Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.6 (thromboplastin)
 13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A high molecular form of bovine factor X has been isolated from freshly collected bovine blood by BaSO4 absorption, exhaustive washing with 0.001 M BaCl2 and chromatographed on DEAE-cellulose column employing a linear salt gradient. This isolated factor X showed a single protein band on analytical polyacrylamide gel disc electrophoresis. Only one single protein peak was observed in the chromatogram of DEAE-Sephadex A-50 chromatography conducted at 3 degrees C. Sedimentation equilibrium analysis of this bovine factor X revealed no apparent heterogeneity or self association-dissociation phenomena. It yielded a weight-average molecular weight of 74 000 for the native factor X. In the absence of any reducing agent, factor X migrated in dodecyl sulfate gel electrophoresis as a single component with an estimated molecular weight of 74 300. Both dodecyl sulfate gel electrophoresis in the presence of 2-mercaptoethanol and agarose gel chromatography in 6 M guanidinium chloride revealed that this native factor X is composed of two polypeptide chains of molecular weights of 56 000 and 22 100. Factor X can be converted to the enzymatically active factor Xa by Russell's viper venom and in the presence of Ca2+. Factor Xa was purified by DEAE-cellulose chromatography. This Russell's viper venom activated factor Xa also showed a single protein band upon analytical polyacrylamide gel disc electrophoresis. Sedimentation equilibrium analysis of this factor Xa yields a weight-average molecular weight of 59 000 with no apparent heterogeneity or self-association phenomena. In the absence of any reducing agent, factor Xa migrated as a single component in dodecyl sulfate gel electrophoresis with an estimated molecular weight of 58 500. From the results of dodecyl sulfate gel electrophoresis in the presence of 2-mercaptoethanol as well as agarose gel chromatography in 6 M guanidinium chloride, factor Xa is also composed of two polypeptide chains of molecular weights of 36 700 and 22 800. Therefore, the heavy and light chains of both native factor X and factor Xa are linked together by disulfides. Great care was taken in washing the BaSO4 precipitate and it is this effective washing which enabled us to isolate the higher molecular from of bovine factor X.
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PMID:Isolation of the high molecular form of bovine factor X and some of its physical properties. 83 78

High-molecular-weight kininogen has been isolated from rat plasma in three steps in a relatively high yield. The purified preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of 2-mercaptoethanol, and the apparent Mr was estimated as 100,000. On incubation with rat plasma kallikrein, rat high Mr kininogen yielded a kinin-free protein consisting of a heavy chain (Mr = 64,000) and a light chain (Mr = 46,000), liberating bradykinin. The kinin-free protein was S-alkylated, and its heavy and light chains were separated by a zinc-chelating Sepharose 6B column. The amino acid compositions of rat high Mr kininogen and its heavy and light chains were very similar to those of bovine high Mr kininogen and its heavy and fragment 1.2-light chains, respectively. A high histidine content in the light chain of rat high Mr kininogen indicated the presence of a histidine-rich region in this protein as in bovine high Mr kininogen, although this region was not cleaved by rat plasma kallikrein. Rat high Mr kininogen corrected to normal values the prolonged activated partial thromboplastin time of Brown-Norway Katholiek rat plasma known to be deficient in high Mr kininogen and of Fitzgerald trait plasma. The kinin-free protein had the same correcting activity as intact high Mr kininogen. Rat high Mr kininogen also accelerated approximately 10-fold the surface-dependent activation of rat factor XII and prekallikrein, which was mediated with kaolin, amylose sulfate, and sulfatide. These results indicate that rat high Mr kininogen is quite similar to human and bovine high Mr kininogens in terms of biochemical and functional properties.
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PMID:Rat plasma high-molecular-weight kininogen. A simple method for purification and its characterization. 384 94

Most nuclearly encoded mitochondrial proteins are synthesized with an amino-terminal leader peptide that is cleaved by the mitochondrial processing peptidase (MPP). Purified rat liver MPP, like the Neurospora and yeast enzymes, consists of two nonidentical subunits, alpha (55 kDa) and beta (50 kDa). To confirm the functional authenticity of the recently cloned and sequenced cDNAs for the alpha- and beta-MPP subunits from rat liver and to study each subunit's participation in MPP activity, we have subcloned and expressed separately in Escherichia coli the mature sequence of each subunit as a fusion protein with the maltose-binding protein. After induction, about 80% of each expressed fusion protein was insoluble in aggregates or inclusion bodies, and 20% remained soluble in the supernatant. The fusion proteins in the soluble fraction were purified by affinity chromatography and treated with factor Xa, and the MPP subunits were purified to homogeneity. When mixed together, these subunits showed no activity, suggesting that they might be misfolded. Therefore, a reconstitution protocol was developed which consisted of denaturation in urea, dithiothreitol, and 2-mercaptoethanol, followed by renaturation by dilution and dialysis under reducing conditions. With this procedure, active MPP was recovered from the mixed subunits, and it could be demonstrated that both alpha- and beta-MPP subunits were necessary for activity. Reconstituted recombinant MPP resembled the native rat liver enzyme as judged by its molecular weight, its inhibition by EDTA, and its ability to process a variety of mitochondrial precursor proteins appropriately to either an intermediate or a mature form.
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PMID:Rat liver mitochondrial processing peptidase. Both alpha- and beta-subunits are required for activity. 813 65

Factor XII Tenri (Y34C), a rare cross-reacting material (CRM)-negative factor XII deficiency, was identified in a 71-yr-old Japanese woman with angina pectoris. In the patient's plasma, factor XII activity and antigen levels were only 1.6% and 5.0%, respectively, of those seen in a normal subject. Immunoblot analysis showed that the secreted factor XII Tenri existed not only as a monomer (76 kDa), but also in complexes with apparent molecular weights of approximately 115, 140, 190, 215, and 225 kDa. After reduction with 2-mercaptoethanol, the factor XII Tenri contained in the complexes was completely converted to monomeric form on immunoblot patterns. It appeared that some of the secreted factor XII Tenri formed several types of disulfide-linked complexes, including a factor XII-alpha1-microglobulin complex, through a newly generated Cys residue. The monomeric form of factor XII Tenri, like normal factor XII, was degraded into 2 major fragments with molecular weights of approximately 45 kDa and 30 kDa following mixing with activated partial-thromboplastin-time measuring reagent (cephalin and ellagic acid), whereas the factor XII Tenri that formed the complexes was not. This indicates that the factor XII Tenri present in disulfide-linked complexes with other proteins (and itself) is not converted to active forms, suggesting that attached proteins obstruct or delay the activation of factor XII via an inhibition of its binding to a negatively charged surface in vitro.
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PMID:Characterization of factor XII Tenri, a rare CRM-negative factor XII deficiency. 1522 38