bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and biochemical characterization of non-myristoylated recombinant pp60c-src kinase
Authors: Lydon N B , Gay B , Mett H , Murray B , Liebetanz J , Gutzwiller A , Piwnica-Worms H , Roberts T M , McGlynn E
Published: August 2015

To obtain sufficient material for the biochemical and biophysical study of pp60c-src, we have utilized a recombinant pp60c-src baculovirus lacking the myristoylation site at codon 2. On infection of Sf9 cells, this virus produced large amounts of soluble non-myristoylated pp60c-src. The use of non-myristoylated pp60c-src (1) increases production of pp60c-src compared with the wild-type protein, (2) facilitates purification, (3) yields a stable product and (4) allows biochemical studies in the absence of detergents. Up to 20 mg of pp60c-src of greater than 95% purity has been purified from 6 litres of Sf9 cells grown in a bioreactor. One major and multiple minor forms of pp60c-src were separated by Mono Q f.p.l.c. Isoelectric focusing of purified pp60c-src species revealed heterogeneity, some of which could be attributed to differences in the tyrosine phosphorylation state of the enzyme. Kinetic analysis of non-myristoylated pp60c-src kinase in the presence of Mg2+ gave Km values for angiotensin II and ATP of 2 mM and 30 microM respectively and a Vmax. of 620 nmol/min per mg. The kinetic constants and metal ion preferences of a number of copolymers and peptide substrates have been compared. Polylysine and poly(GLAT), which was not phosphorylated by the pp60c-src kinase, dramatically activated autophosphorylation of Tyr-416, suggesting a conformation modulation of pp60c-src by charged polymers. This finding implies that Tyr-527 dephosphorylation is not sufficient for full activation of pp60c-src in vitro.
Stimulation of neutrophil adhesion by antibodies recognizing CD15 (LeX) and CD15-expressing carcinoembryonic antigen-related glycoprotein NCA-160
Authors: Stocks S C , Kerr M A
Published: August 2015

The carbohydrate antigen, CD15 (Le(X)), and its sialylated derivative have recently been shown to be involved in the binding of neutrophils to the endothelial lectins, E-selectin and P-selectin. Neutrophil NCA-160, a carcinoembryonic antigen (CEA)-related glycoprotein, is the major carrier of CD15, which is also expressed on the common beta 2 chain of leucocyte integrins. Rabbit IgG antibodies directed against CEA, which cross-react with neutrophil NCAs, increase the adhesion of neutrophils to plastic. This effect is also observed with F(ab')2 and Fab antibody fragments and a monoclonal antibody (mAb) recognizing the same antigen. Anti-CD15 mAbs inhibit adhesion at higher concentrations, but augment adhesion at lower concentrations. Anti-CEA and anti-CD15 antibodies cause the homotypic adhesion of neutrophils demonstrable by light microscopy and flow cytometry. Anti-(integrin beta 2 chain) mAbs inhibit both adhesion to plastic and homotypic adhesion. These results suggest that binding of ligand to NCA-160 is able to trigger neutrophil adhesion events which have been shown to be integrin mediated. Anti-CD15 mAbs do not, however, induce a respiratory burst from neutrophils.
First determination of the secondary structure of purified factor VIII light chain
Authors: Bihoreau N , Fontaine-Aupart M P , Lehegarat A , Desmadril M , Yon J M
Published: August 2015

The first analysis of the secondary structure of human factor VIII light chain was performed by c.d. spectroscopy. The purification process described in this paper allowed us to obtain the large amounts of purified factor VIII light chains required for c.d. experiments. Since this 80 kDa protein is non-covalently associated with a heavy chain to form the active molecule, isolated factor VIII light chains were obtained after immunoadsorption and dissociation of the immobilized active complexes by EDTA. Furthermore, factor VIII light chains were discriminated from the residual active complexes and the free heavy chains by a final ion-exchange-chromatography step. This f.p.l.c. analysis showed that factor VIII light chains were less electronegative than the active complexes. The results of conformational analysis by c.d. show that the protein possesses a high degree of regular secondary structure (58%) with approx. 22% of alpha-helix and 36% of beta-strand structures. The protein was completely unfolded by 3 M-guanidine hydrochloride. The results obtained from the analysis of c.d. spectra were compared with those predicted from three different statistical methods based on amino-acid sequence. The secondary structure information obtained from these methods was in good agreement with the c.d. results. These results were comparable with the secondary structure prediction of ceruloplasmin, a protein known to show sequence identity to factor VIII.
The fate of the prosegment in the acute-phase and programmed synthesis of sapecin, an antibacterial peptide of the flesh fly (Sarcophaga peregrina)
Authors: Homma K , Matsuyama K , Komano H , Natori S
Published: August 2015

The nucleotide sequence of sapecin cDNA suggested that this antibacterial peptide of the flesh fly (Sarcophaga peregrina) is produced from preprosapecin by post-translational processing. We examined the production of sapecin and its prosegment by radioimmunoassay under two different physiological conditions in which its gene is activated, assuming that the prosegment has some biological role. Results suggested that the prosegment is degraded selectively during production of sapecin. We also found that imaginal discs synthesize sapecin when cultured in the presence of 20-hydroxyecdysone.
Erythroid expression and DNAaseI-hypersensitive sites of the carbonic anhydrase 1 gene
Authors: Sowden J , Edwards M , Morrison K , Butterworth P H W , Edwards Y H
Published: August 2015

The carbonic anhydrase 1 gene is expressed in adult human and mouse erythroid cells and colon epithelia from two distinct promoters. We have explored the erythroid promoter for cis-acting sequences involved in transcription using DNAaseI as a probe. Two DNAaseI-hypersensitive sites (DHS-1 and DHS-2) have been identified in the distal erythroid promoter in CA1-expressing erythroleukaemic cells. These sites are present at low levels in K562 cells, which have a foetal/embryonic phenotype and do not express CA1. DHS-1 and DHS-2 are not present in non-erythroid cells, including colon cells, which express CA1 from the proximal colon promoter. DHS-1 and DHS-2 were also generated in an heterologous CA1 gene containing 5 kb of erythroid promoter sequence after transfection into erythroid cells, including K562 cells. These transfection studies showed that both this fragment, and an abbreviated 817 bp promoter fragment which contains only DHS-1, were sufficient to confer erythroid-specific expression to a reporter gene. These promoters were active in cell lines expressing CA1 and in K562 cells. This latter observation implies that a developmental repressor factor is both present in K562 cells and binds to a cis-acting sequence that is absent from the sequence 5 kb upstream of the erythroid transcription start site.
The control of cellular shape and motility. Mg2+ and tropomyosin regulate the formation and the dissociation of microfilament bundles
Authors: Grazi E , Cuneo P , Cataldi A
Published: August 2015

At pH 7.14 and 37 degrees C, in 7.2% (w/v) poly(ethylene glycol) 6000, tropomyosin-regulated actin filaments are converted into filament bundles by increasing the free Mg2+ concentration to 1.7-2.0 mM. When free Mg2+ concentration is decreased below 1.7 mM, bundles dissociate back into tropomyosin-regulated actin filaments. Pure actin filaments are insensitive to this mechanism of control and are found as filament bundles in all the range of free Mg2+ concentrations tested (1.37-2.2 mM). The mechanism of regulation described above is likely to operate in the cell, where the concentration of free Mg2+ is linked to the energy charge of the adenine nucleotide system.
Glutamine is a good substrate for glycogen synthesis in isolated hepatocytes from 72 h-starved rats, but not from 24 h- or 48 h-starved rats
Authors: Mouterde O , Claeyssens S , Chedeville A , Lavoinne A
Published: August 2015

In isolated hepatocytes from 24 h-starved rats, no glycogen synthesis was observed in the presence of glutamine. By contrast, glutamine was the best gluconeogenic substrate to induce glycogen synthesis in isolated hepatocytes from 72 h-starved rats. The effect of glutamine on glycogen synthesis was not accompanied by parallel changes in glucose or lactate production. Glutamine activated glycogen synthase independently of the starvation period; however, the extent of synthase activation was 2-fold higher in isolated hepatocytes from 72 h-starved rats than in hepatocytes from 24 h-starved rats. This increase in synthase activation was associated with increased cell swelling. The rate of glutamine transport was not significantly different in hepatocytes from 24 h- and 72 h-starved rats. By contrast, the intracellular glutamate concentration was 1.5-fold higher after 3 days of starvation in hepatocytes incubated with 5 mM-glutamine. We propose that glutamine may play a key role in the glycogen synthesis observed in vivo after 3 days of starvation.
Purification, characterization and function of dihydrolipoamide dehydrogenase from the cyanobacterium Anabaena sp. strain P.C.C. 7119
Authors: Serrano A
Published: August 2015

A dihydrolipoamide dehydrogenase (dihydrolipoamide: NAD+ oxidoreductase, EC 1.8.1.4) (DLD) has been found in the soluble fraction of cells of both unicellular (Synechococcus sp. strain P.C.C. 6301) and filamentous (Calothrix sp. strain P.C.C. 7601 and Anabaena sp. strain P.C.C. 7119) cyanobacteria. DLD from Anabaena sp. was purified 3000-fold to electrophoretic homogeneity. The purified enzyme exhibited a specific activity of 190 units/mg and was characterized as a dimeric FAD-containing protein with a native molecular mass of 104 kDa, a Stokes' radius of 4.28 nm and a very acidic pI value of about 3.7. As is the case with the same enzyme from other sources, cyanobacterial DLD showed specificity for NADH and lipoamide, or lipoic acid, as substrates. Nevertheless, the strong acidic character of the Anabaena DLD is a distinctive feature with respect to the same enzyme from other organisms. The presence of essential thiol groups was suggested by the inactivation produced by thiol-group-reactive reagents and heavy-metal ions, with lipoamide, but not NAD+, behaving as a protective agent. The function and physiological significance of Anabaena DLD are discussed in relation to the fact that 2-oxoacid dehydrogenase complexes have not been detected so far in filamentous cyanobacteria. Glycine decarboxylase activity, which might be involved in photorespiratory metabolism, has been found, however, in cell extracts of Anabaena sp. strain P.C.C. 7119 as the present study demonstrates.
Site-directed mutagenesis and substrate-induced inactivation of β-lactamase I
Authors: Thornewell S J , Waley S G
Published: August 2015

The substrate-induced inactivation of beta-lactamase I from Bacillus cereus 569/H has been studied. Both the wild-type enzyme and mutants have been used. The kinetics follow a branched pathway of the type recently analysed [Waley (1991) Biochem. J. 279, 87-94]. The substrate cloxacillin (a penicillin) formed an acyl-enzyme (characterized by m.s.), and it was probably the instability of this intermediate that brought about inactivation. A disulphide bond was introduced into beta-lactamase I (the wild-type enzyme lacks this bond) by site-directed mutagenesis: Ala-77 and Ala-123 were replaced by cysteine. Spontaneous oxidation yielded the disulphide. The activity of this newly cross-linked enzyme was a little diminished, but the stability towards inactivation by cloxacillin was not increased. A second mutant of beta-lactamase I was studied: this mutant lacked the first 17 residues, i.e. the first alpha-helix. The mutant had reduced activity towards ordinary (non-inactivating) substrates and no hydrolysis of cloxacillin could be detected. These mutant enzymes were expressed in Bacillus subtilis, and were purified from the extracellular medium.
Muscarinic stimulation of SK-N-BE(2) human neuroblastoma cells elicits phosphoinositide and phosphatidylcholine hydrolysis: relationship to diacylglycerol and phosphatidic acid accumulation
Authors: Pacini L , Limatola C , Frati L , Luly P , Spinedi A
Published: August 2015

Muscarinic stimulation of the human neuroblastoma cell line SK-N-BE(2) elicits hydrolysis of phosphoinositides and phosphatidylcholine (PtdCho) and produces a rapid and sustained elevation of diacylglycerol (DG) mass. PtdIns(4,5)P2 cleavage by phospholipase C (PLC) occurred immediately after carbachol (CCh) addition, and phosphoinositide hydrolysis was then sustained for at least 5 min. Cell stimulation, after extensive PtdCho labelling by long-term [3H]choline administration, resulted in an enhanced release of [3H]phosphocholine (PCho) into the external medium; enhanced [3H]PCho release, which occurred with a 15 s delay with respect to CCh addition, was particularly pronounced within the first minute of stimulation and proved to be caused by PtdCho-specific PLC activation. In fact, when cells were exposed to [3H]choline for a short period, to extensively label the intracellular PCho pool but not PtdCho, stimulation did not result in an enhanced release of [3H]PCho into the medium. PtdCho-specific phospholipase D (PLD) activation was documented by the accumulation of [3H]phosphatidylethanol in cells prelabelled with [3H]myristic acid and stimulated in the presence of 1% (v/v) ethanol; this metabolic pathway, however, proved to be a minor one leading to generation of phosphatidic acid (PtdOH) during cell stimulation, whereas DG production by the sequential action of PtdCho-specific PLD and PtdOH phosphohydrolase was not observed. Studies on cells which were double-labelled with [3H]myristic acid and [14C]arachidonic acid indicated that within 15 s of stimulation DG is uniquely derived from PtdIns(4,5)P2, whereas PtdCho is the major source at later times. Evidence is provided that rapid and selective conversion of phosphoinositide-derived DG into PtdOH may play an important role in determining the temporal accumulation profile of DG from the above-mentioned sources.
Blockade of mevalonate production by lovastatin attenuates bombesin and vasopressin potentiation of nutrient-induced insulin secretion in HIT-T15 cells. Probable involvement of small GTP-binding proteins
Authors: Li G , Regazzi R , Roche E , Wollheim C B
Published: August 2015

Small G-proteins (SMGs) require isoprenylation for their association with membranes. We have examined protein isoprenylation, subcellular distribution of SMGs, cytosolic Ca2+ changes and insulin secretion in HIT-T15 cells after treatment with lovastatin, which inhibits the production of isoprenoids by blocking mevalonate production by 3-hydroxy-3-methylglutaryl-CoA reductase. Numerous proteins in the 20-70 kDa range were found to be isoprenylated. Most of these proteins co-migrated with SMGs (21-27 kDa). Lovastatin treatment (25 microM, 24 h) decreased protein isoprenylation and affected the distribution of several SMGs, causing a large accumulation in the cytosol and a detectable decrease in membranes. Lovastatin selectively attenuated the potentiating action of bombesin and vasopressin, which activate phospholipase C in these cells, on insulin secretion stimulated by nutrients (glucose + leucine + glutamine). This lovastatin effect was overcome by mevalonate. Insulin secretion stimulated by nutrients alone or insulin release in the presence of the potentiating agents forskolin or phorbol myristate acetate remained unaffected. As the modulation of insulin secretion by isoprenaline and somatostatin were not altered by lovastatin, the drug does not non-selectively affect the binding of ligands to their receptors. Lovastatin did not interfere with the activation of phospholipase C by bombesin and vasopressin, since the rise in cytosolic Ca2+ induced by these agents was not changed. Limonene, proposed to block specifically prenyl-protein transferases of SMGs, did not alter protein isoprenylation patterns, but inhibited the stimulated insulin secretion. In conclusion, lovastatin selectively attenuated the potentiation of nutrient-induced insulin secretion by bombesin and vasopressin without affecting their activation of phospholipase C. The concomitant changes in SMG isoprenylation and their subcellular distribution after lovastatin treatment suggest that SMGs could play an important role in the bombesin and vasopressin action on insulin secretion.
Cytochrome bo from Escherichia coli: identification of haem ligands and reaction of the reduced enzyme with carbon monoxide
Authors: Cheesman M R , Watmough N J , Pires C A , Turner R , Brittain T , Gennis R B , Greenwood C , Thomson A J
Published: August 2015

Inner membranes were prepared from Escherichia coli strain RG 145, which is deficient in cytochrome bd, but overexpresses cytochrome bo [Au and Gennis (1987) J. Bacteriol. 169, 3237-3242]. The latter was purified 7-fold by extracting the membranes with octyl beta-D-glucopyranoside, followed by chromatography on DEAE-Sepharose, yielding 150 mg of protein/150 g wet weight of cells. Optical e.p.r. and low-temperature m.c.d. (magnetic circular dichroism) spectroscopies were used to investigate the nature of the protein ligands to the two haems in cytochrome bo from E. coli. Low-spin ferric haem b, the origin of a rhombic e.p.r. spectrum with g = 2.98, 2.26 and 1.50, gives rise to a charge-transfer band in the near-i.r. m.c.d. spectrum at 1622 nm. It is therefore concluded that haem b is co-ordinated by two histidine residues. The low-temperature m.c.d. spectrum of dithionite-reduced cytochrome bo comprises bands due both to low-spin ferrous haem b and to high-spin ferrous haem o. The bands arising from haem o show a direct correspondence with those in the m.c.d. spectrum of five-co-ordinate histidine-ligated ferrous haems such as myoglobin, implying that the protein residue liganding haem o is also histidine. This assignment was confirmed by measuring the e.p.r. spectrum of the nitric oxide derivative of fully reduced cytochrome bo. This showed a rhombic spectrum with g = 2.098, 2.008 and 1.987, and nuclear hyperfine splitting consistent with the co-ordination of ferrous haem by NO and histidine. The hyperfine splittings observed were 1.95 +/- 0.05 mT for the 14N of the NO ligand and 0.75 +/- 0.05 mT for the 14N of the proximal histidine. The e.p.r. spectrum of some samples of oxidized cytochrome bo show, at temperatures below 15 K, broad signals at g = 7.6, 3.6 and 2.8, and other preparations in the presence of glycerol yield signals at g = 10.8, 3.2 and 2.6. These signals, which are abolished by the addition of cyanide, are assigned to the binuclear centre, cytochrome o-CuB, suggesting that the binuclear site may display heterogeneity. Carbon monoxide reacts with the reduced enzyme with a stoichiometry of 1:1, and the dissociation constant for this reaction was determined to be 1.7 x 10(-6)M. The second-order rate constants for this reaction were measured and shown to be similar to those determined for bovine cytochrome aa3 [Gibson and Greenwood (1963) Biochem. J. 86, 541-554].
The effects of thimerosal on calcium uptake and inositol 1,4,5-trisphosphate-induced calcium release in cerebellar microsomes
Authors: Sayers L G , Brown G R , Michell R H , Michelangeli F
Published: August 2015

Thimerosal inhibits calcium uptake in skeletal muscle sarcoplasmic reticulum and rat cerebellar microsomes by inhibiting the Ca(2+)-ATPase. In the presence of 5 mM dithiothreitol (DTT), Ca2+ uptake and ATPase activity were not inhibited by thimerosal, indicating that thimerosal modifies cysteine residues of the Ca(2+)-ATPase. Low thimerosal concentrations (2 microM) sensitize the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ channel, making it open at lower InsP3 concentrations. Higher concentrations of thimerosal, however, cause inhibition of InsP3-induced Ca2+ release. Both sensitization and inhibition of the InsP3 receptor by thimerosal can be prevented by DTT. The binding and metabolism of InsP3 by cerebellar microsomes is not affected by thimerosal. The amount of InsP3-induced Ca2+ release is co-operatively linked to the InsP3 concentration with a Hill coefficient of 2.0 +/- 0.3. This is decreased to 1.0 +/- 0.2 at inhibitory concentrations of thimerosal. Under our experimental conditions, we observed no dependence of quantal Ca2+ release on intraluminal Ca2+ concentration.
Inhibition of 3-hydroxy-3-methylglutaryl-CoA synthase and cholesterol biosynthesis by β-lactone inhibitors and binding of these inhibitors to the enzyme
Authors: Greenspan M D , Bull H G , Yudkovitz J B , Hanf D P , Alberts A W
Published: August 2015

The beta-lactones L-659,699 [(E,E)-11-[3-(hydroxymethyl)-4-oxo-2- oxetanyl]-3,5,7-trimethyl-2,4-undecadienoic acid) and its radioactive derivative 3H-L-668,411 (the 2,3-ditritiated methyl ester of L-659,699) inhibited a partially purified preparation of rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase with an IC50 of 0.1 microM. These compounds were also found to inhibit the incorporation of [14C]acetate into sterols in cultured Hep G2 cells with an IC50 of 3 microM. New kinetic evidence indicated that inhibition of the isolated enzyme was irreversible. In contrast, sterol biosynthesis in cultured Hep G2 cells was rapidly restored upon removal of the compound from the medium of inhibited cultures, suggesting reversibility of inhibition in the cells. Radioactivity was found to be associated with a single cytoplasmic protein by SDS/PAGE of the cytoplasm of Hep G2 cells after incubation of the cells with the inhibitor 3H-L-668,411. This protein was identified as cytoplasmic HMG-CoA synthase. Binding of the radioactive compound to the enzyme was decreased with time if the radioactive inhibitor was removed from the medium. Exposure of a gel containing the radioactive enzyme-inhibitor complex to neutral hydroxylamine also resulted in a loss of radioactivity from the gel. The purified rat liver enzyme reacted with the 3H-ligand to form a stable enzyme-inhibitor complex which could be isolated by h.p.l.c. Radioactivity was also subsequently lost from this complex when it was incubated with neutral hydroxylamine. Incorporation of [14C]acetate into cholesterol in mouse liver was inhibited in a reversible manner after oral administration of the beta-lactone inhibitor. These studies, as well as the kinetic evidence presented, suggest that the beta-lactone inhibitors acylate HMG-CoA synthase in a reaction which appears to be irreversible in vitro, but is easily reversed in cultured cells and in animals.
Characterization of the antiplasmin activity of human thrombospondin-1 in solution
Authors: Anonick P K , Yoo J K , Webb D J , Gonias S L
Published: August 2015

These studies demonstrate relatively rapid association of plasmin with thrombospondin and the effects of this interaction on plasmin activity towards D-Val-L-Leu-L-Lys p-nitroanilide hydrochloride (S-2251) and the proteinase inhibitors alpha 2-antiplasmin (alpha 2AP) and alpha 2-macroglobulin (alpha 2M). Binding of plasmin to thrombospondin reached an apparent reversible equilibrium within 3 min at 22 degrees C. The amidase activity of bound plasmin was inhibited. An analysis of S-2251 hydrolysis indicated that thrombospondin is a linear mixed-type plasmin inhibitor. The dissociation constant (KD) for the binding of plasmin to thrombospondin was 0.5 microM, assuming one plasmin binding site per thrombospondin homotrimer. Plasmin and miniplasmin slowly cleaved thrombospondin, yielding products which were comparable with those generated by other proteinases. Tranexamic acid inhibited the digestion of thrombospondin by plasmin and miniplasmin, suggesting an important role for the kringle-5 domain in this process. When plasmin was incubated first with thrombospondin and then with alpha 2AP, plasmin that was apparently bound to thrombospondin reacted with alpha 2AP at a decreased rate; however, within 20 min, all of the plasmin was recovered in complex with alpha 2AP. Similar results were obtained with alpha 2M. Transfer of plasmin from thrombospondin to alpha 2AP or alpha 2M probably required plasmin-thrombospondin-complex dissociation. A low level of reaction of alpha 2AP with thrombospondin-associated plasmin could not be ruled out. These results demonstrate that the activity of plasmin, when bound to thrombospondin, is greatly diminished or eliminated. The plasmin-thrombospondin complex, which is formed within 3 min, is fully reversible and the associated plasmin is in a latent form protected from proteinase inhibitors. Therefore, thrombospondin may regulate plasmin activity in a manner which is distinct from conventional proteinase inhibitors and other extracellular-matrix proteins.
Phorbol esters inhibit insulin-induced receptor down-regulation in cultured human lymphocytes: association with diminished insulin receptor autophosphorylation
Authors: Torossian K , Nower P , Schwartz T , Fantus I G
Published: August 2015

Exposure of cells to phorbol 12-myristate 13-acetate (PMA) has been reported to result in resistance to the acute biological effects of insulin and an associated reduction in insulin-receptor tyrosine kinase activity. To investigate the relationship of insulin receptor autophosphorylation with a longer-term action of insulin the effect of PMA on insulin-stimulated receptor down-regulation was examined in cultured human lymphocytes (IM-9). Lymphocytes bound [3H]phorbol dibutyrate specifically with characteristics typical of binding to protein kinase C (PKC). Acute exposure (30 min) to PMA resulted in a transient decrease of insulin binding which is consistent with a decrease in receptor number. Chronic (18 h) exposure to PMA (5 nM) resulted in inhibition of insulin-induced down-regulation of its cognate receptor. Sphingosine, an inhibitor of PKC, or chronic pre-exposure to a high concentration of PMA (1 microM), which is known to inactivate PKC, blocked the effect of PMA. PMA inhibited insulin-stimulated receptor internalization by 26% and receptor degradation by 82%. Exposure of intact cells to PMA followed by insulin treatment inhibited insulin-receptor autophosphorylation subsequently assayed in vitro, as well as beta-subunit tyrosine phosphorylation in situ. In summary, PMA inhibited insulin-stimulated receptor down-regulation via activation of PKC. This was associated with an inhibition of both receptor internalization and receptor degradation. There was a concomitant inhibition of receptor tyrosine autophosphorylation consistent with a requirement of receptor kinase activation for both short-term and long-term biological effects of insulin.
Organization of the phosphoinositide cycle. Assessment of inositol transferase activity in purified plasma membranes
Authors: Santiago O M , Rosenberg L I , Monaco M E
Published: August 2015

Experiments were carried out to determine whether or not CDP-diacylglycerol:myo-inositol 3-phosphatidyltransferase (IT) activity (EC 2.7.8.11) could be detected in purified plasma-membrane fractions from WRK-1 rat mammary tumour cells. These cells have previously been shown to have a very active phosphoinositide cycle. Sucrose-density-gradient-purified plasma membranes contained no IT activity that could not be accounted for by endoplasmic-reticulum contamination. However, we also determined that the relative amount of IT activity in endoplasmic reticulum and plasma-membrane fractions could be altered by changing the concentration of detergent in the assay system.
Role of the glycosaminoglycan component of thrombomodulin in its acceleration of the inactivation of single-chain urokinase-type plasminogen activator by thrombin
Authors: de Munk G A W , Parkinson J F , Groeneveld E , Bang N U , Rijken D C
Published: August 2015

Thrombomodulin (TM), a membrane proteoglycan on endothelial cells, binds thrombin in a 1:1 complex, accelerates the protein C activation by thrombin, promotes the thrombin inactivation by antithrombin III and inhibits the procoagulant properties of thrombin. The inactivation of single-chain urokinase-type plasminogen activator (scu-PA) by thrombin is accelerated about 70-fold by TM [De Munk, Groeneveld and Rijken (1991) J. Clin. Invest. 88, 1680-1684]. The present study investigates the role of the O-linked glycosaminoglycan moiety of TM in the latter reaction. In the presence of an excess of a fully-glycosylated soluble recombinant human TM mutant (high-Mr rec-TM), 0.11 nM thrombin inactivated 50% of 4.4 nM scu-PA in 45 min at 37 degrees C. In the presence of a soluble recombinant TM mutant lacking the glycosaminoglycans (low-Mr rec-TM), 1.9 nM thrombin was needed to inactivate 50% scu-PA, as compared with 4.7 nM thrombin in the absence of TM. Using the scu-PA inactivation assay the dissociation constant for the thrombin-TM interaction was found to be 0.4 nM for high-Mr rec-TM and 14 nM for low-Mr rec-TM. Treatment of high-Mr rec-TM with chondroitinase ABC to digest the glycosaminoglycans decreased the accelerating effect to the level of low-Mr rec-TM. A similar decrease was observed after treatment of solubilized rabbit TM with chondroitinase ABC. As expected, chondroitinase ABC had no influence on the accelerating effect of low-Mr rec-TM. The free glycosaminoglycans obtained by alkaline treatment of TM or chondroitin sulphate A also accelerated the inactivation of scu-PA by thrombin, but about 1000-fold higher concentrations than with TM were needed to obtain the same acceleration. It is concluded that the major glycosaminoglycan of TM plays a pivotal role in the inactivation of scu-PA by the TM-thrombin complex, both in the formation and in the activity of the complex.
Separation of native and truncated forms of poliovirus protease 3C produced in Escherichia coli
Authors: Polgár L , Erdélyi F , Hajnal E , Löw M , Gráf L , Korant B D
Published: August 2015

Poliovirus protease 3C is a cysteine enzyme that is essential for the processing of the viral precursor polyprotein containing structural proteins and enzymes, including the protease itself. We have constructed the plasmid pSD/PV3C which produced protease 3C as inclusion bodies when expressed in Escherichia coli. In addition to the full-length protease, a truncated form was also generated, starting from an internal initiation site (Met-27). The enzyme was renatured by dilution of a 6 M guanidinium chloride solution of the inclusion bodies, and the proteins were precipitated from the diluted solution with ammonium sulphate. By extracting the precipitate with a buffer solution, the full-length enzyme could be completely separated from its N-terminally truncated form. Size-exclusion chromatography of the extracted protease 3C resulted in an active enzyme which appeared homogeneous by SDS/PAGE. For measuring the activity of the protease, a spectrofluorimetric method was devised to monitor the hydrolysis continuously, which is simpler and more precise than the h.p.l.c. technique used previously.
Escherichia coli and herpes-simplex-virus ribonucleotide reductase R2 subunit. Compared reactivities of the redox centres
Authors: Atta M , Lamarche N , Battioni J P , Massie B , Langelier Y , Mansuy D , Fontecave M
Published: August 2015

Protein R2, the small subunit of ribonucleotide reductase, contains a diferric centre and a tyrosyl radical absolutely required for enzyme activity. The reduction of the tyrosyl radical and the mobilization of the iron centre result in the inhibition of the enzyme and thus of DNA synthesis. The chemical reactivity of the iron-radical centre of Escherichia coli and herpes simplex virus has been studied by u.v.-visible and e.p.r. spectroscopies. The tyrosyl radical is efficiently scavenged by hydroxamic acids and phenols during reactions controlled by steric hindrance and hydrophobic interactions. The reaction with o-disubstituted phenols yields the corresponding diphenoquinones. The reactivity of the bacterial radical greatly contrasts with that of the viral radical, and the iron centre in herpes-simplex-virus R2 is much more labile than that in E. coli R2, as shown from the facile mobilization of iron by chelators such as catechol. These results suggest that the active sites of the two enzymes are significantly different and might be useful for designing new antiviral agents.
Extracellular-matrix degradation at acid pH. Avian osteoclast acid collagenase isolation and characterization
Authors: Blair H C , Teitelbaum S L , Grosso L E , Lacey D L , Tan H L , McCourt D W , Jeffrey J J
Published: August 2015

Osteoclasts degrade bone matrix, which is mainly type I collagen and hydroxyapatite, in an acidic extracellular compartment. Thus we reasoned that osteoclasts must produce an acid collagenase. We purified this enzyme, a 31 kDa protein, from avian osteoclast lysates (in 100 mM acetate/1 mM CHAPS/1 mM dithiothreitol, pH 4.4), fractionated by (NH2)2SO4 precipitation, gelatin-affinity, cation exchange, and gel filtration. Fraction activity was measured using diazotized collagen or 3H-labelled cross-linked collagen (decalcified and trypsin-treated metabolically L-[4,5-3H]proline-labelled bone) as substrates. Iodoacetate, leupeptin, antipain, pepstatin and mercurials inhibited collagenolysis by the isolated proteinase; mercurial derivatives could not be re-activated by dithiothreitol. Collagen degradation was maximal at pH 4.4; purified proteinase reproduced the collagenolytic activity of cell lysates. The N-terminal amino acid sequence from the isolated protein and its CNBr degradation fragments showed sequence similarity to mammalian cathepsin Bs, and near-identity with avian liver cathepsin B. Peptide substrate specificity of the osteoclastic enzyme resembled those of mammalian cathepsin B and its avian liver counterpart, but degradation of low-molecular-mass substrates by the osteoclastic enzyme was slower, reflecting generally lower kcat. values. Further, kcat/Km varied less between arginine-containing substrates than for previously reported cathepsin Bs, indicating different substrate specificity of the osteoclast enzyme. Polyclonal antibody raised to a 25 kDa fragment of the enzyme recognized a single 31 kDa band in SDS/PAGE of osteoclast lysates blotted to poly(vinylidene difluoride), adsorbed collagenolytic activity of osteoclast lysates, and stained avian osteoclasts in tissue sections. Degenerate sense- and antisense-oligonucleotide primers, predicted from segments of primary amino acid sequence, amplified a 486 bp DNA fragment; this was cloned and sequenced. Of 162 amino acids encoded, 77% are identical with those of human cathepsin B; hybridization identified a 2.4 kb RNA in osteoclast lysates. We conclude that the major avian osteoclast collagenolytic enzyme is a cathepsin B, whose activity varies from other enzymes of its class.
The hepatic glucose-6-phosphatase system in Ehrlich-ascites-tumour-bearing mice
Authors: Lucius R W , Waddell I D , Burchell A , Nordlie R C
Published: August 2015

To examine the effects of the presence of Ehrlich ascites tumours on both the catalytic unit and the substrate/product translocase components of the glucose-6-phosphatase system in vivo, we isolated microsomes from the livers of control and tumour-bearing mice. Samples were analysed immunochemically for the quantity of catalytic unit, stabilizing protein and translocases T2 and T3 proteins. In comparison experiments, a variety of kinetic studies were performed. The most striking findings in tumour-bearing mice were: a 2.5-fold increase in the quantity of translocase T2 protein; increases in the Km and Vmax. for glucose 6-phosphate phosphohydrolase; and a decrease in the Km value for carbamoyl phosphate (carbamoyl-P) of carbamoyl-P:glucose phosphotransferase, all with intact microsomes. The percentage latency at Vmax. decreased for PPi phosphohydrolase and for glucose 6-phosphate phosphohydrolase, but was unaffected for carbamoyl-P:glucose phosphotransferase. These observations support a tumour-related increase in translocase T2 capacity in vivo, as it transports Pi from the microsomal lumen to the medium and carbamoyl-P or PPi from the medium to the microsomal lumen.
Localization and hormonal stimulation of phosphorylation sites in the LNCaP-cell androgen receptor
Authors: Kuiper G G J M , de Ruiter P E , Trapman J , Boersma W J A , Grootegoed J A , Brinkmann A O
Published: August 2015

Phosphorylation of the androgen receptor in human prostate tumour cells (LNCaP) is increased by addition of androgens to intact cells. Double-label studies, using [35S]methionine incorporation into receptor protein, and [32P]P(i) to label metabolically receptor phosphorylation sites, have enabled us to determine the phosphate content, relative to receptor protein, of both nontransformed and transformed and androgen receptors generated in intact LNCaP cells. No net change in the phosphorylation of the intact 110 kDa steroid-binding component of the androgen-receptor complex was found upon transformation to the tight nuclear binding form in the intact cell. Partial proteolysis of androgen receptor protein metabolically labelled with [32P]P(i) and photolabelled with [3H]R1881 (methyltrienolone) revealed that phosphorylation occurs mainly in the N-terminal trans-activation domain, whereas no phosphorylation was detected in the steroid- and DNA-binding domains. The location of most (> 90%) of the hormonally regulated phosphorylation sites in the N-terminal trans-activation domain suggests a role of phosphorylation of the androgen receptor in transcription regulation.
Reconstitution of thromboxane A2 receptor-stimulated phosphoinositide hydrolysis in isolated platelet membranes: involvement of phosphoinositide-specific phospholipase C-β and GTP-binding protein Gq
Authors: Baldassare J J , Tarver A P , Henderson P A , Mackin W M , Sahagan B , Fisher G J
Published: August 2015

Activation of human platelets by the arachidonic acid metabolite thromboxane A2 and the thromboxane A2 mimic U46619 is mediated through phosphoinositide-specific phospholipase C-catalysed hydrolysis of phosphoinositides. We have established conditions to reconstitute U46619-stimulated phosphoinositide breakdown by addition of guanine nucleotides and soluble platelet phospholipase C activities to isolated 32P-labelled membranes. Receptor-activated phosphoinositide hydrolysis was observed in the presence of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) or GTP plus U46619. Phosphoinositide hydrolysis was dependent on both GTP and U46619, with half-maximal stimulation observed at 5 microM and 500 nM respectively. Phospholipase C isoenzymes beta, gamma 1, gamma 2 and delta were purified from platelet cytosol and their ability to reconstitute GTP[S]-dependent and GTP/U46619-dependent phosphoinositide hydrolysis determined. Phospholipase C-beta and -delta, but not phospholipase C-gamma 1 or -gamma 2, catalysed phosphoinositide breakdown in the presence of GTP[S]. In contrast, only phospholipase C-beta was able to reconstitute GTP-dependent U46619-induced hydrolysis. The participation of GTP-regulatory proteins in the reconstitution of GTP[S]- and GTP/U46619-induced phosphoinositide hydrolysis was examined using antibodies to the C-terminals of the alpha-subunits of three of the heterotrimeric GTP-binding proteins expressed in human platelets Gq, Gi2 and Gi3. Anti-Gq antibody, but not anti-Gi2 or Gi3 antibody, inhibited both GTP[S]- and GTP/U46619-dependent reconstitution of phosphoinositide hydrolysis with phospholipase C-beta. In contrast GTP[S]-stimulated hydrolysis by phospholipase C-delta was not inhibited by any of the G-protein antibodies. These results show the functional specificity of GTP-binding proteins and phospholipase C isoenzymes in mediating agonist-induced phosphoinositide hydrolysis in human platelets.
Purification, sequence and antibacterial activity of two novel sapecin homologues from Sarcophaga embryonic cells: similarity of sapecin B to charybdotoxin
Authors: Yamada K , Natori S
Published: August 2015

Two sapecin homologues were purified from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina (flesh fly). These homologues contained six cysteine residues with exactly the same disulphide pairings as those in sapecin. The amino acid sequence of one of them, sapecin C, was also very similar to that of sapecin. The other homologue, sapecin B, was less similar to sapecin but showed significant similarity to charybdotoxin, an inhibitor of K+ channels isolated from a scorpion venom. Like sapecin, these homologues repressed the growth of various Gram-positive bacteria.
Phosphorylation of human topoisomerase I by protein kinase C in vitro and in phorbol 12-myristate 13-acetate-activated HL-60 promyelocytic leukaemia cells
Authors: Cardellini E , Durban E
Published: August 2015

Topoisomerase I was phosphorylated in vitro by protein kinase C (PKC) purified from rat brain with high affinity (Km about 0.1 microM). Tryptic phosphopeptide mapping indicated that two major topoisomerase I peptides phosphorylated in vivo were comigrating with minor peptides phosphorylated by PKC in vitro. Topoisomerase I phosphorylation was stimulated 3-fold in HL-60 cells exposed to the tumour promoter phorbol 12-myristate 13-acetate. The results suggest that topoisomerase I phosphorylation in HL-60 cells is indirectly controlled by PKC.
Binding of the calcium antagonist flunarizine to phosphatidylcholine bilayers: charge effects and thermodynamics
Authors: Thomas P G , Seelig J
Published: August 2015

We have examined the partitioning/transfer of the Ca2+ antagonist flunarizine from the aqueous phase into phospholipid bilayers. We show that the binding of the cationic amphiphilic drug flunarizine to phospholipid bilayers displays traditional linear concentration-dependent characteristics once unmasked of electrostatic effects. The coefficient for the binding/partitioning of flunarizine to phosphatidylcholine was found to be 28700 M-1, supporting the notion that this drug may be particularly membrane-active. The thermodynamics of the partitioning/transfer process have also been studied using high-sensitivity titration calorimetry. Binding was found to be predominantly enthalpy-driven with only a small entropic contribution; delta H = -22.1 kJ.mol-1 (-5.3 kcal.mol-1) at 27 degrees C. This is in conflict with established ideas of entropy-driven partitioning of drugs into phospholipid membranes as a result of the ‘hydrophobic effect’. The strong enthalpic nature of binding is interpreted as being indicative of strong lipophilic interactions between the drug and the phospholipid phase.
Regulation of the ovine intestinal Na+/glucose co-transporter (SGLT1) is dissociated from mRNA abundance
Authors: Lescale-Matys L , Dyer J , Scott D , Freeman T C , Wright E M , Shirazi-Beechey S P
Published: August 2015

We have investigated the mechanisms of regulation of the Na+/glucose co-transporter (SGLT1) in a ruminant animal, which is an exceptional model system for studying intestinal glucose transport. Pre-ruminant lambs absorb glucose, produced by hydrolysis of the milk sugar lactose, in the intestine via apical SGLT1 and basolateral facilitative glucose transporters (GLUT2). Weaning coincides with the development of the rumen, and consequently the amount of hexoses reaching the small intestine of the ruminant sheep is undetectable. During development, SGLT1 activity and abundance in intestinal brush-border membranes decreased by over 200-fold, and either maintaining lambs on a milk replacer diet or infusing sheep intestine with D-glucose restored co-transporter activity and expression. We have measured ovine intestinal SGLT1 mRNA levels during development, with changes in diet and after direct infusion of D-glucose or methyl alpha-D-glucopyranoside into the intestinal lumen, in order to determine the level of regulation. During development, mRNA levels decreased only 4-fold. Lambs maintained on a milk replacer diet showed no change in mRNA levels relative to age-matched controls. Finally, upon infusion of the intestine of the ruminant sheep with sugars, D-glucose infusion increased SGLT1 mRNA, but only by 2-fold, compared with a 60-90-fold increase in co-transporter number and activity. Since the change in Na(+)-dependent glucose transport activity is correlated with SGLT1 protein abundance, and since changes in mRNA levels do not account for the dramatic changes in protein abundance, we conclude that the principal level of SGLT1 regulation by luminal sugar is translational or post-translational.
Effects of hyperbilirubinaemia on glutathione S-transferase isoenzymes in cerebellar cortex of the Gunn rat
Authors: Johnson J A , Hayward J J , Kornguth S E , Siegel F L
Published: August 2015

The glutathione S-transferases (GSTs) are a family of isoenzymes involved in the detoxication of a variety of electrophilic xenobiotics. The present investigation demonstrates that GST activity and the concentration of cytosolic GSTs in cerebellar cortex of Gunn rats were increased in hyperbilirubinaemic animals compared with non-jaundiced controls. Age-dependent and region-specific increases in GST isoenzymes were seen in three regions of the cerebellar cortex of jaundiced Gunn rats, whereas GST concentrations were not altered in the brainstem, thalamus/hypothalamus, cortex or liver. Cytosolic GST activity was increased 1.3-fold in the flocculus and lateral hemispheres of 20-day-old and 1.7-fold in the flocculus, lateral hemispheres and vermis of 60-day-old jaundiced (jj; homozygous) Gunn rats compared with non-jaundiced (Jj; heterozygous) Gunn rats. H.p.l.c. was used to determine the GST subunit protein concentrations in cytosolic fractions isolated from liver and brain regions of jaundiced and non-jaundiced animals. In all regions of the cerebellum from 20-day-old animals, the levels of Alpha-class GST subunits 2 (Yc1; 3.0-fold) and 8 (Yk; 2.0-fold) were increased in jaundiced rats. In 60-day-old animals, the concentrations of Alpha-class GST subunits 2 (Yc1; 5.0-fold) and 8 (Yk; 3.0-fold), Mu-class subunit 11 (Yo; 2.5-fold) and Pi-class subunit 7 (Yp; 2.0-fold) were increased in all regions of cerebellar cortex of jaundiced animals. In cerebellum of 10-, 20- and 60-day-old non-jaundiced and jaundiced Gunn rats, the flocculus had the highest concentration of Mu-class GST subunit 4 (Yb2) and vermis the lowest; hyperbilirubinaemia increased the concentration of subunit 4 (Yb2; 3- to 5-fold) in the flocculus and lateral hemispheres, but not the vermis, of 20- and 60-day-old rats. Intraperitoneal injection of sulphadimethoxine, a long-acting sulphonamide which displaces bilirubin from its albumin-binding sites and increases the bilirubin levels in tissues, further increased the already elevated concentrations of GST subunits in the lateral regions of cerebellar cortex of hyperbilirubinaemic rats. For example, the concentration of subunit 4 (Yb2) was increased 2.2-fold (compared with non-jaundiced controls) in Gunn rats injected with saline and 7.4-fold in rats injected with 100 mg of sulphadimethoxine/kg body weight. In contrast, GSTs in the vermis of jaundiced animals were not affected by sulphadimethoxine injection. Sulphadimethoxine had no effect on GST concentrations in lateral regions and vermis of heterozygous (Jj) Gunn rats.(ABSTRACT TRUNCATED AT 400 WORDS)