Location of gamma-carboxyglutamyl residues in partially carboxylated prothrombin preparations.
Prothrombin contains 10 gamma-carboxyglutamyl (Gla) residues in the N-terminal (fragment 1) domain of the protein. Following anticoagulant administration, a spectrum of undercarboxylated, physiologically less active forms of prothrombin is secreted into bovine or human plasma. The sites of undercarboxylation in these prothrombin species have now been investigated. Plasma containing a mixture of partially carboxylated forms of prothombin was obtained from a dicoumarol-treated bovine, and three pools of partially carboxylated (four, six, or eight Gla) species were purified by adsorption onto barium citrate and barium oxalate, ammonium sulfate fractionation, and chromatography. Fragment 1 obtained from these variants was equilibrated with 3H2O and heated in a dry state to decarboxylate Gla and incorporate 3H into the resulting Glu residues. This peptide was then sequenced by Edman degradation, and the specific radioactivity of PTH-Glu was determined for each potential Gla-containing site. Data obtained from normal prothrombin fragment 1 fit a linear model when the log of specific activity of PTH-Glu was plotted against the cycle number. Analysis of the 80% variant showed a decrease in carboxylation only in the last two Gla residues, while data obtained from the 60% variant indicated a general decrease in carboxylation from the most amino- to the more carboxyl-terminal Gla residues. In the 40% Gla variant, all but the most amino-terminal of the Gla residues appeared to be undercarboxylated. These data indicate that the gamma-carboxylation of glutamyl residues in prothrombin does not occur randomly but instead with preferential carboxylation of the most amino-terminal Gla residues. When carboxylation is limited, the impairment of carboxylation is more severe at the more carboxyl-terminal residues.
Liska
D J
DJ
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Suttie
J W
JW
D015055:1-Carboxyglutamic Acid / Q000032:analysis*; D000818:Animals; D002417:Cattle; D001728:Dicumarol / Q000494:pharmacology; D005260:Female; D010446:Peptide Fragments / Q000302:isolation & purification; D011516:Prothrombin / Q000302:isolation & purification / Q000378:metabolism*; D013917:Thrombin
D010446:Peptide Fragments; D015055:1-Carboxyglutamic Acid; D001728:Dicumarol; D011516:Prothrombin; D013917:Thrombin