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, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (M(r) = 62,000) contains 23% carbohydrate and is composed of a light chain (M(r) = 21,000) and a heavy chain (M(r) = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human alpha-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity.
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PMID:Human plasma protein C: isolation, characterization, and mechanism of activation by alpha-thrombin. 46 91

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

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

Thrombomodulin is an endothelial glycoprotein that serves as a cofactor for protein C activation. To examine the ligand specificity of human thrombomodulin, we performed equilibrium binding assays with human thrombin, thrombin S205A (wherein the active site serine is replaced by alanine), meizothrombin S205A, and human factor Xa. In competition binding assays with CV-1(18A) cells expressing cell surface recombinant human thrombomodulin, recombinant wild type thrombin and thrombin S205A inhibited 125I-diisopropyl fluorophosphate-thrombin binding with similar affinity (Kd = 6.4 +/- 0.5 and 5.3 +/- 0.3 nM, respectively). However, no binding inhibition was detected for meizothrombin S205A or human factor Xa (Kd greater than 500 nM). In direct binding assays, 125I-labeled plasma thrombin and thrombin S205A bound to thrombomodulin with Kd values of 4.0 +/- 1.9 and 6.9 +/- 1.2 nM, respectively. 125I-Labeled meizothrombin S205A and human factor Xa did not bind to thrombomodulin (Kd greater than 500 nM). We also compared the ability of thrombin and factor Xa to activate human recombinant protein C. The activation of recombinant protein C by thrombin was greatly enhanced in the presence of thrombomodulin, whereas no significant activation by factor Xa was detected with or without thrombomodulin. Similar results were obtained with thrombin and factor Xa when human umbilical vein endothelial cells were used as the source of thrombomodulin. These results suggest that human meizothrombin and factor Xa are unlikely to be important thrombomodulin-dependent protein C activators and that thrombin is the physiological ligand for human endothelial cell thrombomodulin.
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PMID:Ligand specificity of human thrombomodulin. Equilibrium binding of human thrombin, meizothrombin, and factor Xa to recombinant thrombomodulin. 131 33

To elucidate the role of the COOH-terminal region of antithrombin III, we studied the effects of synthetic peptides corresponding to its sequence on the amidolytic and proteolytic activities of thrombin and Factor Xa in the presence or absence of the inhibitor, antithrombin III. The peptides ANRPFLVFI and IIFMGRVANP corresponding to residues Ala404 to Ile412 and Ile420 to Pro429, respectively, blocked the inhibition by antithrombin III. The effect of IIFMGRVANP was reduced in the presence of heparin. Both peptides at a concentration of 1 mM blocked complex formation between antithrombin III and thrombin or Factor Xa. The two peptides, particularly IIFMGRVANP, directly enhanced the amidolytic activity of thrombin and Factor Xa on the synthetic substrate Boc-Ala-Gly-Arg-MCA (where Boc is t-butoxycarbonyl and MCA is 4-methylcoumarin), which corresponds to residues P3-P1 of the reactive site of antithrombin III, and also on other substrates due to increased Vmax. IIFMGRVANP also shortened the thrombin-induced fibrinogen clotting time, whereas ANRPFLVFI inhibited the thrombin-catalyzed activation of protein C both in the presence and absence of thrombomodulin. The direct effect of ANRPFLVFI and IIFMGRVANP on thrombin was confirmed by enhancement of the incorporation of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide into thrombin. These findings suggest that the COOH-terminal region of antithrombin III interacts with thrombin and Factor Xa to increase the reactivity of the enzyme, which may enhance acyl-bond formation between the inhibitor and the enzyme.
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PMID:The role of the COOH-terminal region of antithrombin III. Evidence that the COOH-terminal region of the inhibitor enhances the reactivity of thrombin and factor Xa with the inhibitor. 133 Oct 47

This study investigates type II protein C deficiency in a family with manifestations of both arterial and venous thrombosis. Of 64 members of the kindred, 14 have been tested and 7 have PC deficiency. Among affected individuals (n = 7), mean protein C levels by different assays were as follows: enzyme-linked immunosorbent assay (ELISA), 3.8 micrograms/mL (2.1 to 4.3 micrograms/mL); amidolytic with venom activator, 115% (60% to 140%); clotting with venom activator, 42% (23% to 59%). The mean ratio of clotting to amidolytic assays for the affected individuals was 0.37 compared with a normal range of 0.8 to 1.2. Thus, the affected individuals have normal total protein C and their activated protein C has a normal active site assessed by chromogenic substrate; however, they have markedly diminished clotting activity. Immunoassay and chromatography data suggested an abnormality of carboxylation in the gamma carboxyglutamic acid (Gla) domain. Polymerase chain reaction amplification and direct DNA sequencing of exon 2 from genomic DNA of affected individuals showed two nucleotide substitutions. One of the mutations (A----C) results in Glu20----Ala, thereby eliminating a site for vitamin K-dependent gamma-carboxylation. The other substitution (G----A) results in a Val34----Met mutation. DNA sequencing of the other exons from affected individuals has shown no further difference from that of the wild-type gene. The former mutation also removes a Bgl II restriction endonuclease site, which has allowed us to confirm the mutation in affected individuals by direct digestion and Southern hybridization of genomic DNA from family members. This is the first reported family with documented Gla domain mutations in the protein C gene.
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PMID:Protein CVermont: symptomatic type II protein C deficiency associated with two GLA domain mutations. 134 6

We report a C/T dimorphism in the thrombomodulin (TM) gene that predicts an Ala455----Val replacement in the sixth EGF-like domain of TM. This dimorphism has allelic frequencies of 82 (Ala) and 18% (Val) in a normal population. In a group of protein C deficient patients and in a group of subjects with unexplained thrombophilia the allelic frequencies were found to be the same as in the normal population. This indicates that with respect to thrombophilia the dimorphism is essentially neutral.
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PMID:A frequent thrombomodulin amino acid dimorphism is not associated with thrombophilia. 165 67

The last three consecutive epidermal growth factor (EGF)-like structures of human thrombomodulin constitute the functional domain for protein C-activating cofactor activity and anticoagulant activity. Using site-directed deletion mutagenesis, we found that amino acid Asp349 of TME456, a recombinantly produced protein consisting of EGF-like structures 4, 5, and 6, is essential for retaining full protein C-activating cofactor activity. To investigate the role of Asp349 in the protein C-activating cofactor activity of human thrombomodulin, we have constructed two mutants of TMD123, a recombinantly produced protein consisting of domains D1, D2, and D3 of thrombomodulin, using site-directed point mutagenesis of the thrombomodulin coding sequence. In mutant TMD123A, the Asp349 codon was replaced with an Ala codon and in mutant TMD123E, the Asp349 codon was replaced with a Glu codon. The partially purified mutant proteins were assayed for their protein C-activating cofactor activity at various Ca2+ concentrations. TMD123 and TMD123E protein showed similar high levels of cofactor activity and similar patterns of Ca2+ dependence, while TMD123A had lower cofactor activity and did not show any Ca2+ dependence. We concluded that Asp349 in the fourth EGF-like structure of human thrombomodulin plays a role in its Ca(2+)-mediated binding to protein C.
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PMID:Aspartic acid 349 in the fourth epidermal growth factor-like structure of human thrombomodulin plays a role in its Ca(2+)-mediated binding to protein C. 165 4

Interphotoreceptor retinoid-binding protein (IRBP), a retinal-specific Ag, induces experimental autoimmune uveitis in a variety of animals. We have previously shown that sequence 1169-1191 of bovine IRBP is the immunodominant epitope of this protein in Lewis rats and is highly immunogenic and uveitogenic in these rats. The active site of peptide 1169-1191 was determined by testing its truncated forms. The shortest peptide to be immunologically active was found to be 1182-1190 (WEGVGVVPD). To determine the role of individual residues of this sequence, we have tested the immunologic activities of nine analogs of peptide 1181-1191, in which each of residues 1182-1190 was substituted with alanine (A). The tested activities included the capacity to induce experimental autoimmune uveitis and cellular responses in immunized rats, as well as the capability to stimulate lymphocytes sensitized against IRBP or the parent peptide 1181-1191. Analogs that did not stimulate these lymphocytes were also tested for their capacity to competitively inhibit the proliferative response to 1181-1191. Analogs A(1184), A(1186), and A(1187) resembled 1181-1191 in their activities, whereas the other analogs exhibited remarkably reduced activities, with several patterns being noticed. Analog A(1182) was inactive in all tests. Analog A(1190) was very weakly uveitogenic and non-immunogenic, but it stimulated lymphocytes sensitized against IRBP or 1181-1191 when added at exceedingly high concentrations. Analogs A(1183) and A(1185) resembled A(1190) in being weakly uveitogenic and A(1185) was also found to be poorly immunogenic. In addition, relatively high concentrations of A(1183) and A(1185) were needed to stimulate lymphocytes sensitized against IRBP or 1181-1191. However, a different pattern of activities was exhibited by analogs A(1188) and A(1189). These peptides were uveitogenic and immunogenic, but failed to stimulate lymphocytes sensitized to IRBP or 1181-1191. Furthermore, A(1188) and A(1189), but not A(1182), also inhibited the response to 1181-1191 of a cell line specific toward this parent peptide. The data are interpreted to show that residues 1188 and 1189 are involved in the interaction of the peptide with the TCR, whereas residues 1182 and 1190 and, perhaps, 1183 and 1185, are pivotal for the binding of peptide 1181-1190 to the MHC molecules on APC.
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PMID:Analysis of the pivotal residues of the immunodominant and highly uveitogenic determinant of interphotoreceptor retinoid-binding protein. 170 28

We report on a computer algorithm capable of predicting the location of T-helper-cell epitopes in protein antigen (Ag) by analysing the Ag amino acid sequence. The algorithm was constructed with the aim of identifying segments in Ag which are resistant to proteolytic degradation by the enzymes cathepsin B, L, and D. These are prominent enzymes in the endocytic pathway through which soluble protein Ag enter APC, and resistant segments in Ag may, therefore, be expected to contain more T-cell determinants than susceptible segments. From information available in the literature on the substrate specificity of the three enzymes, it is clear that a cysteine is not accepted in any of the S2, S1, S1', and S2' subsites of cathepsin B and L, and not in the S1 and S1' subsites of cathepsin D. Moreover, we have noticed that cysteine-containing T-cell determinants in a number of protein Ag are particularly rich in the amino acids alanine, glycine, lysine, leucine, serine, threonine, and valine. By searching protein Ag for clusters of amino acids containing cysteine and two of the other amino acids we were able to predict 17 out of 23 empirically known T-cell determinants in the Ag with a relatively low number of false (positive) predictions. Furthermore, we present a new principle for searching Ag for potential amphipatic alpha-helical protein segments. Such segments accord well with empirically known T-cell determinants and our algorithm produces a lower number of false positive predictions than the principle based on discrete Fourier transformations previously described.
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PMID:T-helper-cell determinants in protein antigens are preferentially located in cysteine-rich antigen segments resistant to proteolytic cleavage by cathepsin B, L, and D. 171 25


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