bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and characterization of matrix metalloproteinase 9 from U937 monocytic leukaemia and HT1080 fibrosarcoma cells
Authors: Morodomi T , Ogata Y , Sasaguri Y , Morimatsu M , Nagase H
Published: August 2015

The precursor of matrix metalloproteinase 9 (proMMP-9), also known as ‘92 kDa progelatinase/type IV procollagenase’, was purified from the conditioned medium of U937 monocytic leukaemia and HT1080 fibrosarcoma cell lines stimulated with phorbol 12-myristate 13-acetate. ProMMP-9 in these culture media is non-covalently complexed with the 29 kDa tissue inhibitor of metalloproteinases (TIMP), but free proMMP-9 was separated from the TIMP-proMMP-9 complex by chromatography on Green A Dyematrex gel. The final product was homogeneous on SDS/PAGE, with a molecular mass of 88 kDa without reduction and 92 kDa with reduction. Treatment of proMMP-9 with 4-aminophenylmercuric acetate converted the 88 kDa precursor into 80 kDa and 68 kDa forms. Gelatin-containing zymographic analysis showed zones of lysis associated with all three species. However, only the 68 kDa species was shown to be catalytically active by its ability to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, only the 80 kDa species was generated by treatment with 4-aminophenylmercuric acetate, but no enzyme activity was detected. This indicates that TIMP binds to the 80 kDa intermediate and inhibits the generation of the active 68 kDa species. Eight endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, cathepsin G, neutrophil elastase and thermolysin) were tested for their ability to activate proMMP-9. Of them, trypsin was the most effective activator of proMMP-9. Only partial activation (10-30%) was observed with plasmin, cathepsin G and chymotrypsin. The active forms generated by trypsin were identified as 80 kDa, 74 kDa and 66 kDa by their abilities to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, proMMP-9 was also converted into the same molecular-mass species by trypsin, but they were not proteolytically active. This suggests activated MMP-9 is inhibited by TIMP. Activated MMP-9 digested gelatin, type-V collagen, reduced carboxymethylated transferrin and, to a lesser extent, type-IV collagen and laminin A chain. The specific activity against gelatin was estimated to be 15,000 units/mg (1 unit = 1 microgram of gelatin degraded/min at 37 degrees C) by titration with alpha 2-macroglobulin. Comparative studies on digestion of gelatin and collagen types IV and V by MMP-9 and MMP-2 indicated that both enzymes degrade these substrates into similar fragments. However, the susceptibilities of laminin, fibronectin and reduced carboxymethylated transferrin to these two MMPs were sufficiently different to indicate differences in substrate specificities between these two closely related proteinases.
Renaturation and partial peptide sequencing of mitogen-activated protein kinase (MAP kinase) activator from rabbit skeletal muscle
Authors: Wu J , Michel H , Rossomando A , Haystead T , Shabanowitz J , Hunt D F , Sturgill T W
Published: August 2015

Mitogen-activated protein kinase (MAP kinase) activator was purified 2000-fold from skeletal muscle, and proteins which co-purified with the activator were analysed after SDS/PAGE by renaturation and partial sequencing. Activity for tyrosine and threonine phosphorylation of MAP kinase was present in two bands of approx. 48 and 46 kDa, which have sequence similarity to small GTP-binding protein p25 GDP dissociation inhibitor and protein kinases (PBS2, SPK1+, STE7, BYR1) respectively.
Substrate specificity of the bovine and feline neutral α-mannosidases
Authors: De Gasperi R , al Daher S , Winchester B G , Warren C D
Published: August 2015

Neutral alpha-mannosidases were prepared from bovine and cat liver. The activities were distinguished from lysosomal and Golgi alpha-mannosidases by their neutral pH optima, relatively low Km for their synthetic substrate p-nitrophenyl alpha-D-mannoside, inhibition by Zn2+ and absence of inhibition by Co2+, EDTA, low concentrations of swainsonine, or deoxymannojirimycin. The cytosolic alpha-mannosidases were not retained by concanavalin A-Sepharose. They were able to degrade efficiently a variety of oligosaccharides with structures corresponding to certain high-mannose glycans or the oligomannosyl parts of hybrid and complex glycans. However, unlike lysosomal alpha-mannosidases from the same species these enzymes were not able to degrade Man9GlcNAc2 efficiently, and the bovine neutral alpha-mannosidase was not able to degrade a hexasaccharide with a structure analogous to Man5GlcNAc2-PP-dolichol. Sharp differences were noted for the bovine and cat enzymes with regard to the specificity of degradation. The bovine neutral alpha-mannosidase degraded the substrates by defined pathways, but the cat neutral alpha-mannosidase often produced complex mixtures of products, especially from the larger oligosaccharides. Therefore the bovine enzyme resembled the rat and human cytosolic alpha-mannosidases, but the cat enzyme did not. The bovine and cat neutral alpha-mannosidases, unlike the corresponding lysosomal activities, did not show specificity for the hydrolysis of the (1----3)- and (1----6)-linked mannose residues in the N-linked glycan pentasaccharide core.
Cloning and expression of a hepatic microsomal glucose transport protein. Comparison with liver plasma-membrane glucose-transport protein GLUT 2
Authors: Waddell I D , Zomerschoe A G , Voice M W , Burchell A
Published: August 2015

Antibodies raised against a 52 kDa rat liver microsomal glucose-transport protein were used to screen a rat liver cDNA library. Six positive clones were isolated. Two clones were found to be identical with the liver plasma-membrane glucose-transport protein termed GLUT 2. The sequence of the four remaining clones indicates that they encode a unique microsomal facilitative glucose-transport protein which we have termed GLUT 7. Sequence analysis revealed that the largest GLUT 7 clone was 2161 bp in length and encodes a protein of 528 amino acids. The deduced amino acid sequence of GLUT 7 shows 68% identity with the deduced amino acid sequence of rat liver GLUT 2. The GLUT 7 sequence is six amino acids longer than rat liver GLUT 2, and the extra six amino acids at the C-terminal end contain a consensus motif for retention of membrane-spanning proteins in the endoplasmic reticulum. When the largest GLUT 7 clone was transfected into COS 7 cells the expressed protein was found in the endoplasmic reticulum and nuclear membrane, but not in the plasma membrane. Microsomes isolated from the transfected COS 7 cells demonstrated an increase in their microsomal glucose-transport capacity, demonstrating that the GLUT 7 clone encodes a functional endoplasmic-reticulum glucose-transport protein.
Cell-free synthesis of enzymically active tissue-type plasminogen activator. Protein folding determines the extent of N-linked glycosylation
Authors: Bulleid N J , Bassel-Duby R S , Freedman R B , Sambrook J F , Gething M J H
Published: August 2015

Tissue-type plasminogen activator (t-PA) is synthesized in mammalian cells as a mixture of two forms that differ in their extent of N-linked glycosylation. We have investigated the mechanism underlying this variation in glycosylation, using a cell-free system that consists of a rabbit reticulocyte lysate optimized for the formation of disulphide bonds and supplemented with dog pancreas microsomal membranes. Molecules of human t-PA synthesized in vitro are enzymically active and responsive to natural activators and inhibitors, and are glycosylated in a pattern identical with that of the protein produced in vivo. This demonstrates that t-PA synthesized in vitro folds into the same conformation as the protein synthesized in vivo. We show that the extent of glycosylation of individual t-PA molecules is dependent on the state of folding of the polypeptide chain, since the probability of addition of an oligosaccharide side chain at Asn-184 is decreased under conditions that promote the formation of enzymically active molecules. This variation in glycosylation is independent of the rate of protein synthesis.
Tissue-specific expression of a rat intestinal mucin-like peptide
Authors: Xu G , Wang D , Huan L J , Cutz E , Forstner G G , Forstner J F
Published: August 2015

Expression of the gene for a rat intestinal mucin-like peptide (MLP) was studied by Northern-blot analyses of RNA prepared from a panel of rat tissues. Four probes (A-D) were constructed so as to span a 3.5 kb-long cDNA for rat MLP, and used for hybridization. Positive signals were obtained in intestine and colon, whereas lung, liver, stomach, submandibular gland and spleen were negative. The only transcript detected was approx. 9.5 kb in size. No mRNA splice variants were found. Hybridization in situ using probe B1, which corresponds to a cysteine-rich region near the C-terminus of MLP, confirmed that the gene for MLP is expressed by goblet cells of rat intestine and colon.
Purification and characterization of eight glutathione S-transferase isoenzymes of hamster. Comparison of subunit composition of enzymes from liver, kidney, testis, pancreas and trachea
Authors: Bogaards J J P , van Ommen B , van Bladeren P J V
Published: August 2015

Eight dimeric isoenzymes of glutathione S-transferase (GST) were purified from liver, kidney and testis of the Syrian golden hamster, using S-hexylglutathione affinity chromatography and chromatofocusing. The isoenzymes were characterized according to their substrate selectivity, physical properties and amino acid sequence analysis. Thus a classification into Alpha, Mu and Pi classes was made in analogy with GSTs of other species. Two Alpha-class GSTs were purified, termed A1A1 (pI 8.9) and A1A2 (pI 8.6). Four Mu-class subunits were detected (M1-M4), all forming homodimers, with M2 and M3 also forming a heterodimer. The isoelectric points ranged from 5.9 to 8.6. One Pi-class isoenzyme was purified and termed P1P1 (pI 6.8). Using h.p.l.c. analysis, the subunit composition was determined in a number of organs. The major subunits in liver were A1 and M1. Subunit A1 was also the major subunit in the kidney. Subunit M1 was not detected in kidney, while subunit P1 was not found in the liver. Pancreas and trachea contained predominantly the Pi-class subunit, P1. GST in the testis was mostly of the Mu class. The major subunit was M4, and subunits M2 and M3 were exclusively detected in the testis.
Modification of plasma proteins by cigarette smoke as measured by protein carbonyl formation
Authors: Reznick A Z , Cross C E , Hu M L , Suzuki Y J , Khwaja S , Safadi A , Motchnik P A , Packer L , Halliwell B
Published: August 2015

Exposure of human plasma to gas-phase (but not to whole) cigarette smoke (CS) produces oxidative damage to lipids [Frei, Forte, Ames & Cross (1991) Biochem. J. 277, 133-138], which is prevented by ascorbic acid. The ability of CS to induce protein damage was measured by the carbonyl assay and by loss of enzyme activity and protein -SH groups. Both whole and gas-phase CS caused formation of carbonyls in human plasma, which was partially inhibited by GSH but not by ascorbic acid or metal-ion-chelating agents. Isolated albumin exposed to CS showed much faster carbonyl formation (per unit protein) than did whole plasma; damage to isolated albumin was partially prevented by chelating agents. Isolated creatine kinase (CK) lost activity upon exposure to CS much faster than did CK in plasma. Direct addition to plasma of mixtures of some or all of the aldehydes reported to be present in CS caused protein carbonyl formation and inactivation of CK, but neither occurred to the extent produced by CS exposure.
Production, processing and partial purification of functional G protein βγ subunits in baculovirus-infected insect cells
Authors: Robishaw J D , Kalman V K , Proulx K L
Published: August 2015

As a result of the inability to resolve the heterogeneous mixture of G protein beta gamma subunits present in tissues, it has not been possible to compare different beta gamma subunits of the G proteins in terms of their proposed roles in receptor-effector coupling. This study was undertaken to establish the utility of the baculovirus expression system in producing homogeneous beta gamma subunits of defined composition for the comparative analysis of these subunits in reconstitution systems. In this study we report the expression, and appropriate post-translational processing, of recombinant beta 2, gamma 2 and gamma 3 subunits. In addition, we show that the recombinant beta gamma subunits can be readily purified, and can functionally interact with the alpha subunits of the G proteins.
Comparison of diglyceride production from choline-containing phosphoglycerides in human neutrophils stimulated with N-formylmethionyl-leucylphenylalanine, ionophore A23187 or phorbol 12-myristate 13-acetate
Authors: Chabot M C , McPhail L C , Wykle R L , Kennerly D A , McCall C E
Published: August 2015

The turnover of choline-containing phosphoglycerides (PC) in response to agonist stimulation is well documented in human neutrophils. We have now compared the enzymic pathways of N-formylmethionyl-leucylphenylalanine (fMLP)-, A23187- and phorbol-12-myristate 13-acetate (PMA)-induced diglyceride (DG) and phosphatidic acid (PA) generation in these cells. In order to distinguish between phospholipase C- and D-mediated PC breakdown, human neutrophils were radiolabelled with 1-O-[3H]alkyl-2-acyl-glycero-3-phosphocholine and stimulated in the presence of ethanol or propranolol. The addition of 0.5% ethanol to the incubation mixture resulted in the production of phosphatidylethanol, indicative of phospholipase D activation, in response to all three stimuli. Concomitant with phosphatidylethanol formation was a partial block of PA production. The production of DG was also partially blocked by addition of ethanol. Propranolol (200 microM) was also used to assess the contributions of phospholipases C and D toward DG generation. Inhibition of PA phosphohydrolase by propranolol resulted in the complete abolition of DG generation when neutrophils were stimulated with fMLP. In contrast, propranolol only partially inhibited DG generation in response to A23187 and PMA. These results suggested that DG production in response to fMLP stimulation is mediated via the activation of phospholipase D, whereas A23187- or PMA-induced DG generation may involve more than one pathway. However, examination of the water-soluble choline metabolites produced indicated that phospholipase D was responsible for the production of PA and DG in response to all three stimuli.
Binding characteristics of Mn2+, Co2+ and Mg2+ ions with several d-xylose isomerases
Authors: Van Bastelaere P B M , Callens M , Vangrysperre W A E , Kersters-Hilderson H L M
Published: August 2015

D-Xylose isomerases are metal-ion (Mn2+, Co2+, Mg2+)-requiring tetrameric enzymes. Both the stoichiometry and the binding constants have been determined by titrating the metal-ion-free enzymes from five organisms (Actinomycetaceae and more divergent bacteria) with the respective metal ions using the enzyme activity as indicator of active complex-formation. The following characteristics have been observed for each specific isomerase: (i) two essential metal ion sites (one structural and one catalytic) exist per subunit; (ii) the metal ion binding at one site does not affect the binding at the other site; (iii) of the four possible configurations E, aE, Eb and aEb, only the double-occupied enzyme is active; (iv) the metal ion activation is a time-dependent process; (v) the dissociation constants for both the structural and catalytic sites may be identical or may differ by one or higher orders of magnitude; (vi) metal ion binding is stronger in the order Mn2+ greater than Co2+ much greater than Mg2+; (vii) pronounced increases in Km values concomitant with decreasing equivalents of metal ion added are only observed in the presence of Mg2+ ions.
Modification of vertebrate and algal prolyl 4-hydroxylases and vertebrate lysyl hydroxylase by diethyl pyrocarbonate. Evidence for histidine residues in the catalytic site of 2-oxoglutarate-coupled dioxygenases
Authors: Myllylä R , Günzler V , Kivirikko K I , Kaska D D
Published: August 2015

A search for conserved amino acid residues within the cDNA-derived amino acid sequences of 2-oxoglutarate-coupled dioxygenases revealed the presence of two distinct motifs, spaced 49-71 amino acids apart, toward the C-terminal regions of these proteins. Each of the two common motifs contains an invariant histidine residue at a conserved position. The 2-oxoglutarate-coupled dioxygenases function in diverse processes, including the post-translational hydroxylation of proline and lysine residues in vertebrate collagens and the biosynthesis of microbial cephalosporins, yet they have a common reaction mechanisms, which requires the binding of Fe2+, 2-oxoglutarate, O2 and ascorbate at the catalytic site. The two regions of homology, and specifically the identical histidines, potentially represent functionally important sites related to their catalytic activity. Modification of histidine residues by diethyl pyrocarbonate inactivated vertebrate and algal prolyl 4-hydroxylase and vertebrate lysyl hydroxylase, indicating that histidine residues function in the catalytic site of these 2-oxoglutarate-coupled dioxygenases. Inactivation was prevented by the presence of co-substrates, but not by the peptide substrate. It is proposed that the histidine residues in the conserved motifs may function as Fe(2+)-binding ligands.
Conformational stability of bovine α-crystallin. Evidence for a destabilizing effect of ascorbate
Authors: Santini S A , Mordente A , Meucci E , Miggiano G A D , Martorana G E
Published: August 2015

Short-term incubation of bovine alpha-crystallin with ascorbate alters the protein conformational stability. The denaturation curves with urea and guanidinium-chloride show different patterns, suggesting a deviation from a two-state mechanism owing to the presence of one or more intermediates in the unfolding of ascorbate-modified alpha-crystallin. Furthermore, the latter protein profiles are shifted to lower denaturant concentrations indicating a destabilizing action of ascorbate, which is capable of facilitating protein dissociation into subunits as demonstrated by gel filtration with 1.5 M-urea. The decrease in conformational stability cannot be ascribed to any major structural alteration, but rather to localized changes in the protein molecule. In fact, no difference between native and ascorbate-treated alpha-crystallin can be detected by amino acid analysis but perturbation of the tryptophan and tyrosine environment is indicated by alterations in intrinsic fluorescence. Furthermore, turbidity and light-scattering measurements suggest an involvement of the lysine side chains, since aggregability patterns with acetylsalicylic acid are significantly altered. The ascorbate-destabilizing effect on the conformational stability of alpha-crystallin, probably exerted through oxidative modification of amino acid residues and/or the formation of covalent adducts, provokes unfavourable steric interactions between residues along the polypeptide chains, thus favouring aggregation and insolubilization of crystallins which can lead to cataract formation, as also demonstrated by proteolytic digestion patterns which show a lower rate of degradation of the ascorbate-modified alpha-crystallin.
ATPase-inhibitor proteins of brown-adipose-tissue mitochondria from warm- and cold-acclimated rats
Authors: Yamada E W , Huzel N J , Bose R , Kates A L , Himms-Hagen J
Published: August 2015

1. A group of male Sprague-Dawley rats (5-6 weeks old) was cold-acclimated at 4 degrees C for 4 weeks. Warm-acclimated controls remained at 24 degrees C. Total protein content of brown adipose tissue (BAT) increased more than 3-fold and total uncoupling protein (UCP) content increased more than 6-fold upon cold-acclimation. The concentration of UCP in isolated BAT mitochondria almost doubled. 2. Specific ATPase activity of the non-thermogenic BAT mitochondria (from warm-acclimated controls) was low and increased about 6-fold on addition of 1 microM-Ca2+, which raised free Ca2+ levels (measured by Fura-2) in the incubation media from 1.32 +/- 0.28 microM (mean +/- S.E.M.) to 2.29 +/- 0.39 microM [at which the Ca(2+)-binding ATPase-inhibitor protein (CaBI) is inactivated]. Correspondingly, the specific ATP synthetase activity of the non-thermogenic BAT mitochondria was high and was decreased by 74% by addition of 1 microM-Ca2+. 3. In contrast, specific ATPase activity of thermogenic BAT mitochondria (from cold-acclimated rats) was 5 times that of the control group, and addition of Ca2+ had only a small stimulatory response. Correspondingly, the specific ATP synthetase activity of the thermogenic BAT mitochondria was low, and the decrease by Ca2+ was small, albeit significant. 4. Extracts of BAT mitochondria from both groups of animals contained significant amounts of the ATPase-inhibitor protein of Pullman and Monroy (PMI) as well as of CaBI, as shown by gel electrophoresis. Kinetic studies of inhibition of mitochondrial ATPase activity showed that PMI activity was unaltered in extracts from the thermogenic BAT mitochondria, whereas CaBI activity was slightly but significantly increased. 5. The presence of active ATPase-inhibitor proteins in BAT mitochondria was shown for the first time. We conclude that uncoupling of oxidative phosphorylation occurs in thermogenic BAT mitochondria, even in the presence of the ATPase-inhibitor proteins.
Nitric oxide and cyclic GMP formation induced by interleukin 1β in islets of Langerhans. Evidence for an effector role of nitric oxide in islet dysfunction
Authors: Corbett J A , Wang J L , Hughes J H , Wolf B A , Sweetland M A , Lancaster J R , McDaniel M L
Published: August 2015

Treatment of pancreatic islets with interleukin 1 (IL-1) results in a time-dependent inhibition of glucose-stimulated insulin secretion which has recently been demonstrated to be dependent on the metabolism of L-arginine to nitric oxide. In this report IL-1 beta is shown to induce the accumulation of cyclic GMP (cGMP) in a time-dependent fashion that mimics the time-dependent inhibition of insulin secretion by IL-1 beta. The accumulation of cGMP is dependent on nitric oxide synthase activity, since NG-monomethyl-L-arginine (a competitive inhibitor of nitric oxide synthase) prevents IL-1 beta-induced cGMP accumulation. cGMP formation and nitrite production induced by IL-1 beta pretreatment of islets are also blocked by the protein synthesis inhibitor, cycloheximide. The formation of cGMP does not appear to mediate the inhibitory effects of IL-1 beta on insulin secretion since a concentration of cycloheximide (1 microM) that blocks IL-1 beta-induced inhibition of glucose-stimulated insulin secretion and nitric oxide formation does not prevent cGMP accumulation, thus dissociating the two events. By using e.p.r. spectroscopy, IL-1 beta is shown to induce the formation of a g = 2.04 iron-nitrosyl feature in islets which is prevented by cycloheximide, demonstrating the requirement of protein synthesis for IL-1 beta-induced nitric oxide formation. Iron-nitrosyl complex-formation by islets confirms that IL-1 beta induces the generation of nitric oxide by islets, and provides evidence indicating that nitric oxide mediates destruction of iron-sulphur clusters of iron-containing enzymes. Consistent with the destruction of iron-sulphur centres is the finding that pretreatment of islets with IL-1 beta results in an approx. 60% inhibition of mitochondrial oxidation of D-glucose to CO2. Inhibition of islet glucose oxidation appears to be mediated by nitric oxide since both NMMA and cycloheximide prevent IL-1 beta-induced inhibition of glucose oxidation. These results show that IL-1 beta-induced nitric oxide formation parallels the ability of IL-1 beta to inhibit glucose-stimulated insulin secretion by islets, and that protein synthesis is required for IL-1 beta-induced nitric oxide formation. These results also suggest that nitric oxide mediates IL-1 beta-induced inhibitory effects on the pancreatic beta-cell by functioning as an effector molecule responsible for the destruction of iron-sulphur centres of iron-containing proteins, resulting in an impairment of mitochondrial function.
Activity of carnitine palmitoyltransferase in mitochondrial outer membranes and peroxisomes in digitonin-permeabilized hepatocytes. Selective modulation of mitochondrial enzyme activity by okadaic acid
Authors: Guzmán M , Geelen M J H
Published: August 2015

A procedure is described for the rapid measurement of the activity of mitochondrial-outer-membrane carnitine palmitoyltransferase (CPTo) and peroxisomal carnitine palmitoyltransferase (CPTp) in digitonin-permeabilized hepatocytes. CPTo activity was determined as the tetradecylglycidate (TDGA)-sensitive malonyl-CoA-sensitive CPT activity, whereas CPTp activity was monitored as the TDGA-insensitive malonyl-CoA-sensitive CPT activity. Under these experimental conditions, the respective contributions of CPTo and CPTp to total hepatocellular malonyl-CoA-sensitive CPT activity were 74.6 and 25.4%, which correlated well with the values of 76.9 and 23.1% for the respective contributions of the mitochondrial and the peroxisomal compartment to total hepatocellular palmitate oxidation. The sensitivity of CPTo to inhibition by malonyl-CoA was very similar to that of CPTp; thus 50% inhibition of CPTo and CPTp activities was achieved with malonyl-CoA concentrations of 2.6 +/- 0.5 and 3.0 +/- 0.4 microM respectively. Short-term incubation of hepatocytes with the phosphatase inhibitor okadaic acid (i) increased the activity of CPTo and the rate of mitochondrial palmitate oxidation, (ii) decreased the affinity of CPTo for palmitoyl-CoA substrate, and (iii) decreased the sensitivity of CPTo to inhibition by malonyl-CoA. By contrast, neither the properties of CPTp nor the rate of peroxisomal palmitate oxidation were changed upon incubation of cells with okadaic acid. Results indicate therefore that CPTo, but not CPTp, may be regulated by a mechanism of phosphorylation/dephosphorylation. The physiological relevance of these findings is discussed.
Potent inhibition of endopeptidase 24.16 and endopeptidase 24.15 by the phosphonamide peptide N-(phenylethylphosphonyl)-Gly-l-Pro-l-aminohexanoic acid
Authors: Barelli H , Dive V , Yiotakis A , Vincent J P , Checler F
Published: August 2015

A phosphonamide peptide, N-(phenylethylphosphonyl)-Gly-L-Pro-L-aminohexanoic acid, previously shown to block Clostridium histolyticum collagenases, was examined as a putative inhibitor of endopeptidase 24.16 and endopeptidase 24.15. Hydrolysis of two endopeptidase 24.16 substrates, i.e. 3-carboxy-7-methoxycoumarin (Mcc)-Pro-Leu-Gly-Pro-D-Lys-dinitrophenyl (Dnp) and neurotensin, were completely and dose-dependently inhibited by the phosphonamide inhibitor with KI values of 0.3 and 0.9 nM respectively. In addition, the phosphonamide peptide inhibited the hydrolysis of benzoyl (Bz)-Gly-Ala-Ala-Phe-(pAB) p-aminobenzoate and neurotensin by endopeptidase 24.15 with about a 10-fold lower potency (KI values of 5 and 7.5 nM respectively). The selectivity of this inhibitor towards several exo- and endo-peptidases belonging to the zinc-containing metallopeptidase family established that a 1 microM concentration of this inhibitor was unable to affect leucine aminopeptidase, carboxypeptidase A, angiotensin-converting enzyme and endopeptidase 24.11. The present paper therefore reports on the first hydrophilic highly potent endopeptidase 24.16 inhibitor and describes the most potent inhibitory agent directed towards endopeptidase 24.15 developed to date. These tools should allow one to assess the contribution of endopeptidase 24.16 and endopeptidase 24.15 to the physiological inactivation of neurotensin as well as other neuropeptides.
Characterization of mutant forms of the quinoprotein methanol dehydrogenase lacking an essential calcium ion
Authors: Richardson I W , Anthony C
Published: August 2015

Methanol dehydrogenase (MDH) from Methylobacterium extorquens, Methylophilus methylotrophus, Paracoccus denitrificans and Hyphomicrobium X all contained a single atom of Ca2+ per alpha 2 beta 2 tetramer. The role of Ca2+ was investigated using the MDH from Methylobacterium extorquens. This was shown to be similar to the MDH from Hyphomicrobium X in having 2 mol of prosthetic group (pyrroloquinoline quinine; PQQ) per mol of tetramer, the PQQ being predominantly in the semiquinone form. MDH isolated from the methanol oxidation mutants MoxA-, K- and L- contained no Ca2+. They were identical with the enzyme isolated from wild-type bacteria with respect to molecular size, subunit configuration, pI, N-terminal amino acid sequence and stability under denaturing conditions (low pH, high urea and high guanidinium chloride) and in the nature and content of the prosthetic group (2 mol of PQQ per mol of MDH). They differed in their lack of Ca2+, the oxidation state of the extracted PQQ (fully oxidized), absence of the semiquinone form of PQQ in the enzyme, reactivity with the suicide inhibitor cyclopropanol and absorption spectrum, which indicated that PQQ is bound differently from that in normal MDH. Incubation of MDH from the mutants in calcium salts led to irreversible time-dependent reconstitution of full activity concomitant with restoration of a spectrum corresponding to that of fully reduced normal MDH. It is concluded that Ca2+ in MDH is directly or indirectly involved in binding PQQ in the active site. The MoxA, K and L proteins may be involved in maintaining a high Ca2+ concentration in the periplasm. It is more likely, however, that they fill a ‘chaperone’ function, stabilizing a configuration of MDH which permits incorporation of low concentrations of Ca2+ into the protein.
Purification and properties of a phosphatase in French bean (Phaseolus vulgaris L.) leaves that hydrolyses 2′-carboxy-d-arabinitol 1-phosphate
Authors: Kingston-Smith A H , Major I , Parry M A J , Keys A J
Published: August 2015

An enzyme that releases P(i) from 2-carboxy-D-arabinitol 1-phosphate, a naturally occurring tightly binding inhibitor of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), was purified from leaves of French bean seedlings. It was a monomeric protein of M(r) about 56,000. Catalytic activity was stimulated by increased concentrations of inorganic salts to a maximum at an ionic strength above 0.2. NADPH and D-fructose 1,6-bisphosphate increased the activity of the enzyme in both the presence and absence of 0.2 M-KCl. The pure enzyme did not require dithiothreitol for activity. The pH optimum was 7, the Km for 2-carboxy-D-arabinitol 1-phosphate was 0.43 mM and the specific activity 6.8 mumol/min per mg of protein. The enzyme had little or no activity against phosphate ester intermediates of photosynthetic metabolism and glycolysis but hydrolysed the 1,5-bisphosphates of 2′-carboxy-D-ribitol and 2′-carboxy-D-arabinitol more rapidly than 2′-carboxy-D-arabinitol 1-phosphate.
Hepatic effects of endothelin. Receptor characterization and endothelin-induced signal transduction in hepatocytes
Authors: Gandhi C R , Behal R H , Harvey S A K , Nouchi T A , Olson M S
Published: August 2015

Endothelin, a potent vasoactive peptide originally isolated from the vascular endothelial cells, exerts glycogenolytic and vasoconstrictive actions in the perfused rat liver. In this paper we demonstrate high-affinity binding sites for endothelin-1 (ET-1) on rat hepatocytes. Upon incubation at 37 degrees C, association of ET-1 with hepatocytes occurred in a time-dependent manner, was maximal between 3 and 6 h, and subsequently declined; at this temperature ET-1 was rapidly internalized with the internalized ligand exceeding the surface-bound ligand at all time points. The rate of association of 125I-ET-1 with hepatocytes was much slower when the binding assay was performed at 4 degrees C; sequestration of ET-1 in hepatocytes was also substantially reduced at this temperature. ET-1 was extremely potent in stimulating phosphoinositide metabolism in hepatocytes, with significant activation of this signal transduction process occurring at ET-1 concentrations as low as 0.1 pM, with an EC50 of 1 pM. The effect of ET-1 was coupled via a pertussis toxin-sensitive G-protein. Cholera toxin did not affect ET-1-mediated phosphoinositide metabolism and neither toxin influenced the association of 125I-ET-1 with hepatocytes. PAGE of hepatocyte membranes following exposure of the cells to 125I-ET-1 and cross-linking revealed labelling of three major proteins with apparent molecular masses of 32, 49 and 72 kDa. 125I-ET-1 labelling of each of these proteins was inhibited by unlabelled ET-1, whereas unlabelled ET-3 inhibited the labelling of only the 32 and 49 kDa proteins. 125I-ET-3 labelled the 49 kDa protein and this labelling was inhibited by both unlabelled ET-1 and ET-3. Each of these receptors appears to be functional, since both ET-1 and ET-3 stimulated phosphoinositide metabolism in hepatocytes. Down-regulation of ET-1 association and desensitization of ET-1-induced phosphoinositide metabolism occurred upon incubation of hepatocytes with the homologous ligand. Following down-regulation, the ET-1 receptor was restored to the surface of the hepatocyte by prolonged incubation, although the ET-1-stimulated phosphoinositide response remained inhibited even after complete recovery of the ET-1 association capability. These results demonstrate the presence of multiple high-affinity receptors for ET-1 on hepatocytes and the direct action of this peptide on hepatic parenchymal cells via the phosphoinositide signal transduction pathway.
CD3 and CD2 antigen-mediated CD3 γ-chain phosphorylation in permeabilized human T cells. Regulation by cytosolic phosphatases
Authors: Alexander D R , Brown M H , Tutt A L , Crumpton M J , Shivnan E
Published: August 2015

The role of cytosolic and membrane-associated phosphatases in regulating dephosphorylation of the CD3 antigen gamma-chain has been investigated using streptolysin-O-permeabilized T lymphoblasts and Jurkat T leukaemia cells. Permeabilization of T cells caused a rapid extrusion of cytosolic type 2A phosphatases, but a membrane-associated phosphorylase phosphatase activity remained inside the cells. This activity had the properties characteristic of type 2A phosphatases, being resistant to inhibition by type 1 phosphatase inhibitors, though it was inhibited in a time-dependent manner by ATP or by non-hydrolysable ATP analogues, but not by GTP, CTP, ITP or PPi. The membrane-associated type 2A phosphatase in permeabilized cells did not dephosphorylate the CD3 antigen gamma-chain, suggesting that cytosolic phosphatases dephosphorylate the gamma-chain in situ. Cross-linking the CD2 and CD3 antigens with a bivalent monoclonal antibody in the absence of cytosolic phosphatases induced marked phosphorylation of the CD3 gamma-chain, immunoprecipitated using a novel gamma-chain peptide analogue directed antiserum (TG1). Phosphorylation was inhibited by a protein kinase C (PKC) pseudosubstrate inhibitor, indicating that CD2/CD3-induced gamma-chain phosphorylation is a PKC-mediated event. Activation of T cells either with phorbol 12,13-dibutyrate or by CD2-CD3 cross-linking caused [32P]Pi incorporation into the same gamma-chain Ser residues. The site-mapping data suggested that PKC in situ may incorporate phosphate at the CD3 gamma-chain Ser-123 and Ser-126 residues, but that phosphate is rapidly lost from Ser-123 by cytosolic phosphatase action. Our findings underline the importance of the dual actions of kinases and phosphatases as potential regulators of T cell antigen-receptor complex function.
Guanine nucleotide is essential and Ca2+ is a modulator in the exocytotic reaction of permeabilized rat mast cells
Authors: Lillie T H W , Gomperts B D
Published: August 2015

Exocytosis from metabolically depleted permeabilized rat mast cells was measured in response to provision of Ca2+ and guanine nucleotide [GTP or guanosine 5′-[gamma-thio]triphosphate (GTP[S])]. For cells permeabilized in simple salt solutions (NaCl), both of these effectors were required to induce secretion. Exclusion of Mg2+ caused an increase in both the sensitivity of the system to GTP and the extent of secretion elicited, while having no such effects on secretion induced by GTP[S]. The effect of Mg2+ depletion on the ability of GTP to stimulate secretion is probably due to the dependence on Mg2+ of the GTPase activity of GE (a postulated GTP-binding protein which mediates exocytosis). This argues that a persistent stimulus to the G-protein is required to support secretion. Affinity for both GTP[S] and GTP is enhanced when the cells are permeabilized in zwitterionic electrolytes (glutamate, gamma-aminobutyric acid, glycine) instead of NaCl. Under these conditions, secretion occurs in response to provision of either GTP[S] [in the effective absence of Ca2+ (pCa 9)] or Ca2+ (in the absence of guanine nucleotide). Secretion induced by GTP[S] is strongly promoted by the presence of Mg2+ at concentrations in the millimolar range; this promotion by Mg2+ declines as the concentration of Ca2+ is elevated towards pCa 7. At pCa 6, Mg2+ is without effect. Ca(2+)-induced secretion requires the provision of MgATP. Since this is further enhanced by low concentrations (< 100 microM) and then inhibited by high concentrations of GDP, the essential role of ATP is likely to be in the maintenance of GTP via transphosphorylation by a nucleoside diphosphate kinase reaction. Thus, under conditions of high affinity (glutamate environment), GTP[S] alone is capable of inducing exocytosis. Ca2+ acts in concert with guanine nucleotides: it enhances the rate and extent of secretion and increases the affinity for Mg2+ and guanine nucleotides in the activation of the GTP-binding protein (GE) which regulates exocytosis.
Involvement of intracellular Ca2+ and K+ in dissipation of the mitochondrial membrane potential and cell death induced by extracellular ATP in hepatocytes
Authors: Zoeteweij J P , van de Water B , de Bont H J , Mulder G J , Nagelkerke J F
Published: August 2015

Isolated rat hepatocytes were incubated with extracellular ATP to induce a prolonged increase in intracellular Ca2+ ([Ca2+]i) and a loss of viability within 2 h. By using video-intensified fluorescence microscopy, the effects of exposure to extracellular ATP on [Ca2+]i, mitochondrial membrane potential (MMP) and cell viability were determined simultaneously in individual living hepatocytes. The increase in [Ca2+]i on exposure to ATP was followed by a decreasing MMP; there were big differences between individual cells. Complete loss of the MMP occurred before cell death was observed. Omission of K+ from the incubation medium decreased the cytotoxicity of ATP; under these conditions, intracellular K+ was decreased by more than 80%. Treatment with nigericin also depleted intracellular K+ and decreased ATP-induced toxicity. Protection against loss of viability by means of a decrease in intracellular [K+] was reflected by maintenance of the MMP. These observations suggest that ATP-induced cell death may be caused by a mechanism that has been described for isolated mitochondria: after an increase in Ca2+ levels, a K+ influx into mitochondria is induced, which finally disrupts the MMP and leads to cell death.
Partial purification of a 6-methyladenine mRNA methyltransferase which modifies internal adenine residues
Authors: Tuck M T
Published: August 2015

Two forms of a 6-methyladenine mRNA methyltransferase have been partially purified using a T7 transcript coding for mouse dihydrofolate reductase as an RNA substrate. Both enzyme forms modify internal adenine residues within the RNA substrate. The enzymes were purified 357- and 37-fold respectively from nuclear salt extracts prepared from HeLa cells using DEAE-cellulose and phosphocellulose chromatography. The activity of the first form of the enzyme eluted from DEAE-cellulose (major form) was at least 3-fold greater than that of the second (minor form). H.p.l.c. analysis of the hydrolysed, methylated mRNA substrates demonstrated that both forms of the enzyme produced only 6-methyladenine. The two forms of the enzyme differed in their RNA substrate specificity as well as in the dependence for a 5′ cap structure. The 6-methyladenine mRNA methyltransferase activity was found to be elevated in HeLa nuclei as compared with nuclear extracts from rat kidney and brain. Enzymic activity could not be detected in nuclei from either normal rat liver or regenerating rat liver. In the case of the HeLa cell, activity could only be detected in nuclear extracts, with a small amount in the ribosomal fraction. Other HeLa subcellular fractions were void of activity.
Oscillations of cytosolic free calcium concentration in the presence of intracellular antibodies to phosphatidylinositol 4,5-bisphosphate in voltage-clamped guinea-pig hepatocytes
Authors: Noel J , Fukami K , Hill A M , Capiod T
Published: August 2015

In liver cells, the stimulation of alpha 1-adrenoceptors by noradrenaline induces the production of Ins(1,4,5)P3 through the degradation of membrane polyphosphoinositides [PtdIns(4,5)P2]. InsP3 evokes in turn the release of Ca2+ from internal stores. Our results show that the internal perfusion of single guinea-pig hepatocytes with monoclonal anti-PtdInsP2 antibody blocks the rise in cytosolic free Ca2+ concn. ([Ca2+]i) evoked by noradrenaline, an InsP3-dependent agonist, but not by the monohydroxylated bile acid taurolithocholate 3-sulphate, which is known to permeabilize the endoplasmic reticulum. In these conditions, the bile acid elicited either fast or slow fluctuations of [Ca2+]i independently of any InsP3 production. The responses to the bile acid were also observed in the absence of external Ca2+. The presence of intracellular anti-PtdInsP2 antibody does not affect the response to a photolytic release of InsP3 (1.5 microM final concn.) from a caged precursor.
Ethanol and glycogen synthesis in cardiothoracic and skeletal muscles following glucose re-feeding after starvation in the rat
Authors: Xu D , Thambirajah R , Palmer T N
Published: August 2015

The pattern of glycogen deposition in individual cardiothoracic and skeletal muscles in response to oral and intraperitoneal glucose administration was examined in 40 h-starved rats. Rates of glycogen synthesis were consistently higher in oxidative muscles than in non-oxidative muscles. Intragastric ethanol administration was associated with an impaired glycaemic response and the almost total abolition of glycogen deposition in oxidative muscles in response to oral or intraperitoneal glucose re-feeding. This effect was dose-dependent and differential, in that ethanol produced no equivalent impairment in glycogen deposition in non-oxidative muscles. Ethanol treatment also selectively promoted glycogenolysis in oxidative muscles in the starved state. There was positive correlation (P < 0.001) between the decrease in glycogen levels in soleus and diaphragm muscles in response to increasing ethanol doses and blood glucose and lactate concentrations after intraperitoneal glucose administration, implying that the basis for the impairment in glycogen synthesis may be diminished glucose availability. The mechanism whereby ethanol may differentially compromise carbohydrate metabolism in oxidative muscles is discussed.
Cyclic AMP impairs the rapid effect of insulin to enhance cell-surface insulin-binding capacity in rat adipocytes
Authors: Eriksson J W , Lönnroth P , Smith U
Published: August 2015

The aim of this study was to characterize further the interaction between cyclic AMP (cAMP) and insulin binding and action. Rat adipocytes were preincubated at 37 degrees C for 20 min, and after energy depletion with KCN, cell-surface 125I-insulin binding was measured. As recently reported [Eriksson, Lönnroth & Smith (1992) Diabetes 41, 707-714], preincubation with insulin rapidly increased the number of cell-surface insulin binding sites up to approximately 5-fold through recruitment within the plasma membrane. This was completely abolished by the presence of 4 mM-N6-monobutyryl cAMP (a non-hydrolysable cAMP analogue) or 1 microM-isoprenaline, without any apparent change in receptor internalization. Insulin-stimulated receptor tyrosine kinase activity was attenuated by the cAMP analogue only if the exposure of the adipocytes was prolonged to 60 min. The cellular sensitivity to insulin, assessed as 3-O-methylglucose uptake, was markedly decreased by the cAMP analogue, and this could be attributed to the impaired cell-surface binding. However, evidence for post-receptor interactions between cAMP and insulin was also found: an impairment of maximal insulin-stimulated 3-O-methylglucose transport and a delay in the rate of activation of the glucose transport system by insulin. In conclusion, these data demonstrate that beta-adrenergic stimulation and elevated cAMP levels markedly impair the ability of insulin to enhance cell-surface insulin-binding capacity. This novel interaction may be an important mechanism for the cellular insensitivity to insulin produced by cAMP.
Modulation of aggrecan and link-protein synthesis in articular cartilage
Authors: Curtis A J , Devenish R J , Handley C J
Published: August 2015

The addition of serum or insulin-like growth factor-I (IGF-I) to the medium of explant cultures of bovine articular cartilage is known to stimulate the synthesis of aggrecan in a dose-dependent manner. The half-life of the pool of proteoglycan core protein was measured in adult articular cartilage cultured for 6 days in the presence and absence of 20 ng of IGF-I/ml and shown to be 24 min under both sets of conditions. The half-life of the mRNA pool coding for aggrecan was also determined and shown to be approx. 4 h in cartilage maintained in culture with or without IGF-I. The pool size of mRNA coding for aggrecan core protein increased 5-6-fold in cartilage explants maintained in culture in medium containing 20% (v/v) fetal-calf serum; however, in tissue maintained with medium containing IGF-I there was no increase in the cellular levels of this mRNA. This suggests that aggrecan synthesis is stimulated by IGF-I at the level of translation of mRNA coding for the core protein of this proteoglycan and that other growth factors are present in serum that stimulate aggrecan synthesis at the level of transcription of the core-protein gene. Inclusion of serum or IGF-I in the medium of cartilage explant cultures induced increases in the amounts of mRNA coding for type II collagen and link protein, whereas only serum enhanced the amount of mRNA for the core protein of decorin.
X-ray-crystallographic studies of complexes of pepstatin A and a statine-containing human renin inhibitor with endothiapepsin
Authors: Bailey D , Cooper J B , Veerapandian B , Blundell T L , Atrash B , Jones D M , Szelke M
Published: August 2015

H-189, a synthetic human renin inhibitor, and pepstatin A, a naturally occurring inhibitor of aspartic proteinases, have been co-crystallized with the fungal aspartic proteinase endothiapepsin (EC 3.4.23.6). H-189 [Pro-His-Pro-Phe-His-Sta-(statyl)-Val-Ile-His-Lys] is an analogue of human angiotensinogen. Pepstatin A [Iva(isovaleryl)-Val-Val-Sta-Ala-Sta] is a blocked pentapeptide which inhibits many aspartic proteinases. The structures of the complexes have been determined by X-ray diffraction and refined to crystallographic R-factors of 0.15 and 0.16 at resolutions of 0.18 nm (1.8 A) and 0.2 nm (2.0 A) respectively. H-189 is in an extended conformation, in which the statine residue is a dipeptide analogue of P1 and P'1 as indicated by the conformation and network of contacts and hydrogen bonds. Pepstatin A has an extended conformation to the P'2 alanine residue, but the leucyl side chain of the terminal statine residue binds back into the S'1 subsite, and an inverse gamma-turn occurs between P'1 and P'3. The hydroxy moiety of the statine at P1 in both complexes displaces the solvent molecule that hydrogen-bonds with the catalytic aspartate residues (32 and 215) in the native enzyme. Solvent molecules originally present in the native structure at the active site are displaced on inhibitor binding (12 when pepstatin A binds; 16 when H-189 binds).