The Experts below are selected from a list of 24 Experts worldwide ranked by ideXlab platform
Paul E. Bock - One of the best experts on this subject based on the ideXlab platform.
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Analogs of human plasminogen that are labeled with fluorescence probes at the catalytic site of the zymogen. Preparation, characterization, and interaction with streptokinase.
Journal of Biological Chemistry, 1996Co-Authors: Paul E. Bock, Steven T. Olson, Ingrid M. Verhamme, M. Margarida Bernardo, Joseph D. ShoreAbstract:Abstract Fluorescent analogs of the proteinase zymogen, plasminogen (Pg), which are specifically inactivated and labeled at the catalytic site have been prepared and characterized as probes of the mechanisms of Pg activation. The active site induced non-proteolytically in Pg by streptokinase (SK) was inactivated stoichiometrically with the thioester Peptide Chloromethyl Ketone, Nα-[(acetylthio)acetyl]-(D-Phe)-Phe-Arg-CH2Cl; the thiol group generated subsequently on the incorporated inhibitor with NH2OH was quantitatively labeled with the fluorescence probe, 2-((4′-iodoacetamido)anilino)naphthalene-6-sulfonic acid; and the labeled Pg was separated from SK. Cleavage of labeled [Glu]Pg1 by urokinase-type plasminogen activator (uPA) was accompanied by a fluorescence enhancement (ΔFmax/Fo) of 2.0, and formation of 1% plasmin (Pm) activity. Comparison of labeled and native [Glu]Pg1 as uPA substrates showed that activation of labeled [Glu]Pg1 generated [Glu]Pm1 as the major product, while native [Glu]Pg1 was activated at a faster rate and produced [Lys]Pm1 because of concurrent proteolysis by plasmin. When a mixture of labeled and native Pg was activated, to include plasmin-feedback reactions, the zymogens were activated at equivalent rates. The lack of potential proteolytic activity of the Pg derivatives allowed their interactions with SK to be studied under equilibrium binding conditions. SK bound to labeled [Glu]Pg1 and [Lys]Pg1 with dissociation constants of 590 ± 110 and 11 ± 7 nM, and fluorescence enhancements of 3.1 ± 0.1 and 1.6 ± 0.1, respectively. Characterization of the interaction of SK with native [Glu]Pg1 by the use of labeled [Glu]Pg1 as a probe indicated a ∼6-fold higher affinity of SK for the native Pg zymogen compared to the labeled Pg analog. Saturating levels of e-aminocaproic acid reduced the affinity of SK for labeled [Glu]Pg1 by ∼2-fold and lowered the fluorescence enhancement to 1.8 ± 0.1, whereas the affinity of SK for labeled [Lys]Pg1 was reduced by ∼98-fold with little effect on the enhancement. These results demonstrate that occupation of lysine binding sites modulates the affinity of SK for Pg and the changes in the environment of the catalytic site associated with SK-induced conformational activation. Together, these studies show that the labeled Pg derivatives behave as analogs of native Pg which report functionally significant changes in the environment of the catalytic site of the zymogen.
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Active-site-selective Labeling of Blood Coagulation Proteinases with Fluorescence Probes by the Use of Thioester Peptide Chloromethyl
1992Co-Authors: Paul E. BockAbstract:In a new strategy for labeling the active sites of serine proteinases with fluorescence probes (Bock, P. E. (1988) Biochemistry 27, 6633-6639), a thioester Peptide Chloromethyl Ketone inhibitor is incorporated into the enzyme active center and used to produce a unique thiol group which provides a site for selective chemical modification with any one of many thiolreactive fluorescence probes. This approach was developed to increase the opportunities for identifying fluorescent proteinase derivatives that act as reporters of binding interactions by allowing a large number of derivatives, representing a broad range of probe spectral properties, to be readily prepared. In the studies described here, the specificity of the labeling approach was evaluated quantitatively for the labeling of human a and BIT-thrombin with the thioester Peptide Chloromethyl Ketones, Nu-[(acetylthio)acetyl]-~-Phe-ProArg-CH2Cl and Nu-[(acetylthio)acetyl]-D-Phe-PheArg-CH2Cl, and the thiol-reactive fluorescence probe, 6-(iodoacetamido)fluorescein. Irreversible inactivation of thrombin by the inhibitors was accompanied by incorporation of 0.98 2 0.06 mol/mol of the thioester group into the active site, independent of a 470-fold difference between the thioester Peptide Chloromethyl Ketones in the bimolecular rate constants of a-thrombin affinity labeling. Subsequent mild treatment of the covalent thrombin-inhibitor complexes with NH20H in the presence of 5-(iodoacetamido)fluorescein resulted in generation of the thiol group together with its selective modification and incorporation of 0.96 f 0.07 mol of probelmol of active sites. The incorporated label was localized to a 9000 molecular weight region of (Y and 017-thrombin containing the catalytic-site histidine residue. Evaluation of competing, side reactions showed that they did not significantly compromise the active site specificity of labeling. These results demonstrated equivalent, active-site-selective fluorescence probe labeling of a and @/Bly-thrombin by use of either of the thioester Peptide Chloromethyl Ketones, with a site specificity of 294%.
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Active-site-selective labeling of blood coagulation proteinases with fluorescence probes by the use of thioester Peptide Chloromethyl Ketones. I. Specificity of thrombin labeling.
Journal of Biological Chemistry, 1992Co-Authors: Paul E. BockAbstract:Abstract In a new strategy for labeling the active sites of serine proteinases with fluorescence probes (Bock, P. E. (1988) Biochemistry 27, 6633-6639), a thioester Peptide Chloromethyl Ketone inhibitor is incorporated into the enzyme active center and used to produce a unique thiol group which provides a site for selective chemical modification with any one of many thiol-reactive fluorescence probes. This approach was developed to increase the opportunities for identifying fluorescent proteinase derivatives that act as reporters of binding interactions by allowing a large number of derivatives, representing a broad range of probe spectral properties, to be readily prepared. In the studies described here, the specificity of the labeling approach was evaluated quantitatively for the labeling of human alpha and beta/gamma-thrombin with the thioester Peptide Chloromethyl Ketones, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl and N alpha-[(acetylthio)acetyl]-D-Phe-Phe-Arg-CH2Cl, and the thiol-reactive fluorescence probe, 5-(iodoacetamido)fluorescein. Irreversible inactivation of thrombin by the inhibitors was accompanied by incorporation of 0.98 +/- 0.06 mol/mol of the thioester group into the active site, independent of a 470-fold difference between the thioester Peptide Chloromethyl Ketones in the bimolecular rate constants of alpha-thrombin affinity labeling. Subsequent mild treatment of the covalent thrombin-inhibitor complexes with NH2OH in the presence of 5-(iodoacetamido)fluorescein resulted in generation of the thiol group together with its selective modification and incorporation of 0.96 +/- 0.07 mol of probe/mol of active sites. The incorporated label was localized to a 9000 molecular weight region of alpha and beta/gamma-thrombin containing the catalytic-site histidine residue. Evaluation of competing, side reactions showed that they did not significantly compromise the active site specificity of labeling. These results demonstrated equivalent, active-site-selective fluorescence probe labeling of alpha and beta/gamma-thrombin by use of either of the thioester Peptide Chloromethyl Ketones, with a site specificity of greater than or equal to 94%.
Anne Reiner - One of the best experts on this subject based on the ideXlab platform.
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Characterization of serine proteases of Lumbriculus variegatus and their role in regeneration
Invertebrate Biology, 2012Co-Authors: Kay A. Tweeten, Anne ReinerAbstract:Serine proteases, ubiquitous enzymes known to function in digestion and immune protection in both vertebrates and invertebrates and implicated in regeneration in some species, were investigated in the California blackworm, Lumbriculus variegatus. Several serine proteases, rather than a single enzyme with broad specificity, were present in tissue extracts from the worms. Extracts were treated with a fluorescein-labeled Peptide Chloromethyl Ketone that specifically binds to trypsin/thrombin-like proteases. Denaturing gel electrophoresis of labeled extracts showed several serine proteases with their molecular weight ranging 28,000–38,000 daltons. The trypsin/thrombin-like activity was localized, using the fluorescein-conjugated reagent, to the pharynx and digestive tract of L. variegatus. Movement of cells labeled by the reagent into regenerating tissues suggests that some differentiated endodermal tissues were used for reformation of digestive structures during regeneration in L. variegatus. The types of serine proteases in the extracts were further characterized by inhibitor studies. Presence of plasmin-like activity was indicated by degradation of fibrin by tissue homogenates from the worms and the inhibitory effect of aprotinin on enzymes in these extracts. The ability of L. variegatus extracts to generate clots when incubated with rabbit plasma and partial inhibition of extract activity by phenylmethylsulfonyl fluoride and hirudin indicated presence of thrombin-like activity. Consistent with the detection of trypsin, chymotrypsin, and plasmin-like enzymes in the extracts was partial inhibition of L. variegatus serine protease activity by aminoethyl benzenesulfonyl fluoride and soybean trypsin inhibitor. Selective inhibition of chymotrypsin-like activity by N-tosyl-l-phenylalanine Chloromethyl Ketone and chymostatin as well as trypsin-like activity by N-tosyl-l-lysine Chloromethyl Ketone was observed. A potential role during regeneration for serine proteases is suggested by blockage of formation of head and tail structures by aminoethyl benzenesulfonyl fluoride, an inhibitor of these proteases.
Steven T. Olson - One of the best experts on this subject based on the ideXlab platform.
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Apparent Formation of Sodium Dodecyl Sulfate-stable Complexes between Serpins and 3,4-Dichloroisocoumarin-inactivated Proteinases Is Due to Regeneration of Active Proteinase from the Inactivated Enzyme
Journal of Biological Chemistry, 1997Co-Authors: Steven T. Olson, Richard Swanson, Philip A. Patston, Ingemar BjörkAbstract:Abstract Protein proteinase inhibitors of the serpin family were recently reported to form SDS-stable complexes with inactive serine proteinases modified at the catalytic serine with 3,4-dichloroisocoumarin (DCI) that resembled the complexes formed with the active enzymes (Christensen, S., Valnickova, Z., Thogersen, I. B., Pizzo, S. V., Nielsen, H. R., Roepstorff, P., and Enghild, J. J. (1995) J. Biol. Chem. 270, 14859–14862). The discordance between these findings and other reports that similar active site modifications of serine proteinases block the ability of serpins to form SDS-stable complexes prompted us to investigate the mechanism of complex formation between serpins and DCI-inactivated enzymes. Both neutrophil elastase and β-trypsin inactivated by DCI appeared to form SDS-stable complexes with the serpin, α1-proteinase inhibitor (α1PI), as reported previously. However, several observations suggested that such complex formation resulted from a reaction not with the DCI enzyme but rather with active enzyme regenerated from the DCI enzyme by a rate-limiting hydrolysis reaction. Thus (i) complex formation was blocked by active site-directed Peptide Chloromethyl Ketone inhibitors; (ii) the kinetics of complex formation indicated that the reaction was not second order but rather showed a first-order dependence on DCI enzyme concentration and zero-order dependence on inhibitor concentration; and (iii) complex formation was accompanied by stoichiometric release of a Peptide having the sequence SIPPE corresponding to cleavage at the α1PI reactive center P1-P1′ bond. Quantitation of kinetic constants for DCI and α1PI inactivation of human neutrophil elastase and trypsin and for reactivation of the DCI enzymes showed that the observed complex formation could be fully accounted for by α1PI preferentially reacting with active enzyme regenerated from DCI enzyme during the reaction. These results support previous findings of the critical importance of the proteinase catalytic serine in the formation of SDS-stable serpin-proteinase complexes and are in accord with an inhibitory mechanism in which the proteinase is trapped at the acyl intermediate stage of proteolysis of the serpin as a substrate.
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Analogs of human plasminogen that are labeled with fluorescence probes at the catalytic site of the zymogen. Preparation, characterization, and interaction with streptokinase.
Journal of Biological Chemistry, 1996Co-Authors: Paul E. Bock, Steven T. Olson, Ingrid M. Verhamme, M. Margarida Bernardo, Joseph D. ShoreAbstract:Abstract Fluorescent analogs of the proteinase zymogen, plasminogen (Pg), which are specifically inactivated and labeled at the catalytic site have been prepared and characterized as probes of the mechanisms of Pg activation. The active site induced non-proteolytically in Pg by streptokinase (SK) was inactivated stoichiometrically with the thioester Peptide Chloromethyl Ketone, Nα-[(acetylthio)acetyl]-(D-Phe)-Phe-Arg-CH2Cl; the thiol group generated subsequently on the incorporated inhibitor with NH2OH was quantitatively labeled with the fluorescence probe, 2-((4′-iodoacetamido)anilino)naphthalene-6-sulfonic acid; and the labeled Pg was separated from SK. Cleavage of labeled [Glu]Pg1 by urokinase-type plasminogen activator (uPA) was accompanied by a fluorescence enhancement (ΔFmax/Fo) of 2.0, and formation of 1% plasmin (Pm) activity. Comparison of labeled and native [Glu]Pg1 as uPA substrates showed that activation of labeled [Glu]Pg1 generated [Glu]Pm1 as the major product, while native [Glu]Pg1 was activated at a faster rate and produced [Lys]Pm1 because of concurrent proteolysis by plasmin. When a mixture of labeled and native Pg was activated, to include plasmin-feedback reactions, the zymogens were activated at equivalent rates. The lack of potential proteolytic activity of the Pg derivatives allowed their interactions with SK to be studied under equilibrium binding conditions. SK bound to labeled [Glu]Pg1 and [Lys]Pg1 with dissociation constants of 590 ± 110 and 11 ± 7 nM, and fluorescence enhancements of 3.1 ± 0.1 and 1.6 ± 0.1, respectively. Characterization of the interaction of SK with native [Glu]Pg1 by the use of labeled [Glu]Pg1 as a probe indicated a ∼6-fold higher affinity of SK for the native Pg zymogen compared to the labeled Pg analog. Saturating levels of e-aminocaproic acid reduced the affinity of SK for labeled [Glu]Pg1 by ∼2-fold and lowered the fluorescence enhancement to 1.8 ± 0.1, whereas the affinity of SK for labeled [Lys]Pg1 was reduced by ∼98-fold with little effect on the enhancement. These results demonstrate that occupation of lysine binding sites modulates the affinity of SK for Pg and the changes in the environment of the catalytic site associated with SK-induced conformational activation. Together, these studies show that the labeled Pg derivatives behave as analogs of native Pg which report functionally significant changes in the environment of the catalytic site of the zymogen.
Ingemar Björk - One of the best experts on this subject based on the ideXlab platform.
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Apparent Formation of Sodium Dodecyl Sulfate-stable Complexes between Serpins and 3,4-Dichloroisocoumarin-inactivated Proteinases Is Due to Regeneration of Active Proteinase from the Inactivated Enzyme
Journal of Biological Chemistry, 1997Co-Authors: Steven T. Olson, Richard Swanson, Philip A. Patston, Ingemar BjörkAbstract:Abstract Protein proteinase inhibitors of the serpin family were recently reported to form SDS-stable complexes with inactive serine proteinases modified at the catalytic serine with 3,4-dichloroisocoumarin (DCI) that resembled the complexes formed with the active enzymes (Christensen, S., Valnickova, Z., Thogersen, I. B., Pizzo, S. V., Nielsen, H. R., Roepstorff, P., and Enghild, J. J. (1995) J. Biol. Chem. 270, 14859–14862). The discordance between these findings and other reports that similar active site modifications of serine proteinases block the ability of serpins to form SDS-stable complexes prompted us to investigate the mechanism of complex formation between serpins and DCI-inactivated enzymes. Both neutrophil elastase and β-trypsin inactivated by DCI appeared to form SDS-stable complexes with the serpin, α1-proteinase inhibitor (α1PI), as reported previously. However, several observations suggested that such complex formation resulted from a reaction not with the DCI enzyme but rather with active enzyme regenerated from the DCI enzyme by a rate-limiting hydrolysis reaction. Thus (i) complex formation was blocked by active site-directed Peptide Chloromethyl Ketone inhibitors; (ii) the kinetics of complex formation indicated that the reaction was not second order but rather showed a first-order dependence on DCI enzyme concentration and zero-order dependence on inhibitor concentration; and (iii) complex formation was accompanied by stoichiometric release of a Peptide having the sequence SIPPE corresponding to cleavage at the α1PI reactive center P1-P1′ bond. Quantitation of kinetic constants for DCI and α1PI inactivation of human neutrophil elastase and trypsin and for reactivation of the DCI enzymes showed that the observed complex formation could be fully accounted for by α1PI preferentially reacting with active enzyme regenerated from DCI enzyme during the reaction. These results support previous findings of the critical importance of the proteinase catalytic serine in the formation of SDS-stable serpin-proteinase complexes and are in accord with an inhibitory mechanism in which the proteinase is trapped at the acyl intermediate stage of proteolysis of the serpin as a substrate.
Kay A. Tweeten - One of the best experts on this subject based on the ideXlab platform.
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Characterization of serine proteases of Lumbriculus variegatus and their role in regeneration
Invertebrate Biology, 2012Co-Authors: Kay A. Tweeten, Anne ReinerAbstract:Serine proteases, ubiquitous enzymes known to function in digestion and immune protection in both vertebrates and invertebrates and implicated in regeneration in some species, were investigated in the California blackworm, Lumbriculus variegatus. Several serine proteases, rather than a single enzyme with broad specificity, were present in tissue extracts from the worms. Extracts were treated with a fluorescein-labeled Peptide Chloromethyl Ketone that specifically binds to trypsin/thrombin-like proteases. Denaturing gel electrophoresis of labeled extracts showed several serine proteases with their molecular weight ranging 28,000–38,000 daltons. The trypsin/thrombin-like activity was localized, using the fluorescein-conjugated reagent, to the pharynx and digestive tract of L. variegatus. Movement of cells labeled by the reagent into regenerating tissues suggests that some differentiated endodermal tissues were used for reformation of digestive structures during regeneration in L. variegatus. The types of serine proteases in the extracts were further characterized by inhibitor studies. Presence of plasmin-like activity was indicated by degradation of fibrin by tissue homogenates from the worms and the inhibitory effect of aprotinin on enzymes in these extracts. The ability of L. variegatus extracts to generate clots when incubated with rabbit plasma and partial inhibition of extract activity by phenylmethylsulfonyl fluoride and hirudin indicated presence of thrombin-like activity. Consistent with the detection of trypsin, chymotrypsin, and plasmin-like enzymes in the extracts was partial inhibition of L. variegatus serine protease activity by aminoethyl benzenesulfonyl fluoride and soybean trypsin inhibitor. Selective inhibition of chymotrypsin-like activity by N-tosyl-l-phenylalanine Chloromethyl Ketone and chymostatin as well as trypsin-like activity by N-tosyl-l-lysine Chloromethyl Ketone was observed. A potential role during regeneration for serine proteases is suggested by blockage of formation of head and tail structures by aminoethyl benzenesulfonyl fluoride, an inhibitor of these proteases.