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)

The amino acid sequence of the light chain of bovine protein C was determined by sequenator analysis of the carboxymethylated light chain and fragments obtained by cyanogen bromide treatment, tryptic digestion after blocking of lysine residues, and cleavage with 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine (BNPS-skatole). The sequence was (in the standard one-letter code) A-N-S-F-L-X-X-L-R-P-G-N-V-X-R-X-C-S-X-X-V-C-X-F-X-X-A-R-X-I-F-Q-N-T-X-D-T-M-A-F-W-S-K-Y-S-D-G-D-Q-C-E-D-R-P-S-G-S-P-C-D-L-P-C-C-G-R-G-K-C-I-H-G-L-G-G-F-R-C-D-C-A-E-G-W-E-G-R-F-C-L-H-E-V-R-F-S-N-C-S-A-E-B-G-G-C-A-H-Y-C-M-E-E-E-G-R-R-H-C-S-C-A-P-G-Y-R-L-E-D-D-H-Q-L-C-V-S-K-V-T-F-P-C-G-R-L-G-K-R-M-E-K-K-R-K-T-L. The first eleven glutamic acid residues were carboxylated to gamma-carboxyglutamic acid (X). The NH2-terminal, vitamin K-dependent part showed an extensive homology to both prothrombin and factor X, whereas the rest of the chain showed a strong homology to factor X but little similarity to prothrombin.
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PMID:Bovine protein C: amino acid sequence of the light chain. 28 10

Improved methods are described to obtain bovine prothrombin, Factor IX, Protein C, and autoprothrombin III (Factor X, Auto-III) in purified form. The prothrombin had a specific activity of 4,340 Iowa units/mg. Theoretically, a preparation of clean thrombin should have a specific activity of 8,200 U/mg, because 47.08% of the protein in prothrombin is lost when thrombin forms. Such thrombin preparations have been obtained (Arch. Biochem. Biophys. 121, 372 (1967)). The prothrombin concentration of bovine plasma is near 60 mg/liter. Protein C, first isolated by Stenflo (J. Biol. Chem. 251, 355 (1976)), was found to be the precursor of autoprothrombin II-A (Auto-II-A), discovered earlier (Thromb. Diath. Haemorrh. 5, 218 (1960)). Protein C (Factor XIV) was converted to Auto-II-A (Factor XIVa) by thrombin. Digesting purified Auto-III with purified thrombin removed a small glycopeptide from the COOH-terminal end of the heavy chain to yield Auto-IIIm. Auto-III thrombin leads to Auto-IIIm + peptide. Auto-IIIm was not converted to the active enzyme with thromboplastin, and furthermore, inhibited the activation of purified native Auto-III with thromboplastin. Auto-IIIm was also not converted to the active enzymes when the procoagulants consisted of purified Factor VIII, purified Factor IXa, platelet factor 3 and calcium ions. The "activation peptide" released by RVV-X from the NH2-terminal end of the heavy chain and the active enzyme (Auto-Cm) were purified. Auto-III was also activated with purified RVV-X. The same "actid of Auto-Cm. Purified Factor IX developed anticoagulant activity when reacted with an optimum concentration of purified thrombin. A suitable reagent for the assay of Factor IX was prepared by removing prothrombin complex from anticoagulated bovine plasma and restoring the prothrombin and Auto-III concentration with use of the respective purified proenzymes.
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PMID:Improved procedures for the purification of selected vitamin K-dependent proteins. 78 72

Two major proteins, termed proteins A and B, and one minor species, termed protein C, have been purified to homogeneity from dilute acid extracts of dormant spores of Bacillus megaterium. These three species comprise approximately 80% of the protein in the dilute acid extracts and account for 60 to 75% of the protein degraded during spore germination. All three proteins have low molecular weights (7,000 to 10,000), high isoelectric points (greater than 9.8), alanine as the NH2-terminal amino acid, are more hydrophilic than most proteins, and all lack cysteine, cystine, and tryptophan. In addition all three proteins are extremely sensitive to a wide variety of proteolytic enzymes, much more so than "average" proteins such as serum albumin, lysozyme, and hemoglobin. These proteins also bind to both purified DNA and to a nuclear body from dormant spores. Although this binding gives little or no protection to proteins A and B from proteolysis, it does result in elevation of the melting temperature of the DNA by as much as 20degrees.
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PMID:Purification and properties of some unique low molecular weight basic proteins degraded during germination of Bacillus megaterium spores. 80 43

We have generated for the first time monoclonal antibodies (mAbs) specific for topoisomerase I (topo I) from scleroderma patients, and tight skin mice which develop a scleroderma-like syndrome. The epitope specificity of these antibodies has been determined using a series of fusion proteins containing contiguous portions of topo I polypeptide. Western blot analysis demonstrated that both human and mouse mAbs bound strongly to fusion protein C encompassing the NH2-terminal portion of the enzyme, and weakly to fusion proteins F and G containing regions close to the COOH-terminal end of the molecule. This crossreactivity is related to a tripeptide sequence homology in F, G, and C fusion proteins. It is interesting that a pentapeptide sequence homologous to that in fusion protein C was identified in the UL70 protein of cytomegalovirus, suggesting that activation of autoreactive B cell clones by molecular mimicry is possible. Both human and mouse mAbs exhibiting the same antigen specificity, also share an interspecies cross-reactive idiotope. These data suggest that B cell clones producing antitopo autoantibodies present in human and mouse repertoire are conserved during phylogeny, and are activated during the development of scleroderma disease.
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PMID:Antitopoisomerase I monoclonal autoantibodies from scleroderma patients and tight skin mouse interact with similar epitopes. 137 44

Binding Ca2+ to a high affinity site in protein C and 4-carboxyglutamic acid (Gla)-domainless protein C results in a conformational change that is required for activation by the thrombin-thrombomodulin complex, the natural activator of protein C. It has been hypothesized that this high affinity Ca(2+)-binding site is located in the NH2-terminal epidermal growth factor (EGF) homology region of protein C. We have expressed in human 293 cells a deletion mutant of protein C (E2-PD) which lacks the entire Gla region as well as the NH2-terminal EGF homology region of protein C. Ca2+ inhibits activation of E2-PD or Gla-domainless protein C by thrombin with half-maximal inhibition occurring at Ca2+ concentrations of 103 +/- 11 and 70 +/- 7 microM, respectively, but is required for both E2-PD and Gla-domainless protein C activation by the thrombin-thrombomodulin complex with half-maximal acceleration occurring at Ca2+ concentrations of 87 +/- 8 and 89 +/- 8 microM, respectively. Both E2-PD and Gla-domainless protein C exhibit a reversible, Ca(2+)- but not Mg(2+)-dependent decrease (6 +/- 1%) in fluorescence emission intensity with Kd = 38 +/- 3 microM Ca2+. We conclude that the high affinity Ca(2+)-binding site important for the activation of protein C is located outside of the NH2-terminal EGF homology region and that the metal-binding site in the NH2-terminal EGF homology region may not be a high affinity site in intact protein C.
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PMID:The high affinity calcium-binding site involved in protein C activation is outside the first epidermal growth factor homology domain. 160 45

About 30% of human plasma protein C is smaller than the predominant form as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has been suggested that this species, referred to as beta protein C, is a degraded molecule. However, beta protein C is secreted in culture by the HepG2 cell line and is present in plasma collected directly into numerous proteinase inhibitors; the percentage of beta protein C does not change with time during culture or after blood collection. Neither thrombin, activated protein C, nor activated factor X converts the alpha form to beta in the presence or absence of calcium and phospholipids. The NH2-terminal sequences of the heavy chains of both forms are identical, and both release the same dodecapeptide and develop a functional active site when cleaved by thrombin. Both also react with antibodies to a synthetic COOH-terminal peptide. Timed digests with N-glycosidase are consistent with the interpretation that beta protein C has three N-linked oligosaccharide chains whereas alpha protein C has four. It is asparagine 329 that is not glycosylated in beta protein C since antibodies to a synthetic peptide based on the sequence around this amino acid react only with beta protein C. This site is unique in having cysteine instead of serine or threonine 2 residues distal. It is likely that the sulfhydryl group can substitute for the usual hydroxyl group as a hydrogen bond acceptor for the glycosylation reaction only until it forms a disulfide bond. The percentage of protein C that is glycosylated at this site may therefore depend at least in part on the rate of disulfide bond formation which may in turn be related to the rate of protein synthesis.
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PMID:Beta protein C is not glycosylated at asparagine 329. The rate of translation may influence the frequency of usage at asparagine-X-cysteine sites. 169 79

A murine monoclonal antibody (designated VII-M31) directed against bovine factor VII was prepared and characterized. Antibody VII-M31 inhibited the activations of both factors IX and X catalyzed by factor VIIa in the presence of tissue factor, phospholipids, and Ca2+. It possessed a strong affinity for factor VII in the presence of 5 mM Ca2+ (Kd = 1.12 x 10(-10)M). The immunoblotting test of other bovine proteins with the antibody, such as prothrombin, factor X, factor IX, protein C, protein S, and protein Z, in addition to human factor VII, revealed that it recognizes only a Ca2(+)-dependent epitope in bovine factor VII. Furthermore, this antibody VII-M31 covalently coupled with Affi-Gel allowed a simple and rapid purification of bovine factor VII. To localize the antigenic site in factor VII, various segments including a gamma-carboxyglutamic acid (Gla)-domainless protein, a Gla-domain peptide and the fragments isolated from the lysyl endopeptidase digest, were prepared. Among them, the isolated Gla-domain peptide and Gla-domainless factor VII were no longer recognized by antibody VII-M31, indicating that the sequence around the cleavage site by a-chymotrypsin is required for the interaction between the antibody and factor VII. In accordance with this result, the antibody bound specifically to a Gla-containing peptide corresponding to the NH2-terminal 23-50 residues of factor VII, which contains the chymotryptic cleavage site. These results suggest that the specific epitope of this antibody is localized in the carboxy-terminal 28 residues of the Gla-domain constituting the amino-terminal portion of bovine factor VII.
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PMID:Monoclonal antibody (VII-M31) to bovine factor VII: a specific epitope in the gamma-carboxyglutamic acid domain. 170 45

Human factor VIII and factor VIIIa were proteolytically inactivated by activated protein C. Cleavages occurred within the heavy chain (contiguous A1-A2-B domains) of factor VIII and in the heavy chain-derived A1 and A2 subunits of factor VIIIa, whereas no proteolysis was observed in the light chain or light chain-derived A3-C1-C2 subunit. Reactivity to an anti-A2 domain monoclonal antibody and NH2-terminal sequence analysis of three terminal digest fragments from factor VIII allowed ordering of fragments and identification of cleavage sites. Fragment A1 was derived from the NH2 terminus and resulted from cleavage at Arg336-Met337. The A2 domain was bisected following cleavage at Arg562-Gly563 and yielded fragments designated A2N and A2C. A third cleavage site is proposed at the A2-B junction (Arg740-Ser741) since fragment A2C was of equivalent size when derived either from factor VIII or factor VIIIa. The site at Arg562 was preferentially cleaved first in factor VIII(alpha) compared with the site at Arg336, and it was this initial cleavage that most closely correlated with the loss of cofactor activity. Factor VIIIa was inactivated 5-fold faster than factor VIII, possibly as a result of increased protease utilization of the site at Arg562 when the A2 subunit is not contiguous with the A1 domain. When initial cleavage occurred at Arg336, it appeared to preclude subsequent cleavage at Arg562, possibly by promoting dissociation of the A2 domain (subunit) from the A1/light chain dimer. This conclusion was supported by the failure of protease treated A1/A3-C1-C2 dimer to bind A2 subunit and gel filtration analysis that showed dissociation of the A2 domain-derived fragments, A2N and A2C, from the A1 fragment/light chain dimer. These results suggest a mechanism for activated protein C-catalyzed inactivation of factor VIII(alpha) involving both covalent alteration and fragment dissociation.
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PMID:Activated protein C-catalyzed inactivation of human factor VIII and factor VIIIa. Identification of cleavage sites and correlation of proteolysis with cofactor activity. 193 75

Bovine plasma protein C inhibitor was purified; it was then characterized in comparison with human protein C inhibitor. The specific inhibitory activity of the purified inhibitor for bovine activated protein C was 8,500 times that of the inhibitor in plasma. The purified inhibitor showed a single band with Mr 56,000 by SDS-PAGE at pH 7.0, and two bands at pH 8.8, a major one with Mr 56,000 and a minor one with Mr 105,000, under both unreduced and reduced conditions. The pI range of the inhibitor was between 4.4 and 6.1. The Mr of the inhibitor was reduced by treatment with neuraminidase, O-glycanase, and also with glycopeptidase-A, suggesting that the inhibitor has both Asn-linked and Ser/Thr-linked carbohydrate chains. Twenty-seven of the NH2-terminal 49 amino acid residues of the bovine inhibitor, which lacks the first 4 residues from the NH2-terminal amino acid sequence of human inhibitor, were identical to those of the human inhibitor. The bovine inhibitor inhibited bovine and human activated protein C, human thrombin, Factor Xa, Factor XIa, and plasma kallikrein with Ki = 1.0, 5.2, 2.6, 3.0, 1.3 X 10(-8) M, and 4.5 X 10(-9) M, respectively. The inhibitory rates for activated protein C and thrombin were accelerated significantly in the presence of heparin or negatively charged dextran sulfate. However, the acceleration by heparin or dextran sulfate for the inhibition of Factor Xa, Factor XIa, and plasma kallikrein was not significant. The bovine inhibitor did not inhibit human Factor XIIa or plasmin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Bovine plasma protein C inhibitor with structural and functional homologous properties to human plasma protein C inhibitor. 216 Apr 49

To elucidate the binding sites for thrombin and protein C in the six epidermal growth factor (EGF) domains of human thrombomodulin, recombinant mutant proteins were expressed in COS-1 cells. Mutant protein EGF456, which contains the fourth, fifth, and sixth EGF domains from the NH2 terminus of thrombomodulin, showed complete cofactor activity in thrombin-catalyzed protein C activation, as did intact thrombomodulin or elastase-digested thrombomodulin. EGF56, containing the fifth and sixth EGF domains, did not have cofactor activity; but EGF45, containing the fourth and fifth EGF domains, had about one-tenth of the cofactor activity of EGF456. Thrombin binding to attached recombinant thrombomodulin (D123) was inhibited by EGF45 as well as by EGF56. A synthetic peptide (ECPEGYILDDGFICTDIDE), corresponding to Glu-408 to Glu-426 in the fifth EGF domain, inhibited thrombin binding to attached thrombomodulin (D123) with an apparent Ki of 95 microM. At Ca2+ concentrations of 0.25-0.3 mM, intact protein C was maximally activated by thrombin in the presence of EGF45, EGF456, or EGF1-6, which contains the first to sixth EGF domains; but such maximum cofactor activity was not observed when gamma-carboxyglutamic acid-domainless protein C was used. These findings suggest that: 1) thrombin binds to the latter half of the fifth EGF domain; and 2) protein C binds to the fourth EGF domain of thrombomodulin through Ca2+ ions.
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PMID:Further localization of binding sites for thrombin and protein C in human thrombomodulin. 217 98


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