The Experts below are selected from a list of 33 Experts worldwide ranked by ideXlab platform

Jüri Siigur - One of the best experts on this subject based on the ideXlab platform.

  • Isolation, properties and N-terminal amino acid sequence of a factor V activator from Vipera lebetina (Levantine viper) snake venom
    Biochimica et biophysica acta, 1998
    Co-Authors: Ene Siigur, Mari Samel, Külli Tõnismägi, Juhan Subbi, Tõnu Reintamm, Jüri Siigur
    Abstract:

    Abstract A factor V activator (VLFVA) was separated from Vipera lebetina venom by gel filtration on Sephadex G-100 superfine, followed by chromatography on CM-cellulose and on heparin-agarose. This enzyme (VLFVA) with a molecular mass of 28.4 kDa, as determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry, is a single-chain glycoprotein containing seven residues of neutral sugars, seven residues of hexosamines and three residues of neuraminic acid per molecule. The treatment with N-glycosidase F lowered the molecular mass approximately 6%. The N-terminal sequencing of VLFVA up to the 30th residue evidenced a high homology with Vipera russelli factor V activator RVV-Vγ (90% identity). Aside from factor V, no other protein substrate for VLFVA has yet been identified. VLFVA hydrolyzes several synthetic arginine Ester substrates, such as benzoylarginine ethyl Ester (BAEE), Tosylarginine Methyl Ester (TAME) and amide substrates such as Pro-Phe-Arg-MCA. The arginine Ester hydrolase activity of the enzyme is markedly lower than that of the crude venom. The ability of VLFVA to activate factor V and its activity to BAEE and TAME were inhibited by the serine proteinase inhibitor, diisopropylfluorophosphate. VLFVA is thermostable protein, heating for 20 min at 70°C does not alter the arginine Esterase activity of the enzyme.

Steven T. Olson - One of the best experts on this subject based on the ideXlab platform.

  • The pH dependence of serpin-proteinase complex dissociation reveals a mechanism of complex stabilization involving inactive and active conformational states of the proteinase which are perturbable by calcium.
    The Journal of biological chemistry, 2001
    Co-Authors: Sergei V. Calugaru, Richard Swanson, Steven T. Olson
    Abstract:

    Abstract Serpin family protein proteinase inhibitors trap proteinases at the acyl-intermediate stage of cleavage of the serpin as a proteinase substrate by undergoing a dramatic conformational change, which is thought to distort the proteinase active site and slow deacylation. To investigate the extent to which proteinase catalytic function is defective in the serpin-proteinase complex, we compared the pH dependence of dissociation of several serpin-proteinase acyl-complexes with that of normal guanidinobenzoyl-proteinase acyl-intermediate complexes. Whereas the apparent rate constant for dissociation of guanidinobenzoyl-proteinase complexes (k diss, app) showed a pH dependence characteristic of His-57 catalysis of complex deacylation, the pH dependence of k diss, app for the serpin-proteinase complexes showed no evidence for His-57 involvement in complex deacylation and was instead characteristic of a hydroxide-mediated deacylation similar to that observed for the hydrolysis of Tosylarginine Methyl Ester. Hydroxylamine enhanced the rate of serpin-proteinase complex dissociation but with a rate constant for nucleophilic attack on the acyl bond several orders of magnitude slower than that of hydroxide, implying limited accessibility of the acyl bond in the complex. The addition of 10–100 mmCa2+ ions stimulated up to 80-fold the dissociation rate constant of several serpin-trypsin complexes in a saturable manner at neutral pH and altered the pH dependence to a pattern characteristic of His-57-catalyzed complex deacylation. These results support a mechanism of kinetic stabilization of serpin-proteinase complexes wherein the complex is trapped as an acyl-intermediate by a serpin conformational change-induced inactivation of the proteinase catalytic function, but suggest that the inactive proteinase conformation in the complex is in equilibrium with an active proteinase conformation that can be stabilized by the preferential binding of an allosteric ligand such as Ca2+.

Ene Siigur - One of the best experts on this subject based on the ideXlab platform.

  • Isolation, properties and N-terminal amino acid sequence of a factor V activator from Vipera lebetina (Levantine viper) snake venom
    Biochimica et biophysica acta, 1998
    Co-Authors: Ene Siigur, Mari Samel, Külli Tõnismägi, Juhan Subbi, Tõnu Reintamm, Jüri Siigur
    Abstract:

    Abstract A factor V activator (VLFVA) was separated from Vipera lebetina venom by gel filtration on Sephadex G-100 superfine, followed by chromatography on CM-cellulose and on heparin-agarose. This enzyme (VLFVA) with a molecular mass of 28.4 kDa, as determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry, is a single-chain glycoprotein containing seven residues of neutral sugars, seven residues of hexosamines and three residues of neuraminic acid per molecule. The treatment with N-glycosidase F lowered the molecular mass approximately 6%. The N-terminal sequencing of VLFVA up to the 30th residue evidenced a high homology with Vipera russelli factor V activator RVV-Vγ (90% identity). Aside from factor V, no other protein substrate for VLFVA has yet been identified. VLFVA hydrolyzes several synthetic arginine Ester substrates, such as benzoylarginine ethyl Ester (BAEE), Tosylarginine Methyl Ester (TAME) and amide substrates such as Pro-Phe-Arg-MCA. The arginine Ester hydrolase activity of the enzyme is markedly lower than that of the crude venom. The ability of VLFVA to activate factor V and its activity to BAEE and TAME were inhibited by the serine proteinase inhibitor, diisopropylfluorophosphate. VLFVA is thermostable protein, heating for 20 min at 70°C does not alter the arginine Esterase activity of the enzyme.

Sergei V. Calugaru - One of the best experts on this subject based on the ideXlab platform.

  • The pH dependence of serpin-proteinase complex dissociation reveals a mechanism of complex stabilization involving inactive and active conformational states of the proteinase which are perturbable by calcium.
    The Journal of biological chemistry, 2001
    Co-Authors: Sergei V. Calugaru, Richard Swanson, Steven T. Olson
    Abstract:

    Abstract Serpin family protein proteinase inhibitors trap proteinases at the acyl-intermediate stage of cleavage of the serpin as a proteinase substrate by undergoing a dramatic conformational change, which is thought to distort the proteinase active site and slow deacylation. To investigate the extent to which proteinase catalytic function is defective in the serpin-proteinase complex, we compared the pH dependence of dissociation of several serpin-proteinase acyl-complexes with that of normal guanidinobenzoyl-proteinase acyl-intermediate complexes. Whereas the apparent rate constant for dissociation of guanidinobenzoyl-proteinase complexes (k diss, app) showed a pH dependence characteristic of His-57 catalysis of complex deacylation, the pH dependence of k diss, app for the serpin-proteinase complexes showed no evidence for His-57 involvement in complex deacylation and was instead characteristic of a hydroxide-mediated deacylation similar to that observed for the hydrolysis of Tosylarginine Methyl Ester. Hydroxylamine enhanced the rate of serpin-proteinase complex dissociation but with a rate constant for nucleophilic attack on the acyl bond several orders of magnitude slower than that of hydroxide, implying limited accessibility of the acyl bond in the complex. The addition of 10–100 mmCa2+ ions stimulated up to 80-fold the dissociation rate constant of several serpin-trypsin complexes in a saturable manner at neutral pH and altered the pH dependence to a pattern characteristic of His-57-catalyzed complex deacylation. These results support a mechanism of kinetic stabilization of serpin-proteinase complexes wherein the complex is trapped as an acyl-intermediate by a serpin conformational change-induced inactivation of the proteinase catalytic function, but suggest that the inactive proteinase conformation in the complex is in equilibrium with an active proteinase conformation that can be stabilized by the preferential binding of an allosteric ligand such as Ca2+.

Külli Tõnismägi - One of the best experts on this subject based on the ideXlab platform.

  • Isolation, properties and N-terminal amino acid sequence of a factor V activator from Vipera lebetina (Levantine viper) snake venom
    Biochimica et biophysica acta, 1998
    Co-Authors: Ene Siigur, Mari Samel, Külli Tõnismägi, Juhan Subbi, Tõnu Reintamm, Jüri Siigur
    Abstract:

    Abstract A factor V activator (VLFVA) was separated from Vipera lebetina venom by gel filtration on Sephadex G-100 superfine, followed by chromatography on CM-cellulose and on heparin-agarose. This enzyme (VLFVA) with a molecular mass of 28.4 kDa, as determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry, is a single-chain glycoprotein containing seven residues of neutral sugars, seven residues of hexosamines and three residues of neuraminic acid per molecule. The treatment with N-glycosidase F lowered the molecular mass approximately 6%. The N-terminal sequencing of VLFVA up to the 30th residue evidenced a high homology with Vipera russelli factor V activator RVV-Vγ (90% identity). Aside from factor V, no other protein substrate for VLFVA has yet been identified. VLFVA hydrolyzes several synthetic arginine Ester substrates, such as benzoylarginine ethyl Ester (BAEE), Tosylarginine Methyl Ester (TAME) and amide substrates such as Pro-Phe-Arg-MCA. The arginine Ester hydrolase activity of the enzyme is markedly lower than that of the crude venom. The ability of VLFVA to activate factor V and its activity to BAEE and TAME were inhibited by the serine proteinase inhibitor, diisopropylfluorophosphate. VLFVA is thermostable protein, heating for 20 min at 70°C does not alter the arginine Esterase activity of the enzyme.