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 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.
- Research Organization:
- St. Louis Univ. School of Medicine, MO (USA)
- OSTI ID:
- 6908870
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 263:20
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
GLUTAMIC ACID
STOICHIOMETRY
PROTHROMBIN
RECEPTORS
AFFINITY
BIOCHEMICAL REACTION KINETICS
CALCIUM
GADOLINIUM
PH VALUE
PROTEIN DENATURATION
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ULTRACENTRIFUGATION
ALKALINE EARTH METALS
AMINO ACIDS
BLOOD COAGULATION FACTORS
CARBOXYLIC ACIDS
CENTRIFUGATION
COAGULANTS
DRUGS
ELEMENTS
HEMATOLOGIC AGENTS
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
MEMBRANE PROTEINS
METALS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PROTEINS
RARE EARTHS
REACTION KINETICS
SEPARATION PROCESSES
550201* - Biochemistry- Tracer Techniques