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 binding of Ca2+ to a previously described phosphoprotein from human parotid saliva, protein A [Bennick (1975) Biochem J. 145, 557-567] was studied by means of equilibrium dialysis. In 5 mM-Tris/HC1 buffer, pH7.5, protein A bound 664nmol of Ca/mg of protein. Km was determined to be 181 muM and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ apparently occurs to side-chain carboxyl groups in the protein, but protein phosphate is of minor if any importance in calcium binding. Hydrolysis of protein A by trypsin and collagenase or heating of the protein at 60 degrees or 100 degrees C did not affect Ca2+ binding. The Ca2+ binding decreases with increased concentration of the dialysis buffer and on the addition of SrCl2, or MgCl2 or MnCl2 to the dialysis buffer. Protein A does not aggregate in the presence of Ca2+, since the s20,w was identical when determined in the presence (1.30S) and absence (1.35S) of CaCl2. By use of a specific antiserum to protein A it was found that protein C [Bennick & Connell (1971) Biochem. J. 123, 455-464] and perhaps minor related components cross-reacted with protein A. No other salivary proteins showed immunological similarity. Proteins A and C were also present in submandibular saliva. The possible functions of protein A are discussed.
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PMID:The binding of calcium to a salivary phosphoprotein, protein A, common to human parotid and submandibular secretions. 18 Sep 80

The two-way and three-way interactions among active-site-blocked bovine thrombin, bovine protein C, and the elastase fragment of rabbit thrombomodulin (elTM) were examined by analytical ultracentrifugation at 23.3 degrees C in 100 mM NaCl, 50 mM Tris (pH 7.65), and 1 mM benzamidine, in the presence of 0 to 5 mM calcium chloride. Thrombin and elTM form a tight (Kd less than 10(-8) M) 1:1 complex in the absence of Ca2+ that weakens with the addition of Ca2+ (Kd approximately 4 microM in 5 mM Ca2+). Without Ca2+, thrombin and protein C form a 1:1 complex (Kd approximately 1 microM) and what appears to be a 1:2 thrombin-protein C complex. The Kd for the 1:1 complex weakens over 100-fold in 5 mM CaCl2. Protein C and elTM form a Ca(2+)-independent 1:1 complex (Kd approximately 80 microM). Nearly identical binding to thrombin and elTM is observed when active-site-blocked activated bovine protein C is substituted for protein C. Thrombin inhibited by diisopropyl fluorophosphate and thrombin inhibited by a tripeptide chloromethyl ketone exhibited identical behavior in binding experiments, suggesting that the accessibility of protein C to the substrate recognition cleft of these two forms of thrombin is nearly equal. Human protein C binds with lower affinity than bovine protein C. Ternary mixtures also were examined. Protein C, elTM, and thrombin form a 1:1:1 complex which dissociates with increasing [Ca2+]. In the absence of Ca2+, protein C binds to the elTM-thrombin complex with an apparent Kd approximately 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ca2+ dependence of the interactions between protein C, thrombin, and the elastase fragment of thrombomodulin. Analysis by ultracentrifugation. 131 45

Among the vitamin K-dependent plasma proteins, only protein S contains the post-translationally modified amino acid erythro-beta-hydroxyasparagine (Hyn). Protein S also contains erythro-beta-hydroxyaspartic acid (Hya). The function of these unusual amino acids, located in the epidermal growth factor-like domains, is unknown. To determine if these post-translational modifications contribute to the functional integrity of human protein S (HPS), recombinant human protein S lacking Hya and Hyn (rHPSdesHya/Hyn) was purified from the medium of human kidney 293 cells that were transfected with HPS cDNA and grown in the presence of the hydroxylase inhibitor 2,2'-dipyridyl. Solution-phase equilibrium binding studies revealed that rHPSdesHya/Hyn binds C4b-binding protein (C4BP) in a manner indistinguishable from recombinant HPS and plasma-derived HPS, exhibiting a Kd in the presence of 2 mM CaCl2 of approximately 0.7 nM and a Kd in the presence of 4 mM EDTA approximately 10-fold higher. In a purified component system, rHPSdesHya/Hyn displayed normal anticoagulant cofactor activity in the activated protein C-catalyzed inactivation of coagulation factor Va bound in the prothrombinase complex. In addition, digestion of rHPSdesHya/Hyn with thrombin in the presence of EDTA appeared normal, and 2 mM CaCl2 prevented the cleavage. Together these results suggest that the post-translational modifications of Asn and Asp residues are not necessary for the macromolecular or Ca2+ interactions associated with the anticoagulant and C4BP binding characteristics of HPS.
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PMID:beta-Hydroxyaspartic acid and beta-hydroxyasparagine residues in recombinant human protein S are not required for anticoagulant cofactor activity or for binding to C4b-binding protein. 183 48

Murine monoclonal antibodies, developed following immunization with human protein C, were characterized for their ability to bind antigen in the presence of either CaCl2 or excess EDTA. Three stable clones were obtained which produced antibodies that bound to protein C only in the presence of EDTA. All three antibodies bound to the light chain of protein C on immunoblots and also bound to the homologous proteins factor X and prothrombin in solid-phase radioimmunoassays. One antibody, 7D7B10 was purified and studied further. The binding of 7D7B10 to human protein C was characterized by a KD of 1.4 nM. In competition studies, it was found that the relative affinity of the antibody for protein C was 20-40-fold higher than for prothrombin, fragment 1 of prothrombin, or factor X. In contrast, 7D7B10 was unable to bind to factor IX or bovine protein C. The effect of varying Ca2+ concentration on the interaction of the antibody with protein C was complex. Low concentrations of Ca2+ enhanced the formation of the protein C-antibody complex with half-maximal effect occurring at approximately 60 microM metal ion. However, higher concentrations of Ca2+ completely inhibited 7D7B10 binding to protein C with a K0.5 of 1.1 mM. Furthermore, millimolar concentrations of Mn2+, Ba2+, or Mg2+ also completely abolished antibody binding to protein C. The location of the epitope was delineated by immunoblotting and peptide studies and found to be present in the NH2-terminal 15 residues of protein C. Although residues corresponding to positions 10-13 of human protein C were necessary for maximal binding of the antibody, they were not sufficient. No evidence could be found for involvement of the epitope in metal binding per se. Therefore, the effect of Ca2+ on antibody binding is thought to be due to metal-dependent conformational changes in protein C. It seems likely that Ca2+ occupation of a high affinity site, shown by others to be located in the epidermal growth factor-like domain, causes a conformational change in the NH2-terminal region of protein C which is favorable for antibody interaction, whereas Ca2+ binding to the low affinity site(s), known to be present in the gamma-carboxyglutamic acid domain, causes an unfavorable conformational change.
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PMID:Conformational changes in an epitope localized to the NH2-terminal region of protein C. Evidence for interaction of protein C domains. 247 52

A new screening procedure, an easy and specific assay for determining functional Protein S activity, has been developed, with use of Protein C activated by venom activator (Protac). Purified Protein C (100% amidolytic activity) was activated by venom activator (6 units/mL). To a mixture of 100 microL of Protein S-deficient plasma, 20 microL of sample plasma, 100 microL of cephalin (Actin), and 20 microL of activated Protein C we added 100 microL of 25 mmol/L CaCl2 solution and measured the clotting time with a KC 10 coagulometer. The functional Protein S activity correlated well with the concentrations of Protein S antigen measured by enzyme immunoassay and the Laurell rocket technique (r = 0.810 and 0.850, respectively) in normal subjects, patients with myocardial infarction undergoing warfarin therapy, and patients with liver cirrhosis.
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PMID:Functional activity of protein S determined with use of protein C activated by venom activator. 252 95

The employment of a set of truncated dnaK peptides produced by deletion and insertion mutations in the Escherichia coli dnaK gene allowed us to define regions of the dnaK protein which are involved in particular enzymatic functions. The results obtained suggest that the dnaK polypeptide is organized into at least two distinct functional domains. The highly conserved amino-terminal portion is required for the ATPase activity. The carboxyl-terminal portion, characterized by relatively low similarity among species, is responsible for the autophosphorylating activity. The mutant dnaK protein C[74], which lacks amino acid sequences at the extreme carboxyl-terminal portion of the protein, retains both the ATPase and the autophosphorylating activities. The results obtained with the full-length (70-kDa) dnaK756 protein suggest that the thermolabile defect of the dnaK756 mutation affects directly or indirectly the ATPase active site of the enzyme. The autophosphorylating activity of the dnaK+, dnaK756, and C[74] polypeptides was activated at least 10-fold by the addition of CaCl2.
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PMID:Functional domains of the Escherichia coli dnaK heat shock protein as revealed by mutational analysis. 253 44

The physiological role of coagulation cofactor Protein S (PrS) for activated Protein C (APC) has recently been appreciated by the description of patients with PrS-deficiency, suffering from thromboembolism. The present study introduces a one-stage clotting assay for the assessment of PrS functional activity in plasma samples. The assay procedure is based on a factor Xa-initiated clotting test utilizing a mixture of AL(OH)3-adsorbed substrate plasma and patient's plasma supplemented with purified prothrombin (0.15 microM) and APC (0.05 microM), with phospholipids and CaCl2. Owing to the varying concentration of PrS in the sample plasma, clotting times were prolonged up to 25 seconds in the presence of APC, whereas no prolongation occurred in its absence. The test procedure proved to be specific for PrS, since preincubation with monospecific antibodies against PrS abolished the prolongation of clotting time, while reconstitution of adsorbed plasma with purified PrS restored its cofactor activity completely. The functional assay showed an inter-assay and intra-assay variation in the normal range of 11.7% and 10.1%, respectively (n = 20). PrS activity in a group of unselected patients (n = 34), revealing no abnormalities in global coagulation tests, amounted to 95.8 +/- 16.5% (mean +/- S.D.) with a range from 67% to 136% when analyzed in comparison to a plasma pool constituted from healthy volunteers. Patients (n = 32) undergoing oral anticoagulant therapy presented 21.1 +/- 10.8% residual PrS-activity accompanied by a concomitant decrease in PrS-antigen levels to 69.9 +/- 21.2%. The assay described is sensitive, it can be performed on routine basis and allows the detection of patients with PrS-deficiency.
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PMID:A functional test for protein S activity in plasma. 296 29

Substantial thrombomodulin activity could be detected in tissue thromboplastin preparations from placenta or from lung but not from brain. When the amount of these preparations was adjusted to contain 1 unit of tissue factor activity, up to 0.85 units of thrombomodulin activity could be measured, corresponding to the generation of 17 pmol/ml/min of activated protein C when 1.5 microM human protein C was activated by 20 nM human alpha-thrombin in the presence of 5 mM CaCl2. After treatment by phospholipase C, thrombomodulin activity was reduced in these samples. Addition of mixed brain procoagulant phospholipids partially restored thrombomodulin activity in the phospholipase C-treated samples. These results emphasize the role of phospholipids in the expression of optimal thrombomodulin activity in tissue thromboplastin preparations from placenta or from lung.
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PMID:Thrombomodulin activity is found in tissue thromboplastin preparations from placenta and from lung but not from brain. 303 9

Human factor VIII was isolated from commercial factor VIII concentrates and found to consist of multiple polypeptides with molecular weights ranging from 80 000 to 210 000. Immunological and amino acid sequence data identified these polypeptides as subunits of factor VIII. N-Terminal amino acid sequence analysis determined that the Mr 210 000 and 80 000 proteins are derived from the N- and C-terminal portions of factor VIII, respectively; Mr 90 000-180 000 polypeptides are derived from the Mr 210 000 polypeptide by C-terminal cleavages. Treatment of purified factor VIII with thrombin resulted in proteolysis of Mr 80 000-210 000 proteins and the generation of polypeptides of Mr 73 000, 50 000, and 43 000. Maximum coagulant activity of thrombin-activated factor VIII was correlated with the generation of these polypeptides. The proteolysis as well as activation of factor VIII by thrombin was found to be markedly dependent on CaCl2 concentration. Proteolysis of factor VIII with activated protein C (APC) resulted in degradation of the Mr 90 000-210 000 proteins with the generation of an Mr 45 000 fragment. This cleavage correlated with inactivation of factor VIII by APC. The Mr 80 000 protein was not degraded by APC. Factor Xa cleaved the Mr 80 000-210 000 factor VIII proteins, resulting in the generation of fragments of Mr 73 000, 67 000, 50 000, 45 000, and 43 000. Factor Xa was found to initially activate and subsequently inactivate factor VIII.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity. 308 57

We isolated protein C from a barium citrate-adsorbed fresh plasma and human factor IX concentrate by immunoaffinity chromatography on a column of Sepharose coupled with monoclonal antibodies to protein C. The antibodies used were conformation-specific monoclonal antibodies to the calcium-induced structure of protein C. Protein C was bound to antibodies coupled with Sepharose in the presence of calcium ions and was eluted with EDTA. This immunopurification resulted in a 13,000-fold purification of the fully functional zymogen from plasma. The immunoaffinity-isolated protein C was found to have higher amounts of single-chain protein C than conventionally isolated protein C when analyzed by sodium dodecyl sulfate-polyacrylamide gels under reduced conditions. The factor IX concentrate was applied to this Ca2+-dependent antibody JTC-3-immobilized Sepharose in the presence of 5 mM CaCl2, and protein C with its gamma-carboxyglutamic acid (Gla) domain intact was firstly bound to this column and then eluted by metal chelation with EDTA. When flow-through fractions were applied again in the presence of Ca2+ to this column, modified protein C which had lost its N-terminal 42-residue peptide was weakly bound to this column. It was eluted in the absence of Ca2+. However, only a low percentage of modified protein C was detectable by an enzyme-linked immunosorbent assay using Ca2+-dependent monoclonal antibody JTC-3 and peroxidase-labeled immunopurified polyclonal antibody. These results indicate that factor IX concentrate has both Gla-domain-intact and Gla-domainless protein C. Moreover, it suggests that Ca2+-dependent monoclonal antibody JTC-3 may recognize the coupled conformational change of protein C induced by the combined effect of Ca2+ binding to the Gla domain and to other parts of protein C.
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PMID:Immunoaffinity purification of protein C by using conformation-specific monoclonal antibodies to protein C-calcium ion complex. 362 Apr 98


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