Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two different monoclonal antibodies against the heparin-dependent inhibitor of human activated protein C were produced, using cleaved modified inhibitor for immunization and partially purified inhibitor for screening of the hybridomas. One of the antibodies recognized free and complexed forms of the inhibitor in immunoblotting experiments. The other antibody was used to develop an assay for APC-PCI inhibitor complexes. Using the assay the formation of complexes was studied in plasma, both in the presence and absence of heparin. The rate of complex formation was similar to that reported previously for the loss of activated protein C amidolytic activity in plasma. The same antibody was also immobilized on Sepharose and used to purify the inhibitor from fresh human plasma. The purified material appeared as two narrowly spaced bands with Mr about 57,000 in SDS-PAGE. The average yield from 1 liter of fresh plasma was 1 mg of inhibitor. The purified inhibitor formed SDS stable complexes with activated protein C and urokinase that could be identified in immunoblots using specific antibodies.
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PMID:Monoclonal antibodies against the heparin-dependent protein C inhibitor suitable for inhibitor purification and assay of inhibitor complexes. 321 24

A human solvent-detergent (SD)-treated factor IX concentrate has been produced from cryoprecipitate-poor plasma using DEAE-Sepharose CL-6B and heparin-Sepharose CL-6B chromatography. The DEAE eluate was incubated with an SD mixture [0.3% tri(n-butyl) phosphate-1% Tween 80, 6-h at 24 degrees C] which was found to inactivate, in less than 1 h, more than 3.8 log10 of vesicular stomatitis virus and more than 4.8 log10 of Sindbis virus; the SD was removed by a subsequent heparin adsorption step. The specific activity of the concentrate was 10.9 +/- 1.3 IU factor IX: c/mg protein (n = 15). The factor IX coagulant to antigen ratio was 0.7 +/- 0.1. The concentrate was essentially free of factors II, VII and X, and protein C. The usual major contaminants of prothrombin complex concentrate (PCC) were absent: the concentrate contained about 94% alpha-1 proteins, and only 4 major proteins were resolved by SDS-PAGE (respective apparent molecular weight: 130, 86, 76 and 69 kilodaltons), and by crossed immunoelectrophoresis against an anti-PCC serum. The nonactivated partial thromboplastin time was equivalent to that of PCC; the product was devoid of factor IXa, of other activated procoagulant factors and of coagulant-active phospholipids (removed with SD in the heparin breakthrough fraction). Animal studies using the Wessler test and acute-toxicity test in rabbits revealed no adverse side effects. SD treatment could thus be used to inactivate viruses in factor IX concentrate and improve the safety of replacement therapy in hemophilia B.
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PMID:Large-scale production and properties of a solvent-detergent-treated factor IX concentrate from human plasma. 326 37

The effects of human activated protein C (APC) and thrombin on plasminogen activator inhibitor (PAI-1) released from cultured human umbilical endothelial cells, grown in serum-free 35S-methionine containing medium, were studied in two ways: measurement of PAI-1 activity with an amidolytic assay, and immunoprecipitation of the medium with anti-PAI-1 IgG, anti-protein C IgG or anti-thrombin IgG followed by SDS-PAGE and autoradiography. Addition of APC or thrombin to the endothelial cell conditioned medium results in a time and concentration dependent loss of PAI-1 activity and in the degradation of PAI-1 from 46 kD into a 42 kD product. After incubation of the medium with APC in the presence of cells, an additional band of 95 kD was found, which could be immunoprecipitated with both anti-PAI-1 IgG and anti-protein C IgG, indicating the formation of an APC-PAI-1 complex before degradation occurs. No complex could be detected after incubation of the medium with thrombin in the presence of endothelial cells. Blocking the active sites of APC and thrombin prevented both the formation of APC-PAI-1 complexes and the inactivation and degradation of PAI-1. After removal of the active PAI-1 from the medium, no degradation of the inactive PAI-1 by APC or thrombin could be found. It is concluded that both APC and thrombin react with the active PAI-1, resulting in inactivation and degradation of PAI-1.
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PMID:The interaction of activated protein C and thrombin with the plasminogen activator inhibitor released from human endothelial cells. 349 80

Human protein C-inhibitor (PCI) was isolated from human citrated plasma by combining rivanol precipitation, ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on dextran sulfate Sepharose. The purified PCI migrated with the beta-globulins and was free from protein contaminations as judged by immunoelectrophoresis. In SDS-PAGE under reducing and unreducing conditions PCI showed a single band at Mr = 57,000. The specific activity of the inhibitor was 226 units/mg. Surprisingly, the isolated PCI inhibited the amidolytic activity of urokinase (u-PA) on Glu-Gly-Arg-pNA (S-2444) in a time-dependent manner. Heparin, dextran sulfate and pentosanpolysulfate accelerated the reaction catalytically. PCI revealed itself as a non-competitive inhibitor of u-PA. The Ki-value was determined to be 7.9 x 10(-8)M. Inhibition of amidolytic activity was found to be associated with the formation of an 1:1 equimolar complex with a Mr of 110,000 as demonstrated by means of polyacrylamide gel electrophoresis and following Western blotting technique using polyclonal antibodies against u-PA and PCI. The specific activity of the isolated PCI of 226 units/mg, which approximates the theoretical value of pure PCI, indicates a highly purified preparation of PCI. The heparin-dependent inhibition of urokinase by this highly purified protein as well as comparison of the kinetic data and amino-acid composition of both PCI and the recently described plasminogen activator inhibitor (PAI) 3 give high evidence of identity of PCI and PAI-3.
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PMID:Inhibition of urokinase by protein C-inhibitor (PCI). Evidence for identity of PCI and plasminogen activator inhibitor 3. 350 Dec 95

SDS-polyacrylamide gel electrophoresis of anti-glucose-6-phosphate dehydrogenase immunoprecipitates from radiolabeled uterine tissue extracts previously revealed three proteins: A, B and C, which were tentatively identified as a 60-64 kDa precursor form, a 57 kDa predominant form, and a 40-42 kDa nascent peptide form of the enzyme, respectively. A peptide-mapping technique was used to examine structural homologies among A, B and C. Following the labeling of uterine proteins with [35S]methionine, labeled proteins A, B and C were isolated by immunoprecipitation and electrophoresis. Each protein was individually co-digested with authentic, [3H]methionine-labeled glucose-6-phosphate dehydrogenase using papain, the resulting peptides were resolved by isoelectric focusing and the peptides from the two sources on each gel were compared using double-label counting methods. Proteins A, B and C had at least eight peptides in common, both proteins A and C had two additional peptides in common that were not present in protein B, and B protein had two peptides that were either absent or present in reduced amounts in digests of proteins A and C. The extensive structural homology and immunoreactivity of these proteins indicated that proteins A, B and C were all related to glucose-6-phosphate dehydrogenase. The presence of two extra peptides in proteins A and C suggested that these peptides may be derived from a common NH2-terminal leader sequence which was present in both the precursor and nascent peptide chains. The presence of two peptides that were present in protein B and absent from proteins A and C is easiest to explain if they are derived from the two ends of the molecule, with the corresponding peptides in proteins A and C containing additional peptide sequences that are 'normally' removed by endogenous proteolytic processing enzymes. Based on the relative time-course of synthesis of the three glucose-6-phosphate dehydrogenase-related proteins in control and estrogen-treated uteri, it appears that estradiol promotes an increase in the relative rate of transfer of label from protein A into B by stimulating the rate of processing of the precursor to the predominant form of the enzyme and enhances the rate of translational conversion of protein C into higher molecular weight forms.
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PMID:Isolation of a precursor and a nascent chain form of glucose-6-phosphate dehydrogenase from rat uterus and regulation of precursor processing by estradiol. 394 90

Canine parvovirus-2 (CPV-2), Mink enteritis virus (MEV) and feline panleukopenia virus (FPV) were produced using identical cell culture and purification techniques. The distributions of the haemagglutinating activity of the three different parvoviruses in a CsCl gradient were similar with haemagglutinating peaks identified at 1.48-1.49, 1.42, 1.36 and 1.30-1.31 g cm-3. The number and distribution of the viral proteins and the equivalent protein molecular weights are similar for all three viruses in SDS-polyacrylamide gels (10%). Four viral proteins were identified and their molecular weights were determined: protein A (77 500-79 500), protein B (63 000-63 500), protein C (61 500-63 000) and protein D (50 000-55 000). The viral protein D although reported for some other parvoviruses has not previously been demonstrated in CPV-2, MEV or FPV.
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PMID:Comparison of the viral proteins of canine parvovirus-2, mink enteritis virus and feline panleukopenia virus. 631 27

Protein C inhibitor isolated from human plasma inhibited thrombin, factor Xa, trypsin and chymotrypsin as well as activated protein C, but had very little effect on urokinase and plasmin. The inhibition constants (K1) of protein C inhibitor for activated protein C, thrombin and factor Xa were 5.6 X 10(-8) M, 6.7 X 10(-8) M and 3.1 X 10(-7) M, respectively. The second-order rate constant for inhibition of activated protein C by the inhibitor increased about 30-fold in the presence of an optimal heparin concentration (5-10 units/ml). The inhibition of activated protein C by plasma protein C inhibitor was also accelerated by heparin. When activated protein C (Mr = 62,000) was incubated with protein C inhibitor (Mr = 57,000), enzyme-inhibitor complexes with apparent Mr = 102,000 and 88,000 were observed in the nonreduced and the reduced samples, respectively, on SDS-polyacrylamide gel electrophoresis. In addition to these complexes, a band of unbound enzyme and a band with Mr = 54,000 were detected. When 125I-labeled protein C inhibitor was exposed to activated protein C, the inhibitor band was converted to bands with apparent Mr = 102,000 and 54,000 in the nonreduced samples, as determined by autoradiography after gel electrophoresis in SDS. The band with Mr = 54,000 also appeared when the inhibitor reacted with other serine proteases. The activated protein C was released from the inactive complex by treatment with 1 M ammonia or hydroxylamine. This phenomenon was found by SDS-polyacrylamide gel electrophoresis to represent the dissociation of the enzyme-inhibitor complex by ammonia or hydroxylamine into the free enzyme and the proteolytically modified inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanism of inhibition of activated protein C by protein C inhibitor. 632 92

HeLa cells have undetectable tissue factor (thromboplastin) activity when measured by a one-stage coagulation assay. In contrast, these cells accelerated the factor VII-catalyzed cleavage of factor X. The two assays gave similar results after either heating the samples to 100 degrees C for 2 min or exposure to thrombin. Neither of these treatments altered the tissue factor activity of human foreskin fibroblasts, a cell type with high tissue factor activity. HeLa cells contain an inhibitor(s) directed against factor Xa but not thrombin. The inhibitor(s) was inactivated by exposure to thrombin or by heat treatment. Inhibition of factor Xa-catalyzed cleavage of a synthetic peptide was blocked by ethyleneglycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) so the inhibition was apparently dependent on divalent cations. Inhibition was not accelerated by heparin. The inhibitor(s) was not protein C or other serine proteases since it was not inactivated by diisopropylfluorophosphate. The factor Xa inhibitor(s) has been isolated from HeLa cells with an approximate 500-fold increase in specific activity. After SDS-polyacrylamide gel electrophoresis factor Xa-inhibitory activity was recovered from a region corresponding to the major Coomassie-staining band at 43 kDa and in lesser amounts from regions corresponding to 26 and 17 kDa. Cellular inhibitors of coagulation may partially explain the low apparent tissue factor observed in some in vitro cells and may serve a regulatory role in limiting the expression of tissue factor.
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PMID:Alterations in the apparent tissue factor (thromboplastin) expression in HeLa cells by a cellular factor Xa inhibitor. 663 60

Rat submandibular glands undergo a transient stage of differentiation in the perinatal acini. Two morphologically distinct cell types (Type I and Type III) appear and secrete several electrophoretically identifiable proteins. To examine the regulation of the secretion of these proteins and their association with secretion by the two cell types, 4-day-old glands were incubated with secretory agonists and antagonists, and the cultures were evaluated for secreted proteins in the medium and morphologically assayed degranulation of the cells. Stimulation of glands with beta-adrenergic agonists resulted in the secretion of three proteins, A, B1, and B2, at Rf 0.26, 0.36, and 0.40 in native, anionic gels at pH 8.3. Cholinergic stimulation caused the appearance of a different protein, C, at Rf 0.12, but only slight secretion of A, B1, and B2. With both beta-adrenergic and cholinergic stimulation, another protein, D, was seen near the origin. Partial purification of the proteins was effected by gel chromatography followed by preparative IEF (Isoelectric point focusing). Proteins A, B1, and B2 were recovered at isoelectric pH 4.5, 5.4, and 5.1 respectively. SDS gels of IEF fractions and secretion medium indicate Mr 25K, 26K, 27K, 97K, and greater than 150K for A, B1, B2, C, and D respectively. beta-Adrenergic stimulation caused degranulation only of Type I cells and cholinergic stimulation only of Type III cells. We propose the existence of two independently regulated secretory systems in the perinatal acini, with Type I cells secreting protein C on stimulation of cholinergic receptors, and Type III cells secreting A, B1, and B2 through beta-adrenergic receptors. With either stimulation, protein D is secreted, and thus it may serve a general function in the secretion process.
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PMID:Two independently regulated secretory systems within the acini of the submandibular gland of the perinatal rat. 669 33

The chromatin fraction of rat liver exhibited proteolytic activity caused by serine proteases, with maximal activity at pH 8 or 10. They were analyzed by affinity labeling with [3H]diisopropylfluorophosphate followed by SDS-polyacrylamide gel electrophoresis, and partially purified by gel filtration through Sepharose 6B after selective extraction from the chromatin fraction. The following results were obtained. 1. The chromatin fraction contained three DFP-binding proteins with molecular weights of 52,000, 25,000, and 15,000 daltons, and they were tentatively designated proteins A, B, and C, respectively. Unlike proteins A and B, protein C reacted with DFP more strongly at pH 10 than at pH 8. A greater part of protein B was present in the nucleoli, while the others were predominantly distributed in extra-nucleolar chromatin. 2. Proteins A and B were extracted from the chromatin fraction by 5 M urea and 0.7 M NaCl, respectively; while protein C was not extractable by either solution. Proteins A and B were further purified by gel filtration through Sepharose 6B. 3. Protein B was a neutral protease with a maximal activity for 3H-labeled ribosomal proteins at pH 8 and showed high specificity for basic proteins, such as histone and ribosomal proteins. Protein A was an alkaline protease with a maximal activity at pH 10 and showed proteolytic activity not only for basic proteins but also for hydrophobic proteins, such as casein and non-histone proteins of chromatin. 4. Protein A was activated at pH 8 by high concentrations of NaCl, suggesting the presence of some inhibitor(s). Protein A was converted to protein C at pH 10, and also at pH 8 with high concentrations of NaCl. Thus, protein A is suggested to be the complex of protein C and unknown inhibitor(s). 5. When the chromatin fraction was incubated at pH 10, non-histone proteins were degraded much faster than at pH 8, although H1 histone was degraded at similar rates at both pHs. These results indicate that the chromatin fraction of rat liver contains at least two kinds of serine proteases, B and C, and that protease B is probably involved in the metabolism of basic protein, especially H1 histone. Protease C, the greater part of which associates with some inhibitors to form protein A, may play its main role in the metabolism of non-histone proteins.
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PMID:Studies on the serine proteases associated with rat liver chromatin. 675


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