Localization of a fibrinogen calcium binding site between gamma-subunit positions 311 and 336 by terbium fluorescence.
Calcium is required for effective fibrin polymerization. The high affinity Ca2+ binding capacity of fibrinogen was directly localized to the gamma-chain by autoradiography of nitrocellulose membrane blots of fibrinogen subunits incubated with 45Ca2+. Terbium (Tb3+) competitively inhibited 45Ca2+ binding to fibrinogen during equilibrium dialysis, accelerated fibrin polymerization, and limited fibrinogen fragment D digestion by plasmin. The intrinsic fluorescence of Ca2+-depleted fibrinogen was maximally enhanced by Ca2+ and Tb3+, but not by Mg2+, at about 3 mol of cation/mol of fibrinogen. Protein-bound Tb3+ fluorescence at 545 nm was maximally enhanced by resonance energy transfer from tryptophan (excitation at 290 nm) at about 2 mol of Tb3+mol of fibrinogen and about 1 mol of Tb3+/mol of plasmic fragment D94 (Mr 94,000). Fibrinogen fragments D78 (Mr 78,000) and E did not show effective enhancement of Tb3+ fluorescence, suggesting that the Ca2+ site is located within gamma 303 to gamma 411, the peptide which is absent in fragment D78 but present in D94. When CNBr fragments of the carboxyamidated gamma-subunit were assayed for enhancement of Tb3+ fluorescence, peptide CBi (gamma 311-336) bound 1 mol of Tb3+/mol of CBi. Thus, the Ca2+ site is located within this peptide. The sequence between gamma 315 and gamma 329 is homologous to the calmodulin and parvalbumin Ca2+ binding sites.
Dang
C V
CV
Ebert
R F
RF
Bell
W R
WR
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D010320:Parvalbumins / Q000032:analysis; D013050:Spectrometry, Fluorescence; D013725:Terbium
D002147:Calmodulin; D005338:Fibrin Fibrinogen Degradation Products; D010320:Parvalbumins; C015214:fibrinogen D fragment; C018543:fibrinogen fragment E; D013725:Terbium; D004533:Egtazic Acid; D005340:Fibrinogen; D005341:Fibrinolysin; D003488:Cyanogen Bromide; D002118:Calcium