovine adrenal and hepatic microsomal binding sites lose much of their specificity, as InsP6 and Ins(1,3,4,5,6)P5 are equally as potent as Ins(1,3,4,5)P4. 5. Pi (25 mM), which is frequently included in Ins(1,3,4,5)P4 binding assays, had a small inhibitory effect on binding of cerebellar and adrenal microsomes at pH 5.5, but a large effect at pH 7.0, so that a considerable decrease occurs in the amount of specific binding at pH 5.5 compared with that at pH 7.0, if Pi is omitted from the binding assay. 6. Cerebellar and adrenal microsomes were used in a ligand-displacement mass assay (conducted under near-physiological conditions, at pH 7.0) on extracts of cerebral-cortex slices stimulated with agonists, and both preparations faithfully detected the increases in Ins(1,3,4,5)P4 that occurred, implying that Ins(1,3,4,5)P4 is the principal ligand on these binding sites in intact cells. 7. Apparent contradictions in the literature with regard to Ins(1,3,4,5)P4 binding sites in neuronal and peripheral tissues can be largely accounted for by the data, and the properties of the binding sites detected at physiological pH are consistent with the possibility that they are putative receptors for the proposed second-messenger role for Ins(1,3,4,5)P4.
Translation rates of isolated liver mitochondria under conditions of hepatic mitochondrial proliferation
Authors: Brass E P
Published: August 2015

The hepatic mitochondrial content is increased in rats by treatment with the hypolipidaemic drug clofibrate and by administration of the cobalamin analogue hydroxycobalamin[c-lactam] (HCCL), an inhibitor of hepatic L-methylmalonyl-CoA mutase activity. As a first step in defining the mechanisms regulating liver mitochondrial contents in these models, the current studies were designed to test the hypothesis that hepatic mitochondrial proliferation is associated with enhanced translation rates of mitochondrial DNA gene products. Incorporation of [35S]methionine and [3H]leucine into protein was quantified in mitochondria isolated from control, clofibrate- and HCCL-treated rats. Use of multiple amino acid substrate concentrations permitted the maximal rate of translation (Vmax.) to be determined independent of endogenous amino acid concentrations. The Vmax. for methionine incorporation was not different in the models evaluated (0.062, 0.057 and 0.061 pmol/min per mg of mitochondrial protein in control, clofibrate- and HCCL-treated rats respectively). Similar results were obtained for leucine incorporation when absolute fractional radiolabel incorporation rates were analysed and when conventional Lineweaver-Burk analysis was employed. These results demonstrate no change in the intrinsic capacity of mitochondrial translation in these two models of hepatic mitochondrial proliferation.
Isolation and partial characterization of heparan sulphate proteoglycans from human hepatic amyloid
Authors: Magnus J H , Stenstad T , Husby G , Kolset S O
Published: August 2015

Proteoglycans were isolated from human amyloidotic liver by extraction with guanidine, followed by trichloroacetic acid precipitation, DEAE-Sephacel ion-exchange chromatography, and Sepharose CL-6B gel chromatography. A significant portion of the material was found to be free chondroitin/dermatan sulphate chains (30%), whereas the predominant part was heparan sulphate proteoglycan (HSPG) (70%). The approx. molecular mass of the HSPG was 200 kDa, as measured by gel electrophoresis and gel chromatography. The molecular mass of the core protein was shown to be 60 kDa by SDS/PAGE following de-aminative cleavage of the heparan sulphate chains. The heparan sulphate chains were liberated from the core protein by alkali treatment and found to have a molecular mass of approx. 35 kDa by Sepharose CL-6B gel chromatography. The core protein was shown, by immunoblotting, to react with a monoclonal antibody against bovine basement membrane HSPG. The presence of HSPG in amyloid deposits was further confirmed by immunohistochemistry on tissue sections from amyloidotic liver using the same antibody.
Gsα is a substrate for mono(ADP-ribosyl)transferase of NG108-15 cells. ADP-ribosylation regulates Gsα activity and abundance
Authors: Donnelly L E , Boyd R S , MacDermot J
Published: August 2015

NG108-15 neuroblastoma x glioma somatic hybrid cells were permeabilized in the presence of [32P]NAD+ and then cultured for 18 h. Resolution of the cell proteins on polyacrylamide gels revealed [32P]ADP-ribosylation of five major protein species with molecular mass values of 52 kDa, 44 kDa, 35 kDa, 30 kDa and 25 kDa. A similar pattern of labelling was also seen when NG108-15 cell membranes were incubated with [32P]NAD+ and hydrolysis of the product revealed mono(ADP-ribosyl)ation. Immunoprecipitation of these products with anti-Gs alpha antiserum revealed a single band identical to cholera toxin substrate. Culture of [32P]NAD(+)-loaded cells for 18 h in the presence of 50 mM-nicotinamide inhibited the eukaryotic mono(ADP-ribosyl)transferase activity. Inhibition of the eukaryotic enzyme was also accompanied by an increase in the abundance of Gs alpha, whether measured by Western blotting with anti-Gs alpha antibody (two separate antisera) or by cholera toxin-dependent [32P]ADP-ribosylation. There was no accompanying change in the abundance of G beta. The increase in Gs alpha abundance in nicotinamide-treated NG108-15 cells was accompanied by a 2-fold increase in basal adenylate cyclase activity (measured in the presence of GTP), and by a smaller but significant increase in iloprost-dependent activation of adenylate cyclase. Receptor number or affinity was not affected by nicotinamide, since this treatment did not alter the binding parameters of [3H]iloprost to NG108-15 cell membranes. Short-term exposure of cells to nicotinamide for 1 h revealed no significant difference in either basal or agonist-stimulated adenylate cyclase activity. These results reveal that mono(ADP-ribosyl)ation of Gs alpha by eukaryotic ADP-ribosyltransferase modifies the abundance and activity of Gs alpha in NG108-15 cells, and hence may play a role in the hormonal regulation of cell function.
Regulation of pyruvate dehydrogenase kinase activity from pig kidney cortex
Authors: Pawelczyk T , Olson M S
Published: August 2015

The activity of pyruvate dehydrogenase (PDH) kinase in the purified PDH complex from pig kidney is sensitive to changes in ionic strength. The enzyme has optimum activity within a small range of ionic strength (0.03-0.05 M). An increase in ionic strength from 0.04 M to 0.2 M lowers the activity of PDH kinase by 32% and decreases the Km for ATP from 25 microM to 10 microM. At constant ionic strength (0.15 M) the enzyme has optimum activity over a broad pH range (7.2-8.0). The PDH kinase is stimulated 2.2-fold by 20 mM-K+, whereas Na+ even at high concentration (80 mM) has no effect on the enzyme activity. The stimulation of PDH kinase by K+ is not dependent on pH and ionic strength. PDH kinase is inhibited by HPO4(2-) in the presence of K+, whereas HPO4(2-) has no effect on the activity of this enzyme in the absence of K+. HPO4(2-) at concentrations of 2 and 10 mM inhibits PDH kinase by 28% and 55% respectively. The magnitude of this inhibition is not dependent on the ATP/ADP ratio. Inhibition by HPO4(2-) in the concentration range 0-10 mM is non-competitive with respect to ATP, and becomes mixed-type at concentrations over 10 mM. The Ki for HPO4(2-) is 10 mM. When HPO4(2-) is replaced by SO4(2-), the same effects on the activity of PDH kinase are observed. PDH kinase is also inhibited by Cl-. In the presence of 80 mM-Cl- the PDH kinase is inhibited by 40%. The inhibition by Cl- is not dependent on K+. In conclusion, we postulate that changes in phosphate concentrations may play a significant role in the regulation of PDH kinase activity in vivo.
Characterization of a phenobarbital-inducible cytochrome P-450, NADPH-cytochrome P-450 reductase and reconstituted cytochrome P-450 mono-oxygenase system from rat brain. Evidence for constitutive presence in rat and human brain
Authors: Anandatheerthavarada H K , Boyd M R , Ravindranath V
Published: August 2015

Cytochrome P-450 was purified to apparent homogeneity from the brain microsomes of phenobarbital-treated rats. The specific content of the purified P-450 was 12.7 nmol/mg of protein. NADPH-cytochrome P-450 reductase (reductase) was also purified to apparent homogeneity from brain microsomes. The specific content was 34.7 mumol of cytochrome c reduced/min per mg of protein. The reduced carbon monoxide spectrum of purified P-450 exhibited a peak at 450 nm. Both the P-450 and the reductase moved as single bands on SDS/PAGE. The molecular masses of the purified P-450 and the reductase were determined to be 53.3 and 72.0 kDa respectively. The purified brain P-450 cross-reacted with antibodies to rat liver P-450IIB1/IIB2 when examined by Western immunoblotting, but no immunological similarity was observed with rat liver P-450IA1/IA2 or P-450IIE1. Purified rat brain reductase cross-reacted with antibodies to rat liver reductase. Further, immunoblot experiments with untreated rat and human brain microsomes using antisera to the purified rat brain P-450 and reductase indicated that these forms of P-450 and NADPH-cytochrome P-450 reductase exist constitutively in rat and human brain. Purified rat brain P-450 was reconstituted with purified NADPH-cytochrome P-450 reductase, deoxycholate and dilauroyl glyceryl 3-phosphocholine. NADPH-dependent N-demethylation of aminopyrine and morphine was observed in the reconstituted system. The catalytic-centre activities were 80.25 and 38.2 nmol of formaldehyde formed/min per nmol of P-450 respectively. The reconstituted system had a comparatively lower catalytic-centre activity for 7-ethoxycoumarin O-de-ethylase (10.5 nmol of product formed/min per nmol of P-450).
Caeruloplasmin biosynthesis by the human uterus
Authors: Schilsky M L , Stockert R J , Pollard J W
Published: August 2015

The blue copper protein caeruloplasmin is synthesized mainly by hepatocytes. An alternative transcript for caeruloplasmin produced in certain extrahepatic tissues, CP-1, contains an additional 12 nucleotides encoding 4 amino acids not present in the hepatic transcript, CP-2 [Yang, Friedrichs, Cupples, Bonifacio, S Sanford, Horton & Bowman (1990) J. Biol. Chem. 265, 10780-10785]. We have demonstrated transcription of caeruloplasmin mRNA by a well-differentiated human uterine epithelial adenocarcinoma cell line, Ishikawa, and by human uterine endometrium and purified endometrial glands. Identical CP-2 nucleotide sequences were obtained for partial caeruloplasmin transcripts from human liver and Ishikawa cells, indicating that CP-2 transcripts are produced by uterine epithelial lining cells. The synthesis of caeruloplasmin protein was demonstrated for Ishikawa cells and another uterine adenocarcinoma cell line, ECC1. Peptide-mapping analysis indicated that caeruloplasmin secreted by Ishikawa cells was structurally identical with the protein synthesized by the human hepatoblastoma cell line HepG2. The secretion of a 135,000-M(r) caeruloplasmin by Ishikawa and ECC1 cells, comparable with that of the human hepatoblastoma cell line, HepG2, indicated similar processing of uterine and hepatic caeruloplasmin. Incorporation of 67Cu into caeruloplasmin was demonstrated for Ishikawa and ECC1 cells, suggesting that the human uterus produces a bioactive form of caeruloplasmin and possesses the necessary metal transporters and intracellular machinery for copper incorporation into this protein.
Comparative behaviour of calpain and cathepsin B toward peptidyl acyloxymethyl ketones, sulphonium methyl ketones and other potential inhibitors of cysteine proteinases
Authors: Pliura D H , Bonaventura B J , Smith R A , Coles P J , Krantz A
Published: August 2015

Peptidyl acyloxymethyl ketones, previously established as potent inactivators of the lysosomal cysteine proteinase cathepsin B, were evaluated against smooth-muscle calpain, a member of the family of Ca(2+)-dependent cysteine proteinases. Only modest rates of time-dependent inhibition could be achieved, even with peptidyl affinity groups optimized for calpain and linked to a carboxylate leaving group of very low pKa [2,6-(CF3)2PhCOO-, pKa 0.58]. Selective inactivation of cathespin B versus calpain was consistently observed with this type of inhibitor. Examination of other potential inhibitors revealed a rank order of potency against calpain to be: peptidyl sulphonium methyl ketones > fluoromethyl ketones, diazomethyl ketones >> acyloxymethyl ketones, an order which differs sharply from that found for cathespin B.
Expression of active octameric chicken cardiac mitochondrial creatine kinase in Escherichia coli
Authors: Furter R , Kaldis P , Furter-Graves E M , Schnyder T , Eppenberger H M , Wallimann T
Published: August 2015

Sarcomeric mitochondrial creatine kinase (Mib-CK) of chicken was expressed in Escherichia coli as a soluble enzyme by using an inducible phage-T7 promoter. Up to one third of the protein in E. coli extracts consisted of soluble recombinant Mib-CK in an enzymically active form. Approx. 20 mg of nearly-homogenous Mib-CK was isolated in a two-step isolation procedure starting with 1 litre of isopropyl beta-D-thiogalactopyranoside-induced E. coli culture, whereas previous attempts to express other CK genes in E. coli have resulted in 20-fold lower yields and inclusion-body formation. Selection of the Mib-CK expression plasmid on media containing kanamycin rather than ampicillin extended the time period of maximal Mib-CK expression. Recombinant Mib-CK displayed an identical N-terminal amino acid sequence, identical Km for phosphocreatine and Vmax. values, the same electrophoretic behaviour and the same immunological cross-reactivity as the native enzyme isolated from chicken heart mitochondria. The recombinant Mib-CK had the same molecular mass as native chicken Mib-CK in m.s. analysis, indicating that post-translational modification of the enzyme in chicken tissue does not occur. As judged by gel-permeation chromatography and electron microscopy, recombinant enzyme formed predominantly octameric oligomers with the same overall structure as the chicken heart enzyme. Furthermore, the enzymes isolated from both sources formed protein crystals of space group P42(1)2, when grown in the absence of ATP, with one Mi-CK octamer per asymmetric unit. The indistinguishable X-ray-diffraction patterns indicate identical structures for the native and recombinant proteins.
Molecular cloning and characterization of the promoter region of the calcineurin Aα gene
Authors: Chang C D , Takeda T , Mukai H , Shuntoh H , Kuno T , Tanaka C
Published: August 2015

The 5′-flanking region of the calcineurin A alpha gene was isolated from a rat genomic library. It lacked TATA and CAAT boxes but contained G+C-rich regions, and was demonstrated to function as a strong promoter in neuronal cell lines (NG108-15 mouse neuroblastoma x rat glioma hybrid cells or N1E115 mouse neuroblastoma cells), but not in nonneuronal cell lines (C6 rat glioma or L-M mouse fibroblastoid cells) in a transient chloramphenicol acetyltransferase expression assay. Deletion analysis of the 5′-flanking region revealed that the core promoter region, as well as the sequence critical for cell-type-specific-promoter function, reside within the fragment -107 to +157 with respect to the major transcription initiation site.
Basal and phorbol-ester-stimulated turnover of phosphatidylcholine in HeLa cells involve different pathways
Authors: Hii C S T , Edwards Y S , Murray A W
Published: August 2015

HeLa cells prelabelled with [3H]lysoplatelet-activating factor (lyso-PAF) accumulated phosphatidic acid (PtdOH) when incubated in the presence of either propranolol (an inhibitor of PtdOH phosphohydrolase) or phorbol ester. In the presence of ethanol, phorbol ester but not propranolol stimulated the accumulation of phosphatidylethanol, an index of phospholipase D activity. Incubation of cells with [3H]lyso-PAF led to a rapid accumulation of label in diacylglycerol (DG) followed by a delayed accumulation in PtdOH. It is concluded that propranolol-induced PtdOH accumulation is derived from DG by DG kinase and does not involve phospholipase D.
Regulation of 5-lipoxygenase and 5-lipoxygenase-activating protein expression in HL-60 cells
Authors: Bennett C F , Chiang M Y , Monia B P , Crooke S T
Published: August 2015

5-Lipoxygenase performs the first two enzymic reactions in the biosynthetic pathway for the leukotrienes. We have utilized HL-60 cells to study the mechanisms regulating expression of 5-lipoxygenase and the recently described 18 kDa membrane protein, 5-lipoxygenase-activating protein (FLAP). Differentiation of HL-60 cells into granulocyte-like cells with dimethyl sulphoxide (Me2SO), retinoic acid or dibutyryl cyclic AMP (Bt2-cAMP) resulted in a 2-3-fold increase in 5-lipoxygenase enzyme activity and a 4-fold increase in leukotriene B4 synthesis. Differentiation of HL-60 cells into monocyte-like cells with 1,25-dihydroxyvitamin D3 induced 5-lipoxygenase activity 5-fold, but leukotriene B4 production was only increased 2-fold. Differentiation of HL-60 cells with phorbol 12-myristate 13-acetate (PMA) into macrophage-like cells failed to induce 5-lipoxygenase or leukotriene B4 production. Examination of the levels of the transcript encoding 5-lipoxygenase and FLAP demonstrated that differentiation of HL-60 cells into granulocytes resulted in a co-ordinate induction of 5-lipoxygenase and FLAP mRNA. In contrast, differentiation of HL-60 cells into monocytes resulted in discordant regulation of 5-lipoxygenase and FLAP mRNA. Treatment with 1,25-dihydroxyvitamin D3 resulted in a 6-fold increase in 5-lipoxygenase mRNA and a 1.3-fold increase in FLAP mRNA, while treatment with phorbol ester did not induce 5-lipoxygenase mRNA but did increase FLAP mRNA 2-fold. The transcriptional rate of the 5-lipoxygenase and FLAP genes did not change upon Me2SO or 1,25-dihydroxyvitamin D3 treatment, suggesting that the increase of the mRNA coding for these proteins was not due to transcriptional activation of their respective genes. The mRNA half-life for 5-lipoxygenase did not change significantly upon treatment with Me2SO or 1,25-dihydroxyvitamin D3. The FLAP mRNA half-life increased from approx. 3.5 h to 4.5 h in cells treated with either Me2SO or 1,25-dihydroxyvitamin D3. These data suggest that expression of 5-lipoxygenase and FLAP is controlled by a post-transcriptional event other than stabilization of the mRNA.
Cloning of α2 chain of type VI collagen and expression during mouse development
Authors: Ibrahimi A , Bertrand B , Bardon S , Amri E Z , Grimaldi P , Ailhaud G , Dani C
Published: August 2015

We have previously described the molecular cloning of a cDNA probe which detects a 6 kb mRNA termed pOb24. pOb24 mRNA appeared to be a marker of the preadipose state both in vitro and in vivo. A pOb24 genomic fragment was isolated and used to screen cDNA libraries in order to isolate the full-length pOb24 cDNA and to identify the corresponding protein. The screening yielded a new cDNA clone which detected a 3.7 kb mRNA species in addition to the 6 kb mRNA species. Sequences at the 3′ end of the 6 kb and 3.7 kb mRNAs indicate that both mRNAs are generated from the same gene through the use of two different polyadenylation sites. The protein encoded by the 3.7 kb mRNA appeared to be homologous to the human alpha 2 chain of type VI collagen (A2COL6). The expression of the A2COL6 gene was not confined to adipose tissue; mRNA species can be detected in ovaries, adrenal glands and lungs but not in liver and skeletal muscle. The expression appeared specific for initial phase(s) of cell differentiation since it is parallel to that of the MyoD1 gene during muscle embryogenesis in vivo. In the myogenic C2C12 cell line, the A2COL6 gene exhibited the same regulation as MyoD1 and myogenin genes. These results indicate that A2COL6 gene expression is a marker of the preadipose state, but may also be a marker of other differentiation programmes such as that of muscle.
Focal-adhesion components are enriched in ventral membranes isolated from transformed keratinocytes in culture
Authors: Gates R E , Hanks S K , King L E
Published: August 2015

The lack of procedures for isolating or reconstituting intact focal adhesions has hindered studies of how focal adhesions are organized and of how their assembly/disassembly is controlled. A method for isolating large quantities of the ventral portion of plasma membranes from transformed keratinocytes (A-431 cells) in culture is described. Plasma membranes are stabilized using Zn2+ and the ventral portion isolated attached to the culture substratum after the body of the cell has been sheared away. Compared with complete plasma membranes isolated from cells scraped from the dish, these ventral-membrane preparations are enriched 18.5-fold and 5.1-fold in the focal-adhesion components talin and vinculin respectively. While the epidermal-growth-factor receptor-kinase is less abundant in preparations of ventral membranes, a recently described tyrosine kinase which localizes to focal adhesions in mouse fibroblasts is enriched 19.9-fold. Extracellular matrix components, as well as their integrin receptors, are also enriched in these preparations of the ventral portion of plasma membranes compared with preparations of complete plasma membranes.
Substrate selectivities differ for hepatic mitochondrial and peroxisomal β-oxidation in an Antarctic fish, Notothenia gibberifrons
Authors: Crockett E L , Sidell B D
Published: August 2015

Hepatic mitochondrial and peroxisomal beta-oxidation were examined in an Antarctic marine teleost, Notothenia gibberifrons. Enzymic profiles and rates of beta-oxidation by intact organelles were determined by using a range of fatty acyl-CoA substrates to evaluate substrate preferences. Partitioning of beta-oxidation between organelles was estimated. Substrate selectivities are broader for peroxisomal beta-oxidation than for mitochondrial beta-oxidation. Mitochondria show marked preference for the oxidation of a monounsaturated substrate, palmitoleoyl-CoA (C16:1), and two polyunsaturates, eicosapentaenoyl-CoA (C20:5) and docosahexaenoyl-CoA (C22:6). Carnitine palmitoyltransferase activities with palmitoleoyl-CoA (C16:1) are 2.4-fold higher than activities with palmitoyl-CoA (C16:0). Most polyunsaturated acyl-CoA esters measured appear to inhibit by over 40% the oxidation of palmitoyl-CoA by peroxisomes. Our findings suggest that the polyunsaturates, eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), found in high concentrations in Antarctic fishes [Lund and Sidell (1992) Mar. Biol. 112, 377-382], are utilized as fuels to support aerobic energy metabolism. Metabolic capacities of rate-limiting enzymes and beta-oxidation rates by intact organelles indicate that up to 30% of hepatic beta-oxidation in N. gibberifrons can be initiated by the peroxisomal pathway.
The linkage between binding of the C-terminal domain of hirudin and amidase activity in human α-thrombin
Authors: de Cristofaro R , Rocca B , Bizzi B , Landolfi R
Published: August 2015

A method derived from the analysis of viscosity effects on the hydrolysis of the amide substrates D-phenylalanylpipecolyl-arginine-p-nitroaniline, tosylglycylprolylarginine-p-nitroanaline and cyclohexylglycylalanylarginine-p-nitroalanine by human alpha-thrombin was developed to dissect the Michaelis-Menten parameters Km and kcat into the individual rate constants of the binding, acylation and deacylation reactions. This method was used to analyse the effect of the C-terminal hirudin (residues 54-65) [hir-(54-65)] domain on the binding and hydrolysis of the three substrates. The results showed that the C-terminal hir-(54-65) fragment affects only the acylation rate, which is increased approx. 1.2-fold for all the substrates. Analysis of the dependence of acylation rate constants on hirudin-fragment concentration, allowed the determination of the equilibrium binding constant of C-terminal hir-(54-65) (Kd approximately 0.7 microM). In addition this peptide was found to competitively inhibit thrombin-fibrinogen interaction with a Ki which is in excellent agreement with the equilibrium constant derived from viscosity experiments. These results demonstrate that binding of hir-(54-65) to the fibrinogen recognition site of thrombin does not affect the equilibrium binding of amide substrates, but induces only a small increase in the acylation rate of the hydrolysis reaction.