Metal ion blockage of tritium incorporation into gamma-carboxyglutamic acid of prothrombin. Stoichiometry of gamma-carboxyglutamic acid to Gd3+ for the high affinity sites.
Prothrombin possesses two high affinity and four low affinity gamma-carboxyglutamic acid (Gla)-dependent gadolinium binding sites. Earlier work (Price, P. A., Williamson, M. K., and Epstein, D. J. (1981) J. Biol. Chem. 256, 1172-1176) has shown that tritium can be specifically incorporated at the gamma-carbon of Gla in proteins at pH 5. In the present work we show that inclusion of saturating concentrations of Ca2+ in nondenaturing buffer systems ranging from pH 5.5 to 8.5 prevents the exchange of tritium into all 10 Gla residues of prothrombin. Similarly, saturating concentrations of Gd3+ prevent tritium incorporation into Gla at pH 5.5. Positive cooperativity was observed for the binding of Gd3+ to human prothrombin (at pH 5.5) for the two high affinity sites (Kd congruent to 35 nM). The four low affinity sites bind Gd3+ with a Kd congruent to 5 microM. Incubation of prothrombin ranging in concentrations from 10 to 40 microM with 2 eq of Gd3+ at pH 5.5 prevents 5.7 (average of seven determinations) Gla residues from tritium incorporation. Sedimentation velocity experiments conducted at pH 5.5 indicate that prothrombin in the presence of saturating concentrations of Gd3+ polymerizes, most likely, to a trimer. Further, in the presence of 2 eq of Gd3+, calculated percent weight average concentration of monomer prothrombin is congruent to 100% at 10 microM, approximately equal to 95% at 20 microM, and congruento to 80% at 40 microM protein concentration. Thus, it appears that under conditions in which prothrombin primarily exists as a monomer, occupancy of the initial two metal binding sites by Gd3+ involves six Gla residues.
Bajaj
S P
SP
Section of Medicine, Pathology, and Biochemistry, St. Louis University School of Medicine, Missouri 63104.
Saini
R
R
Katz
A
A
Cai
G Z
GZ
Maki
S L
SL
Brodsky
G L
GL
D015055:1-Carboxyglutamic Acid / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D005682:Gadolinium / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D011489:Protein Denaturation; D011516:Prothrombin / Q000378:metabolism*; D014316:Tritium / Q000378:metabolism*; D014461:Ultracentrifugation
D046911:Macromolecular Substances; D014316:Tritium; D015055:1-Carboxyglutamic Acid; D011516:Prothrombin; D005682:Gadolinium; D002118:Calcium