Phospholipid binding properties of bovine prothrombin peptide residues 1-45.
The present study investigates the unique contribution of the NH2-terminal 33 residues of prothrombin, the gamma-carboxyglutamic acid (Gla) domain, to the Ca(II) and phospholipid-binding properties of prothrombin. Two Gla domain peptides, 1-42 and 1-45, produced by chymotryptic cleavage of prothrombin fragment 1 (residues 1-156 of the amino terminus of bovine prothrombin) and isolated by anion-exchange chromatography were utilized to characterize the Gla domain of prothrombin. This investigation utilized several experimental approaches to examine the properties of the Gla domain peptides. These studies were somewhat hampered by the metal ion-induced insolubility of the peptides. However, the 1-45 peptide was specifically radioiodinated, which facilitated the study of this peptide at low concentrations. In contrast to prothrombin fragment 1, the intrinsic fluorescence of both 1-42 and 1-45 was not quenched upon the addition of 1 mM Ca(II) or any concentration of Mg(II). Equilibrium dialysis studies revealed that the 1-42 peptide bound three Ca(II) ions noncooperatively, whereas fragment 1 binds seven Ca(II) ions in a positive cooperative manner. Ca(II)-promoted conformational changes are observed by comparison of electrophoretic mobility changes in the presence of increasing Ca(II) concentrations. Prothrombin, fragment 1, and the Gla domain peptides 1-42 and 1-45 exhibited similar electrophoretic mobility behavior in the presence of Ca(II) ions. The radiolabeled 1-45 peptide was found to comigrate with phospholipid vesicles on gel permeation chromatography in the presence of Ca(II). Fragment 1 was shown to inhibit this Ca(II)-dependent phospholipid binding of 1-45, demonstrating that the 1-45 peptide does possess the necessary phospholipid-binding structure. Furthermore, a metal ion-dependent conformational monoclonal antibody, F9.29, was inhibited from binding fragment 1 by the 1-42 peptide.
Pollock
J S
JS
Department of Chemistry, University of North Carolina, Chapel Hill 27599.
Shepard
A J
AJ
Weber
D J
DJ
Olson
D L
DL
Klapper
D G
DG
Pedersen
L G
LG
Hiskey
R G
RG
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002918:Chymotrypsin; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D011516:Prothrombin / Q000302:isolation & purification / Q000378:metabolism*
D010446:Peptide Fragments; D010743:Phospholipids; C057276:prothrombin (1-45); D011516:Prothrombin; D002918:Chymotrypsin; D008274:Magnesium; D002118:Calcium