DETERMINATION OF THE DISULFIDE BRIDGES IN FACTOR VA HEAVY-CHAIN

被引:33
作者
XUE, JC [1 ]
KALAFATIS, M [1 ]
SILVEIRA, JR [1 ]
KUNG, C [1 ]
MANN, KG [1 ]
机构
[1] UNIV VERMONT,COLL MED,DEPT BIOCHEM,BURLINGTON,VT 05405
关键词
D O I
10.1021/bi00248a021
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The M(r) = 94 000 heavy chain of bovine factor Va contains 10 cysteine residues which are distributed in the 2 A domains which make up this portion of the factor V molecule. The A1 domain contains four cysteines while the A2 domain contains six cysteines. The locations of disulfide bridges and free cysteines in bovine factor Va heavy chain were analyzed using iodo[C-14] acetamide-labeled factor Va heavy chain digested with trypsin, plasmin, V-8 protease, and cyanogen bromide. Following HPLC separation of the resulting peptides, free cysteines were identified by the incorporation of radioactivity while disulfide-containing peptides were detected using an SBD-F fluorometric assay after reduction. All cysteine-containing peptides were analyzed by amino acid sequence analysis. The four cysteines in the A1 domain are associated with two disulfide bonds, Cys(139)-Cys(165) and Cys(220)-Cys(301). One disulfide bond was explicitly identified in the A2 domain; Cys(471)-Cys(497), and a free cysteine was found in the A2 domain at Cys(538). Significant difficulties were encountered in preparing identifiable or soluble peptides which would permit the explicit identification of the three remaining cysteines in the A2 domain. On the basis of homology, it is likely that Cys(589) is a free SH while a disulfide bridge exists between Cys(579) and Cys(660). Thus, three major disulfide bonding patterns, characterized as ''alpha'', ''beta'', and ''gamma'' loops, are found in factor V. Each A domain contains a 26 residue ''alpha loop at positions 139-165, 471-497, and 1684-1710. The A1 and A2 domains each contain 81 amino acid residue ''beta'' loops at 220-301 and 579-660. The C domains contain 2 ''gamma'' loops of 154 (C1) and 155 (C2) residues, respectively, 1866-2020 and 2025-2180.
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页码:13109 / 13116
页数:8
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