A metal ion-binding site in the kringle region of bovine prothrombin fragment 1

• Published in: The Journal of biological chemistry 1992-03, Vol.267 (7), p.4570-4576
• Main Authors: Berkowitz, P, Huh, N W, Brostrom, K E, Panek, M G, Weber, D J, Tulinsky, A, Pedersen, L G, Hiskey, R G
• Format: Article
• Language: English
• Subjects: Animals; calcium; Calcium - metabolism; Cations, Divalent; Cattle; Chromatography, Gel; Chymotrypsin - metabolism; Electrophoresis, Polyacrylamide Gel; Fluorescence; Glycosylation; Hydrolysis; Magnesium - metabolism; metals; Peptide Fragments - metabolism; Peptide Mapping; Phospholipids - metabolism; Protein Precursors - metabolism; Prothrombin - metabolism; Trypsin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)42871-2

45Ca(II) binding studies (equilibrium dialysis) on the kringle domain of bovine prothrombin fragment 1 were conducted using a mixture of peptides (residues 43-156 and 46-156) resulting from limited alpha-chymotryptic hydrolysis of fragment 1. Analysis of the Scatchard plot of these data indicates a single, low affinity Ca(II)-binding site to be present. Similar results were obtained from studies on the decarboxylated fragment 1 derivative, 10-gamma-MGlu-fragment 1. Acetylation of bovine fragment 1 in the absence of Ca(II) or Mg(II) ions results in the loss of the metal ion-promoted quenching of the intrinsic Trp fluorescence of the protein and the Ca(II)-mediated binding to phosphatidylserine/phosphatidylcholine (PS/PC) vesicles. The acetylation of the NH2 alpha-group of Ala-1 has been shown (Welsch, D. J., and Nelsestuen, G. L. (1988) Biochemistry 27, 4946-4952) to abolish the PS/PC binding property of fragment 1. The present study demonstrates that acetylation of a second site possibly Ser-79 or Thr-81 using the conditions described in the preceding paper results in loss of both the fluorescence transition and the Ca(II)-mediated PS/PC binding of the resulting protein derivative. Removal of the O-acetyl group at the Ser-79/Thr-81 site is accomplished by aminolysis with 0.2 M hydroxylamine, pH 10, 50 degrees C; the fluorescence transition is partially restored. PS/PC binding is partially restored if the NH2 alpha-group of Ala-1 is trinitrophenylated but is not restored if the NH2 alpha-group of Ala-1 is acetylated. We conclude that the Ser-79/Thr-81 site may represent a portion of the metal ion-binding site within the kringle domain of fragment 1. Occupancy of this site by a Ca(II) ion appears to be important in the binding of the protein to PS/PC vesicles.