Localization of segments essential for polymerization and for calcium binding in the γ-chain of human fibrinogen

A. Váradi, Harold A. Scheraga

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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 γ-chain remnant consisting of residues 63-411. Fragment D1A was digested further in the presence of Ca2+, yielding fragment D1 (with its γ-chain containing residues 86-411). The digestion of fragment D1 [in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) to complex Ca2+] led to a gradual shortening of the carboxyl-terminal portion of the γ-chain. Fragment D2 (with its γ-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 γ-chain is intact in this preparation of D2, even though it is split in the isolated peptide 7303-355 (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 γ-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 shorter ones reported earlier [Olexa, S. A., & Budzynski, A. Z. (1981) J. Biol. Chem. 256, 3544-3549; Horwitz, B. H., Váradi, A., & 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 possesses a single Ca2+ binding site, as revealed by equilibrium dialysis binding studies. Since fragment D3 (with its γ-chain containing residues 86-302) fails to bind Ca2+, we conclude that segment 7303-355/356 plays a crucial role in Ca2+ binding.

Original languageEnglish
Pages (from-to)519-528
Number of pages10
JournalBiochemistry
Volume25
Issue number3
Publication statusPublished - 1986

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Polymerization
Fibrinogen
Calcium
Egtazic Acid
Peptides
Dialysis
Ethylene Glycol
Fibrinolysin
Disulfides
Ether
Digestion
Binding Sites
Maintenance
Degradation
Acids
fibrinmonomer

ASJC Scopus subject areas

  • Biochemistry

Cite this

Localization of segments essential for polymerization and for calcium binding in the γ-chain of human fibrinogen. / Váradi, A.; Scheraga, Harold A.

In: Biochemistry, Vol. 25, No. 3, 1986, p. 519-528.

Research output: Contribution to journalArticle

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title = "Localization of segments essential for polymerization and for calcium binding in the γ-chain of human fibrinogen",
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 γ-chain remnant consisting of residues 63-411. Fragment D1A was digested further in the presence of Ca2+, yielding fragment D1 (with its γ-chain containing residues 86-411). The digestion of fragment D1 [in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) to complex Ca2+] led to a gradual shortening of the carboxyl-terminal portion of the γ-chain. Fragment D2 (with its γ-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 γ-chain is intact in this preparation of D2, even though it is split in the isolated peptide 7303-355 (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 γ-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 shorter ones reported earlier [Olexa, S. A., & Budzynski, A. Z. (1981) J. Biol. Chem. 256, 3544-3549; Horwitz, B. H., V{\'a}radi, A., & 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 possesses a single Ca2+ binding site, as revealed by equilibrium dialysis binding studies. Since fragment D3 (with its γ-chain containing residues 86-302) fails to bind Ca2+, we conclude that segment 7303-355/356 plays a crucial role in Ca2+ binding.",
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N2 - 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 γ-chain remnant consisting of residues 63-411. Fragment D1A was digested further in the presence of Ca2+, yielding fragment D1 (with its γ-chain containing residues 86-411). The digestion of fragment D1 [in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) to complex Ca2+] led to a gradual shortening of the carboxyl-terminal portion of the γ-chain. Fragment D2 (with its γ-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 γ-chain is intact in this preparation of D2, even though it is split in the isolated peptide 7303-355 (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 γ-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 shorter ones reported earlier [Olexa, S. A., & Budzynski, A. Z. (1981) J. Biol. Chem. 256, 3544-3549; Horwitz, B. H., Váradi, A., & 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 possesses a single Ca2+ binding site, as revealed by equilibrium dialysis binding studies. Since fragment D3 (with its γ-chain containing residues 86-302) fails to bind Ca2+, we conclude that segment 7303-355/356 plays a crucial role in Ca2+ binding.

AB - 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 γ-chain remnant consisting of residues 63-411. Fragment D1A was digested further in the presence of Ca2+, yielding fragment D1 (with its γ-chain containing residues 86-411). The digestion of fragment D1 [in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) to complex Ca2+] led to a gradual shortening of the carboxyl-terminal portion of the γ-chain. Fragment D2 (with its γ-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 γ-chain is intact in this preparation of D2, even though it is split in the isolated peptide 7303-355 (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 γ-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 shorter ones reported earlier [Olexa, S. A., & Budzynski, A. Z. (1981) J. Biol. Chem. 256, 3544-3549; Horwitz, B. H., Váradi, A., & 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 possesses a single Ca2+ binding site, as revealed by equilibrium dialysis binding studies. Since fragment D3 (with its γ-chain containing residues 86-302) fails to bind Ca2+, we conclude that segment 7303-355/356 plays a crucial role in Ca2+ binding.

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