Binding of Ca2+ to normal and dicoumarol-induced prothrombin

Stenflo, Johan; Ganrot, P. O.

doi:10.1016/0006-291x(73)91069-3

Cited by 136 publications

(34 citation statements)

References 12 publications

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“…In addition, the disulfide pairing yet remains to be established, and the identity of residues 38 and 39 must be confirmed. Two of the three vitamin K-dependent coagulation zymogens, factor X and prothrombin, are known to bind calcium ions (21)(22)(23). A calcium-binding site in prothrombin has recently been identified at positions 7 and 8 as y-carboxyglutamic acid (2-amino-4,4'-dicarboxybutyric acid) (24,25).…”

Section: Discussionmentioning

confidence: 99%

Bovine factor X1 (Stuart factor). Primary structure of the light chain.

Enfield¹,

Ericsson²,

Walsh³

et al. 1975

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

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The amino-acid sequence of the light chain of bovine factor X1 is presented. The sequence of 112 of the 140 residues was determined automatically on fragments produced by specific cleavage of arginyl, glutamyl, tryptophanyl, and asparaginyl-glycine bonds. The remainder was determined by conventional procedures. The amino-terminal sequence of the light chain is homologous with the amino-terminal region of bovine prothrombin and, like the latter, appears to contain several residues of a recently discovered unusual amino acid, -y-carboxyglutamic acid. The role of this amino acid in the calciumbinding ability of factor X and prothrombin is discussed.Bovine factor X (Stuart factor) is the zymogen of a protease involved in blood coagulation. Zymogen activation is mediated by activated factor IX (factor IXa), in the presence of factor VIII, calcium, and phospholipid, as well as by Russell's viper venom and by trypsin (1-3). Activated factor X (factor Xa) catalyzes the conversion of prothrombin to thrombin.Purified bovine factor X can be separated chromatographically into two fractions (factors XI and X2) having similar chemical and biological properties (4, 5). As commonly described, factor X is a glycoprotein of molecular weight 54,000, containing about 10% carbohydrate and composed of two polypeptide chains linked by one or more disulfide bonds (4, 6). The light and heavy chains have molecular weights of 16,000 and 38,000, respectively; the carbohydrate is exclusively associated with the heavy chain. The heavy chains of bovine factors X5 and IXa are homologous with trypsin, thrombin, and other mammalian serine proteases (7,8). The homology is particularly apparent in the amino-terminal regions and in the peptide segments surrounding the serine residue of the active site. Furthermore, the amino-terminal sequences of the zymogens, factor IX and prothrombin, and of the light chain of factor X are also homologous (9). These homologies suggest that these three serine proteases involved in blood coagulation and the pancreatic serine proteases have evolved from a common ancestral gene. For this reason, and in order to establish a basis for relating the chemical structure of factor Xa to its function, the determination of its amino-acid sequence was undertaken. This communication presents a preliminary account of the sequence of the light chain. METHODSPurified bovine factor X, was isolated from bovine plasma (4) by Dr. Kazuo Fujikawa. The protein was reduced and pyridylethylated, and the two chains were separated as described (4).Automatic sequence analysis was performed with the Beckman Sequencer (model 890B) on the intact light chain and on five large fragments prepared as shown diagrammatically in Fig. 1. Two fragments were obtained by cleavage of asparaginyl-glycine bonds (10) and one fragment each by cleavage of peptide bonds adjacent to tryptophanyl (11) and glutamyl (12) residues. The fifth fragment was isolated after a cleavage adjacent to arginyl residues, requiring succinylation of E-amino groups (13) and ...

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Section: Discussionmentioning

confidence: 99%

Bovine factor X1 (Stuart factor). Primary structure of the light chain.

Enfield¹,

Ericsson²,

Walsh³

et al. 1975

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

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“…The biosynthesis of prothrombin is vitamin K dependent, and deficiency of this vitamin or administration of the vitamin K antagonist, dicoumarol, gives rise to an abnormal prothrombin which does not function in blood coagulation (1)(2)(3)(4)(5)(6). The activation of prothrombin in vivo requires the binding of Ca2+ (7); abnormal prothrombin does not bind Ca2+ (2,8,9). During the activation of normal prothrombin an NH2-terminal fragment (molecular weight approximately 25,000) is split off; the difference between abnormal and normal prothrombin has been localized to this part of the molecule.…”

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confidence: 99%

Vitamin K Dependent Modifications of Glutamic Acid Residues in Prothrombin

Stenflo

Fernlund

Egan³

et al. 1974

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

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710

291

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A tetrapeptide, residues 6 to 9 in normal prothrombin, was isolated from the NH2-terminal, Ca2+-binding part of normal prothrombin. The electrophoretic mobility of the peptide was too high to be explained entirely by its amino-acid composition. According to 1H nuclear magnetic resonance spectroscopy and mass spectrometry, the peptide contained two residues of modified glutamic acid, -y-carboxyglutamic acid (3-amino-1,1,3-propanetricarboxylic acid), a hitherto unidentified amino acid. This amino acid gives normal prothrombin the Ca2 +_ binding ability that is necessary for its activation. Observations indicate that abnormal prothrombin, induced by the vitamin K antagonist, dicoumarol, lacks these modified glutamic acid residues and that this is the reason why abnormal prothrombin does not bind Ca2+ and is nonfunctioning in blood coagulation.Prothrombin is a plasma glycoprotein that is activated during the process of blood coagulation to the proteolytic enzyme thrombin. The biosynthesis of prothrombin is vitamin K dependent, and deficiency of this vitamin or administration of the vitamin K antagonist, dicoumarol, gives rise to an abnormal prothrombin which does not function in blood coagulation (1-6). The activation of prothrombin in vivo requires the binding of Ca2+ (7); abnormal prothrombin does not bind Ca2+ (2, 8, 9). During the activation of normal prothrombin an NH2-terminal fragment (molecular weight approximately 25,000) is split off; the difference between abnormal and normal prothrombin has been localized to this part of the molecule. Evidence has been produced that the difference is due to the lack of certain prosthetic groups in abnormal prothrombin (10-13).In an endeavour to define the difference between normal and abnormal prothrombin, the NH2-terminal fragments from both proteins were isolated and degraded further. A heptapeptide from normal prothrombin (residues 4 to 10) and a corresponding heptapeptide from abnormal prothrombin were isolated by BrCN degradation and trypsin digestion. The heptapeptide from normal prothrombin differed from the corresponding peptide in abnormal prothrombin in that it had a higher anodal electrophoretic mobility at pH 6.5 (13).By extensive proteolytic digestion, the heptapeptide from normal prothrombin was degraded to a tetrapeptide. This tetrapeptide, containing residues 6 to 9, still had an abnormally high anodal electrophoretic mobility at pH 6.5. This paper reports evidence that each of the two glutamic acid residues of this peptide are modified by replacement of one hydrogen on the -y carbon atom by a carboxyl group. This work will be described in greater detail elsewhere. MATERIALS AND METHODSIsolation of Tetrapeptide. The heptapeptide from normal prothrombin (residues 4 to 10) (ref. 13) was first thoroughly digested with aminopeptidase M (Sigma) and afterwards with carboxypeptidase B (Sigma). A tetrapeptide was isolated from the digest by gel chromatography on Sephadex G-25 superfine and obtained in pure form as judged by high voltage electrophoresis at...

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“…Of particular interest in this regard is the apparent enhanced selectivity for calcium over magnesium when pairs of glutamic acid (Glu) residues are replaced by y-carboxyglutamic acid (Gla) residues (Williams, 1977;Nelsestuen & Suttie, 1973;Esmon, Suttie & Jackson, 1975;Stenflo & Ganrot, 1972); presumably, it is this selectivity which requires the presence of ten Gla residues (and no Glu residues) in the calcium-binding region of the blood protein prothrombin (Magnusson, Stottrup-Jensen, Petersen & Claeys, 1975). * To whom correspondence should be addressed.…”

Section: Introductionmentioning

confidence: 99%

Conformation and structure of α-L-glutamyl-L-glutamic acid

Eggleston¹,

Hodgson²

1982

Acta Crystallogr Sect B

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The crystal structure of the acidic dipeptide o-Lglutamyl-L-glutamic acid, ct-L-Glu-L-GIu, C IoH16N20 7, has been determined from three-dimensional X-ray diffractometer data. The dipeptide crystallizes in the space group P2~ of the monoclinic system with two formula units in a cell of dimensions a = 5.343 (2) 1.38 cm-L The structure was solved by direct methods and refined by least-squares techniques to a final value of the weighted R factor (on F) of 0.050 based on 1632 independent intensities with I ___ 2tr(I). The dipeptide occurs as a zwitterion in the crystal, with the amino terminus protonated and the main-chain carboxyl group deprotonated. The two Glu side chains are on opposite sides of the backbone, and the structure is extended. The peptide linkage is significantly nonplanar, the co torsional angle being 167.6 ° . There is extensive intermolecular hydrogen bonding in the crystals, but no intramolecular hydrogen bonding.

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Binding of Ca2+ to normal and dicoumarol-induced prothrombin

Cited by 136 publications

References 12 publications

Bovine factor X1 (Stuart factor). Primary structure of the light chain.

Bovine factor X1 (Stuart factor). Primary structure of the light chain.

Vitamin K Dependent Modifications of Glutamic Acid Residues in Prothrombin

Conformation and structure of α-L-glutamyl-L-glutamic acid

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