Modifications of bovine prothrombin fragment 1 in the presence and absence of Ca(II) ions. Loss of positive cooperativity in Ca(II) ion binding for the modified proteins.
Chemical modification of bovine prothrombin fragment 1 according to the procedure of D. J. Welsch and G. L. Nelsestuen (1988) [Biochemistry 27, 4946-4952 and ealier papers] provided a series of fragment 1 derivatives in which various nitrogen-containing side chains were N-acetylated and/or N-2,4,6-trinitrophenylated. In addition the des-[Ala-1,Asn-2]- and des-[Ala-1,Asn-2,Lys-3]-fragment 1 derivatives were prepared by limited enzymatic hydrolysis of fragment 1 using cathepsin C and plasmin, respectively. Quantitative studies on the Ca(II) binding of these proteins have been accomplished using 45Ca(II) equilibrium dialysis. Binding of these fragment 1 derivatives to phosphatidylserine/phosphatidylcholine (PS/PC) vesicles (25:75) in the presence of Ca(II) ions has been studied using the light-scattering technique. Acylation of the 5 lysine residues of fragment 1 by the action of acetic anhydride (500-fold molar excess) in the presence of 75 mM Ca(II), pH 8.0, results in loss of positive cooperativity in Ca(II) binding (Scatchard plot) and an increase in the number of Ca(II) ions bound. The Ca(II)-dependent PS/PC binding of the acylated protein is reduced. Removal of 2 and 3 residues from the amino terminus likewise leads to loss of positive cooperativity in Ca(II) binding and reduced binding affinity to PS/PC vesicles. The important role of the amino-terminal 1-10 sequence is discussed. We conclude that positive cooperativity in Ca(II) binding is not a prerequisite for the Ca(II)-dependent binding of bovine prothrombin fragment 1 to PS/PC vesicles.
Weber
D J
DJ
Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290.
Berkowitz
P
P
Panek
M G
MG
Huh
N W
NW
Pedersen
L G
LG
Hiskey
R G
RG
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020789:Cathepsin C; D002413:Cations, Divalent; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D006868:Hydrolysis; D010446:Peptide Fragments / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism*; D011516:Prothrombin / Q000378:metabolism*
D002413:Cations, Divalent; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; D011498:Protein Precursors; C029733:prothrombin fragment 1; D011516:Prothrombin; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D020789:Cathepsin C; D005341:Fibrinolysin; D002118:Calcium