A metal ion-binding site in the kringle region of bovine prothrombin fragment 1.
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.
Berkowitz
P
P
Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290.
Huh
N W
NW
Brostrom
K E
KE
Panek
M G
MG
Weber
D J
DJ
Tulinsky
A
A
Pedersen
L G
LG
Hiskey
R G
RG
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D002417:Cattle; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D006031:Glycosylation; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D010449:Peptide Mapping; D010743:Phospholipids / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D011516:Prothrombin / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D002413:Cations, Divalent; D010446:Peptide Fragments; D010743:Phospholipids; D011498:Protein Precursors; C029733:prothrombin fragment 1; D011516:Prothrombin; D002918:Chymotrypsin; D014357:Trypsin; D008274:Magnesium; D002118:Calcium