Purification and identification of bovine liver gamma-carboxylase.

Abstract

The microsomal y-carboxylase catalyzes modification of a limited set of glutamyl residues to y-carboxyglutamyl residues in a vitamin K-dependent reaction that also utilizes 02 and CO2. We report the purification to apparent homogeneity of the bovine liver microsomal carboxylase. Affinity chromatography exploiting the association of the carboxylase with prothrombin precursor and carboxylase binding to the propeptide sequence were combined with ion-exchange chromatography and fractionation using immobilized lectins. A 3.5 x 105-fold purification was obtained, which is the highest purification, by a factor of 35, yet reported for this enzyme. A single 98-kDa protein is obtained from this isolation. Carboxylase activity is associated with this protein by two different criteria. Antibodies prepared against the carboxylase detected the 98-kDa protein when used in Western analysis. In addition, the single 98-kDa protein was shown to comigrate with activity when electrophoresed in a nondenaturing gel system. The availability of purified preparations of carboxylase will facilitate an increased understanding of the complex biochemical reaction carried out by this protein.The liver microsomal carboxylase catalyzes the posttranslational modification of selected glutamyl residues to Y-carboxylated glutamyl (Gla) residues in a limited set of proteins in a reaction requiring vitamin K hydroquinone, 02, and CO2 (1-3). These vitamin K-dependent proteins contain multiple glutamyl residues clustered at the N terminus in what is referred to as the Gla domain. Carboxylation of glutamyl residues in the Gla domain enables the Ca2+-mediated interaction ofthese proteins with phospholipids and is required for their biological activity. Vitamin K-dependent carboxylase activity has been detected in almost all mammalian tissues assayed and has been observed in one invertebrate as well (4). Its apparent presence in a wide variety of cultured lines can also be inferred from the ability of these lines to secrete carboxylated vitamin K-dependent recombinant proteins (5-10). A major limitation in understanding the mechanism of carboxylation has been the lack of a purified preparation of this enzyme. Attempts pmade using selective detergent extraction, ammonium sulfate fractionation, and conventional chromatography have been successful only in a limited purification (11-13). More recently, the propeptide sequence unique to all vitamin K-dependent proteins has been used in attempts to purify the carboxylase (14-16). This sequence is similar among vitamin K-dependent proteins that share very little other homology, leading to the proposal that the propeptide sequence is important for carboxylase recognition (17). The observations that mutations in protein C (18) or factor IX (19) impair carboxylation supported this concept. Carboxylase purifications have been attempted by using the propeptide as a ligand in affinity chromatography (15,16). The enzyme has also been isolated by virtue of its known association with its vitamin K-dependent prote...

In vitro and in vivo functional characterization of bovine vitamin K-dependent gamma-carboxylase expressed in Chinese hamster ovary cells.

“…We further demonstrate here that expression of y-carboxylase activity correlated with expression of the expected polypeptide. More recently, Berkner et al (27) have described purification of a 98-kDa polypeptide that has 15-fold higher specific activity in vitro over that described by Wu et al (9). Since different assay conditions were employed, it is difficult to directly compare specific activities of the two preparations.…”

Incomplete gamma carboxylation of human coagulation factor VII: differential effects on tissue factor binding and enzymatic activity

“…Coagulation protein factors II, VII, IX, X, protein C and protein S are synthesized in the liver and undergo posttranslational modification by a vitamin K dependent carboxylase enzyme which converts 9-12 glutamic acid residues in the amino termini of these proteins to carboxyglutamic acid residues (GLA) (Furie & Furie, 1990). Both the bovine and human vitamin K dependent carboxylase enzymes have been purified and their mechanism of action described (Esmon et al, 1975;Hubbard et al, 1989;Wu et al, 1991a, b;Berkner et al, 1992;Rehemtulla et al, 1993;Morris et al, 1993;Suttie, 1993). GLA residues have been shown to form low-affinity calcium binding sites (Bajaj et al, 1982) which stabilize the tertiary structure of the GLA domain in these proteins thereby facilitating binding to phospholipid surfaces and regulating coagulation (Soriano-Garcia et al, 1989;Borowski et al, 1985).…”

Expression of bovine vitamin K-dependent carboxylase activity in baculovirus-infected insect cells.

“…For this reason, the evidence that the original cDNA encodes the putative carboxylase remains circumstantial. Furthermore, affinity purification of bovine liver carboxylase, exploiting the association of the enzyme with proprothrombin, has resulted in the purification of a 98-kDa protein thought to represent a vitamin K-dependent carboxylase with higher specific activity than the 94-kDa protein (14). The relationship of this 98-kDa protein to the bovine protein we have expressed is currently unknown.…”