Localization of segments essential for polymerization and for calcium binding in the .gamma.-chain of human fibrinogen
- 11 February 1986
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 25 (3), 519-528
- https://doi.org/10.1021/bi00351a001
Abstract
We have isolated an intermediate plasmic degradation product, D2, of fibrinogen that does not inhibit the polymerization of fibrin monomer but does bind Ca2+. Fibrinogen was digested to a limited extent with plasmin in the presence of Ca2+, and a "large" fragment D (fragment D1A) was isolated with a .gamma.-chain remnant consisting of residues 63-411. Fragment D1A was digested further in the presence of Ca2+, yielding fragment D1 (with its .gamma.-chain containing residues 86-411). The digestion of fragment D1 [in the presence of ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N'',N''-tetraacetic acid (EGTA) to complex Ca2+] led to a gradual shortening of the carboxyl-terminal portion of the .gamma.-chain. Fragment D2 (with its .gamma.-chain containing residues 86-335/356) was isolated from an intermediate digest in the presence of EGTA. The Lys-338-Cys-339 peptide bond of the .gamma.-chain is intact in this preparation of D2, even though it is split in the isolated peptide .gamma.303-355 (with an intact disulfide bond at Cys-326-Cys-339). Fragment D2 does not interfere with the polymerization of fibrin monomer, whereas fragment D1 is a potent inhibitor of this polymerization. We conclude that the .gamma.-chain segment 356/357-411, present in fragment D1 but absent from fragment D2, is essential for maintenance of a polymerization site located in the outer (D) nodule of fibrinogen. This segment (356/357-411) is longer than two short ones reported earlier [Olexa, S.A., and Budzynski, A. Z. (1981) J. Biol. Chem. 256, 3544-3459; Horwitz, B.H., Varadi, A., and Scheraga, H.A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5980-5984]; the data for the earlier reports are reinterpreted here. Finally, fragment D2 possess a single Ca2+ binding site, as revealed by equilibrium dialysis binding studies. Since fragment D3 (with its .gamma.-chain containing residues 86-302) fails to bind Ca2+, we conclude that segment .gamma.303-355/356 plays a crucial role in Ca2+ binding.This publication has 19 references indexed in Scilit:
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