Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein C is a vitamin K dependent protein present in bovine plasma (Stenflo, J. (1976), J. Biol. Chem. 251, 355). It is a glycoprotein (mol wt approximately 62 000) composed of a heavy chain (mol wt 41 000) and a light chain (mol wt 21 000). The heavy chain has an amino-terminal sequence of Asp-Thr-Asn-Gln and contains nearly three-fourths of the carbohydrate. The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe. Incubation of protein C with either factor X activator from Russell's viper venom or trypsin resulted in the cleavage of an Arg-Ile bond between residues 14 and 15 of the heavy chain. Concomitant with this cleavage was the formation of a serine enzyme which was inhibited by diisopropyl phosphorofluoridate. Liberation of the tetradecapeptide decreased the molecular weight of the heavy chain from about 41 000 to 39 000 and resulted in the formation of a new amino-terminal sequence of Ile-Val-Asp-Gly in the heavy chain. No change in the molecular weight of the light chain was observed during the activation reaction. These results indicate that protein C, like the four vitamin K dependent coagulation proteins, exists in plasma in a precursor form and is converted to a serine protease by hydrolysis of a specific Arg-Ile peptide bond. The biological substrate for the enzymatic form of protein C and the physiological mechanism whereby protein C is converted to a serine enzyme are not known.
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PMID:Proteolytic activation of protein C from bovine plasma. 99 Feb 50

The protease from Russell's viper venom that activates factor X (Stuart factor), factor IX (Christmas factor), and protein C was purified by gel filtration on Sephadex G-150 and QAE-Sephadex A-50 column chromatography. The purified enzyme migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 79 000. A minimal molecular weight of 78 500 +/- 800 was determined by sedimentation equilibrium in the presence of 6 M guanidine hydrochloride. Upon reduction with 2-mercaptoethanol, a heavy chain (mol wt 59 000) and a light chain were observed. The light chain migrated as a single band (mol wt 19 000) in 7.5% polyacrylamide-sodium dodecyl sulfate gels but appeared as a doublet (mol wt 18 000 and 20 000) in 10% polyacrylamide-sodium dodecyl sulfate gels. The amino-terminal end of the heavy chain was heterogeneous and contained isoleucine, valine and serine. The amino-terminal sequence of the light chain was Val-Leu-Asp. The factor X activator contained 13% carbohydrate including 6.0% hexose, 1.7% N-acetyleneuraminic acid, and 5.3% galactosamine. Most of the carbohydrate was found to be present in the heavy chain, although some was also observed in both forms of the light chain. The factor X activator had no esterase activity toward benzoyl-Phe-Val-Arg-p-nitroanilide or benzoylarginine ethyl ester and was not inhibited by 0.05 M diisopropyl phosphorofluoridate. These data indicate that factor X activator from Russell's viper venom is a highly specific protease composed of one heavy chain and one light chain, and these chains are held together by a disulfide bond(s).
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PMID:Factor X activating enzyme from Russell's viper venom: isolation and characterization. 99 Feb 51

Activated protein C has been observed to bind to the light chains of factor Va and factor VIII. Fragments of the factor VIII light chain were produced by recombinant DNA techniques and expressed in Escherichia coli. Three fragments of the light chain were studied; L4 (residues 1974-2332), L3.2 (residues 1560-1829 and 2046-2332), and L3.3 (residues 1560-2052). Two fragments, L4 and L3.3, which overlapped sequences between residues 1974-2052, inhibited the anticoagulant activity of activated protein C. Comparison of the sequences of factors V and VIII in this region revealed that residues 2005-2018 in the factor VIII sequence were homologous with residues 1861-1874 in the factor V sequence. The peptides Arg-Ala-Gly-Met-Gln-Thr-Phe-Leu-Ile (RAGMQTPFLI; residues 1865-1874) from the factor V sequence and His-Ala-Gly-Met-Ser-Thr-Leu-Phe-Ile-Val (HAGMSTLFIV; residues 2009-2018) from the factor VIII sequence were synthesized. Both peptides were observed to inhibit the anticoagulant activity of activated protein C and its inactivation of factors Va and VIII. Furthermore RAGMQTPFLI quenched the fluorescence of the dansyl-Glu-Gly-Arg-modified protease. Polyclonal antibodies against RAGMQTPFLI bound to factor Va and inhibited the anticoagulant activity of activated protein C and the inactivation of factor Va. These results indicate that a portion of the binding sites for activated protein C on the light chains of factors V and VIII are contained in the sequences RAGMQTPFLI or HAGMSTLFIV, respectively.
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PMID:Identification of the binding site for activated protein C on the light chain of factors V and VIII. 213 54

A group of leupeptin analogues was found in Streptomyces griseus strain 254, isolated from a soil sample from Fujian Province, China. The inhibitors excreted in the culture filtrate were purified by adsorption on macroporous resin, followed by sequential ion exchange chromatography on DEAE-52 cellulose, CM-32 cellulose and affinity chromatography with immobilized trypsin. The preparation thus obtained was further purified by preparative HPLC. Several major components were found and characterized, which possessed different inhibitory properties toward trypsin. Based upon amino acid and mass spectrophotometric analysis, these peptides were placed in four major structural categories, viz., R-Val-Val-argininal, R-Leu-Leu-argininal, R-Ile-Ile-argininal and R-Thr-Thr-argininal, this latter component representing a newly identified leupeptin analogue. The structural variability of the R-group was partly responsible for the multiplicity of the peaks obtained with HPLC. All peptides displayed varying degrees of inhibitory activity toward proteases involved in blood coagulation and fibrinolysis, including plasmin, factor Xa, activated protein C and thrombin. Among these peptide inhibitors, the molecule containing threonine showed the strongest inhibitory activity.
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PMID:The inhibition of the enzymic activity of blood coagulation and fibrinolytic serine proteases by a new leupeptin-like inhibitor, and its structural analogues, isolated from Streptomyces griseus. 250 6

Factor VIII functions in the intrinsic pathway of coagulation as the cofactor for factor IXa proteolytic activation of factor X. Proteolytic cleavage is required for activation and may be responsible for inactivation of cofactor activity. To identify which of the multiple cleavages are required for activation and inactivation of factor VIII, site-directed DNA-mediated mutagenesis of the factor VIII cDNA was performed and the altered forms of factor VIII were expressed in COS-1 monkey cells and characterized. Conversion of arginine residues to isoleucine residues at the aminoterminal side of the cleavage sites at positions 740, 1648, and 1721 resulted in cleavage resistance at the modified site with no alteration in the in vitro procoagulant activity and the susceptibility to thrombin activation. Similar modification of the thrombin cleavage sites at either position 372 or position 1689 resulted in molecules with residual factor VIII activity but resistant to thrombin cleavage at the modified site and not susceptible to thrombin activation. Modification of the arginine to either an isoleucine or a lysine at residue 336, the site postulated for proteolytic inactivation by activated protein C, resulted in a factor VIII molecule with increased procoagulant activity. This increased activity may result from greater resistance to proteolytic inactivation. A model for the activation and inactivation of factor VIII is proposed.
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PMID:Proteolytic requirements for thrombin activation of anti-hemophilic factor (factor VIII). 312 86

An enzyme capable of activating protein C has been purified 60-fold from the venom of the Southern copperhead snake (Agkistrodon contortrix) by ion-exchange and gel filtration chromatography. The purified enzyme consists of a single polypeptide with an apparent molecular weight of 37,000. The isoelectric point of the protein C activator was determined to be 6.3 when measured by chromatofocusing. The enzyme was inhibited by p-nitrophenyl p-guanidinobenzoate, phenylmethanesulfonyl fluoride, and D-Phe-Pro-Arg-CH2Cl but was not affected by cysteine-directed reagents or by metal chelators. These results suggest that the enzyme is a serine protease. Protein C activator was capable of hydrolyzing the thrombin substrate tosyl-Gly-Pro-Arg-p-nitroanilide (TGPRpNA), and steady-state kinetic studies determined that the Km for amidolysis of this substrate was 1.1 mM while the Vmax was 66 s-1. The activator demonstrated considerable substrate specificity since the amidolysis of D-Phe-Pip-Arg-pNA, D-Ile-Pro-Arg-pNA, Bz-Ile-Glu-Gly-Arg-pNA, D-Val-Leu-Arg-pNA, and pyrGlu-Pro-Arg-pNA was less than 10% of that of TGPRpNA when measured under identical conditions using 1.0 mM substrate concentrations. The enzyme appears to be thrombin-like in its preference for arginyl as compared to lysyl chloromethyl ketones as well as by its inhibition by benzamidine and p-aminobenzamidine. However, the substrate specificity of the activator is distinguished from alpha-thrombin in that it does not clot fibrinogen and does not react with antithrombin III or hirudin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of a protein C activator from the venom of Agkistrodon contortrix contortrix. 338 39

The protease from Southern Copperhead venom that activates protein C was purified to homogeneity by sulfopropyl (SP)-Sephadex C-50 ion-exchange chromatography, Sephadex G-150 gel filtration, and Mono-S fast protein liquid chromatography. The purified enzyme is a glycoprotein containing 16% carbohydrate, and migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 40,000 kDa. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Ile-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His. The purified venom protein C activator hydrolyzed several tripeptide p-nitroanilides. The amidolytic and proteolytic activities of the enzyme were readily inhibited by phenylmethanesulfonyl fluoride, p-amidinophenylmethanesulfonyl fluoride, chloromethyl ketones, and human antithrombin III. Covalent binding of diisopropyl fluorophosphate to the enzyme was confirmed using a tritium-labeled preparation of the inhibitor. The venom protease readily activated human and bovine protein C at 1:1000 enzyme:substrate weight ratio. The protease also cleaved human prothrombin, factor X, factor IX, factor VII, and fibrinogen. Prothrombin coagulant activity decreased upon incubation with the venom protease, and the rate of this reaction was reduced in the presence of calcium. Factor X and factor IX coagulant activity increased upon incubation with the venom protease in the presence of calcium, and decreased in the absence of calcium. Human factor VII clotting activity decreased slightly upon incubation with the venom protease. Although the venom protease did not clot human fibrinogen, it nonetheless cleaved the A alpha chain of fibrinogen, and this cleavage appeared to be associated with a measurable increase in the clottability of the protease-treated fibrinogen by thrombin. These data demonstrate that the protein C activator from Southern Copperhead venom is a typical serine protease with a relatively broad specificity.
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PMID:Characterization of a protein C activator from Agkistrodon contortrix contortrix venom. 362 72

Previous work has shown that the light chain of protein C, an anticoagulant plasma protein, contains an unusual amino acid [Fernlund, P. & Stenflo, J. (1982) J. Biol. Chem. 257, 12170-12179]. To determine the structure of this amino acid a heptapeptide, CMCys-Ile-X-Gly-Leu-Gly-Gly (residues 69-75 in the light chain), was isolated from enzymatic digests of the light chain. According to automatic Edman sequence analysis, 1H NMR spectroscopy, and mass spectrometry the heptapeptide had beta-hydroxyaspartic acid in its third position, which corresponds to position 71 in the light chain of protein C. Analysis of acid and aminopeptidase M hydrolysates of the heptapeptide showed the beta-hydroxyaspartic acid to be the erythro form. Acid hydrolysis of protein C released approximately equal to 1 mol of beta-hydroxyaspartic acid per mol of protein. The function of this amino acid, which, to the best of our knowledge, has not been found previously in proteins, is unknown.
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PMID:beta-Hydroxyaspartic acid in vitamin K-dependent protein C. 657 39

Processing of intracellular proteins yields 8-11 residue peptides that are displayed on the APC surface as peptide/MHC class I complexes. The remarkably precise excision of antigenic peptides from their precursor polypeptides raises the question of whether specific flanking residues influence presentation efficiency. Here we addressed this question by analyzing the generation of OVA/Kb or influenza nucleoprotein/Db complexes in APC expressing precursors with varying N- or C-terminal flanking residues. We find that T cell responses were not significantly affected by varying the N-terminal flanking residue in the precursors. In contrast, presentation of peptide/MHC complexes was inhibited with the addition of a single C-terminal flanking residue. The most dramatic inhibition was observed with isoleucine, leucine, cysteine, and proline as the C-terminal flanking residues. These residue-specific variations in presentation activity could not be accounted for by differences in the stimulatory activity of corresponding synthetic peptides but were proportional to the relative amounts of naturally processed peptides recovered in the cell extracts. These findings suggest differences in the susceptibility of N- vs C-terminal flanking residues to proteolytic cleavage during Ag processing. The strong influence of specific C-terminal flanking residue(s) could be an important factor affecting the choice of peptides presented to T cells on the APC surface.
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PMID:Presentation of endogenous peptide/MHC class I complexes is profoundly influenced by specific C-terminal flanking residues. 759 93

Peptides corresponding to the loop regions of the fourth, fifth, and sixth epidermal growth factor (EGF)-like domains of thrombomodulin (TM) have been synthesized and assayed for thrombin inhibition, as indicated by both inhibition of thrombin-mediated fibrinogen clotting and inhibition of the association of thrombin with TM that results in protein C activation. Peptides from the fifth EGF-like domain showed significant inhibition of fibrinogen clotting and protein C activation, whereas peptides from the fourth and sixth EGF-like domains were weak inhibitors in both assays. Two structural features were important for inhibitory potency of the peptides from the fifth EGF-like domain: cyclization by a disulfide bond and attachment of the "tail" amino acids C-terminal to the disulfide loop. Linear control peptides did not significantly inhibit clotting or protein C activation. The C-terminal loop alone, the "tail" peptide, or a mixture of the two were at least 10-fold less potent inhibitors of clotting or protein C activation. A more constrained peptide analog was designed by deletion of an isoleucine within the C5-C6 disulfide loop, TM52-1 + 5C. This analog was a better inhibitor in both assay systems, having a Ki for protein C activation of 26 microM.
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PMID:Thrombin inhibition by cyclic peptides from thrombomodulin. 761 75


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