Bovine Factor X.  II.  Characterization of purified Factor X.  Alterations in zone electrophoretic and chromatographic behavior occurring during purification

Jackson, Craig M.; Hanahan, Donald J.

doi:10.1021/bi00852a047

Cited by 97 publications

(41 citation statements)

References 31 publications

(41 reference statements)

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“…In its canonical role, VK serves as a coenzyme for VK-dependent carboxylase. This enzyme converts glutamate residues into ␥-carboxyglutamate (Gla) residues in VK-dependent proteins, such as prothrombin, and factors IX and X (6,10,29). Such VK-induced protein modification also occurs in osteocalcin (7,21) and matrix Gla protein (MGP) (22).…”

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confidence: 99%

Vitamin K Induces Osteoblast Differentiation through Pregnane X Receptor-Mediated Transcriptional Control of the Msx2 Gene

Igarashi

Yogiashi

Mihara

et al. 2007

Molecular and Cellular Biology

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Vitamin K is a fat-soluble vitamin that serves as a coenzyme for vitamin K-dependent carboxylase. Besides its canonical action, vitamin K binds to the steroid and xenobiotic receptor (SXR)/pregnane X receptor (PXR) and modulates gene transcription. To determine if the osteoprotective action of vitamin K is the result of the PXR/SXR pathway, we screened by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis the PXR/SXR target genes in an osteoblastic cell line (MC3T3-E1) treated with a vitamin K2 (menaquinone 4 [MK4]). Osteoblastic differentiation of MC3T3-E1 cells was induced by MK4. Msx2, an osteoblastogenic transcription factor, was identified as an MK4-induced gene. Functional analysis of the Msx2 gene promoter mapped a vitamin K-responsive element (PXR-responsive element [PXRE]) that was directly bound by a PXR/retinoid X receptor ␣ heterodimer. In a chromatin immunoprecipitation analysis, PXR was recruited together with a coactivator, p300, to the PXRE in the Msx2 promoter. MK4-bound PXR cooperated with estrogen-bound estrogen receptor ␣ to control transcription at the Msx2 promoter. Knockdown of either PXR or Msx2 attenuated the effect of MK4 on osteoblastic differentiation. Thus, the present study suggests that Msx2 is a target gene for PXR activated by vitamin K and suggests that the osteoprotective action of MK4 in the human mediates, at least in part, a genomic pathway of vitamin K signaling.The K vitamins are a group of fat-soluble vitamins that occur in two natural forms: phyloquinones (K1) and menaquinones (K2). Vitamin K (VK) has classically been associated with blood coagulation (31). In its canonical role, VK serves as a coenzyme for VK-dependent carboxylase. This enzyme converts glutamate residues into ␥-carboxyglutamate (Gla) residues in VK-dependent proteins, such as prothrombin, and factors IX and X (6,10,29). Such VK-induced protein modification also occurs in osteocalcin (7,21) and matrix Gla protein (MGP) (22). Thus, VK may exert beneficial effects on bone formation and remodeling. In fact, animal studies suggest that VK deficiency results in a reduction in bone mass together with hypocarboxylation of osteocalcin (25).Clinically, the most common form of K2, menaquinone 4 (MK4), has been shown to prevent bone fractures (3). This osteoprotective effect is more pronounced in K2 than in K1, and hence MK4 has been used to treat osteoporotic patients in Japan (9,11,28). However, the bone phenotypic abnormalities of mice deficient in osteocalcin and MGP do not fully support the classical view that the osteoprotective action of VK is the result of the modification of skeletal proteins. These mice, which are genetically deficient for osteocalcin or MGP, exhibited bone mass increases instead of losses (4). This suggests that the osteoprotective action of VK is mediated by another pathway.MK4 recently has been shown to act as a ligand for the steroid and xenobiotic receptor (SXR) in human osteoblastic cells (33). It transcriptionally regulates gene expression and represents a ne...

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confidence: 99%

Vitamin K Induces Osteoblast Differentiation through Pregnane X Receptor-Mediated Transcriptional Control of the Msx2 Gene

Igarashi

Yogiashi

Mihara

et al. 2007

Molecular and Cellular Biology

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“…Bovine factor X was purified (12) and activated with the factor X coagulant protein from Russell's viper venom (13).…”

Section: Methodsmentioning

confidence: 99%

A Polypeptide Region of Bovine Prothrombin Specific for Binding to Phospholipids

Gitel

Owen

Esmon

et al. 1973

Proc. Natl. Acad. Sci. U.S.A.

132

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The blood-clotting protein prothrombin can be converted to thrombin in free solution by the proteolytic enzyme, activated factor X. When prothrombin is bound to the surface of phospholipid vesicles, the rate of thrombin generation is increased more than 30-fold over the rate of unbound prothrombin. If the prothrombin activation process is terminated after a time interval in which less than 10% of the expected thrombin has been produced, two major products are found in the activation mixture. These products have been termed intermediate 1, a precursor of thrombin, and fragment 1. The conversion of prothrombin to thrombin in the blood coagulation process is markedly accelerated in the presence of phospholipid vesicles (1-3). Both prothrombin (3, 4) and activated factor X (the enzyme catalyzing the proteolyses involved in thrombin formation) (5) bind to phospholipid particles. This binding requires divalent cations and is reversible by removal of the divalent cations. In contrast to prothrombin, the final active product of the activation process, thrombin, does not bind to phospholipid surfaces (3,4).Investigations into the prothrombin activation mechanism have demonstrated the existence of at least two intermediates between prothrombin and thrombin and two activation fragments (6-10). As the relationship of some of the products described in earlier investigations to those seen by sodium dodecyl sulfate-acrylamide gel electrophoresis (8-10) Intermediate 1 and fragment 1 can be formed by the action of thrombin on prothrombin in both the presence and absence of factor Xa; however, factor Xa is required to form thrombin from prothrombin or either of the intermediates. Interaction between phospholipid vesicles and the activation products from partial activation of prothrombin with factor Xa were investigated in this study. MATERIALS AND METHODSOleic acid (>99% purity), phospholipase D, bovine-serum albumin, Russell's viper (Vipera russellii) venom, and Tris * HCl were obtained from Sigma Chemical Co. DEAESephadex was a product of Pharmacia, and Bio-Gel A 0.5 m was obtained from Bio Rad Laboratories. Dialysis tubing was a product of Union Carbide Corp., and silica gel GF-254 was a product of E. Merck Darmstadt. Preparative thin-layer chromatography (TLC) plates were obtained from Analtech Inc. All common laboratory chemicals were of analytical reagent grade and were obtained from major domestic supply houses. Bovine brain "cephalin" was prepared by the procedure of Folch (11).Bovine factor X was purified (12) 1344Abbreviations: factor Xa, factor X that has been activated by the factor X activating enzyme of Russell's viper venom; TLC, thin-layer chromatography. * To whom requests for reprints should be sent.

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“…It is composed of two polypeptide chains ("heavy" and "light") held together by one or more disulfide bonds. The intact protein can be separated chromatographically into two components (factor Xi and X2) which are identical in molecular weight and amino-acid composition (2)(3)(4). Upon activation by the complex of factors IXa and VIII in the presence of calcium and phospholipid, by factor VII and tissue factor in the presence of calcium, by a protease from Russell's viper venom, or by trypsin, glycopeptides are split, either near the amino or both the amino and carboxyl ends of the heavy chain, and an enzymatically active serine protease, factor Xa, is formed (5)(6)(7)(8).…”

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confidence: 99%

Bovine factor X1 (Stuart factor): amino-acid sequence of heavey chain.

Titani¹,

Fujikawa²,

Enfield³

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

111

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The amino-acid sequence of the heavy chain of bovine blood coagulation factor X1 (Stuart factor) isolated before and after activation has been determined. Sequence analysis was performed on fragments obtained by cleavage with cyanogen bromide and by tryptic digestion. Comparison of the complete sequence with those of other hepatic and pancreatic serine proteases demonstrates homology of the heavy chain of activated factor X1 (factor XI.) with the B chain of bovine thrombin as well as with bovine trypsin, chymotrsins A and B, and porcine elastase. The activation peptide cleaved near the amino terminus by a protease from Russell's viper venom differs in both size and sequence from those of other serine proteases. With three exceptions, all of the residues which are important in the catalytic functions of trypsin and chymotrypsin occur in corresponding loci in the heavy chain of factor Xa. These findings suggest that the three-dimensional structure of the heavy chain is similar to that of the pancreatic serine proteases and that these enzymes have evolved from a common ancestral gene.Factor X (Stuart factor) is the zymogen of a protease which upon activation by a complex of activated factor IX (factor IXa) and factor VIII catalyzes the conversion of prothrombin to thrombin (1). Bovine factor X is a glycoprotein with a molecular weight of 55,100. It is composed of two polypeptide chains ("heavy" and "light") held together by one or more disulfide bonds. The intact protein can be separated chromatographically into two components (factor Xi and X2) which are identical in molecular weight and amino-acid composition (2-4). Upon activation by the complex of factors IXa and VIII in the presence of calcium and phospholipid, by factor VII and tissue factor in the presence of calcium, by a protease from Russell's viper venom, or by trypsin, glycopeptides are split, either near the amino or both the amino and carboxyl ends of the heavy chain, and an enzymatically active serine protease, factor Xa, is formed (5-8). The heavy chain of factor Xa is homologous to pancreatic and certain hepatic serine proteases, as evidenced by similarities of the amino-terminal sequence and the sequence surrounding the serine and histidine residues of the active site (9).The sequence of the light chain of bovine factor X1 (10), for which the pancreatic proteases have no counterpart, is homologous to the amino-terminal sequences of bovine prothrombin and factor IX (11). All three of these factors are vitamin K dependent, and this dependency appears to be expressed in the post-transcriptional synthesis of y-carboxyglutamyl residues (12, 13) in the amino-terminal portions of the zymogens.This communication presents the amino-acid sequence of the heavy chain of bovine factor X1, and an examination of the structural homology of this chain with those of other serine proteases. Abbreviation: CHO, carbohydrate. METHODSFactor X1 was isolated from bovine plasma and converted to the active form (factor Xia) by incubation with a protease from Russel...

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Bovine Factor X. II. Characterization of purified Factor X. Alterations in zone electrophoretic and chromatographic behavior occurring during purification

Cited by 97 publications

References 31 publications

Vitamin K Induces Osteoblast Differentiation through Pregnane X Receptor-Mediated Transcriptional Control of the Msx2 Gene

Vitamin K Induces Osteoblast Differentiation through Pregnane X Receptor-Mediated Transcriptional Control of the Msx2 Gene

A Polypeptide Region of Bovine Prothrombin Specific for Binding to Phospholipids

Bovine factor X1 (Stuart factor): amino-acid sequence of heavey chain.

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