title: Purification and identification of bovine liver gamma-carboxylase.
abstract: The microsomal gamma-carboxylase catalyzes modification of a limited set of glutamyl residues to gamma-carboxyglutamyl residues in a vitamin K-dependent reaction that also utilizes O2 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 10(5)-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.
authors.affiliation: ZymoGenetics Inc., Seattle, WA 98105.
authors.forename: K L
authors.initials: KL
authors.lastname: Berkner
authors.forename: M
authors.initials: M
authors.lastname: Harbeck
authors.forename: S
authors.initials: S
authors.lastname: Lingenfelter
authors.forename: C
authors.initials: C
authors.lastname: Bailey
authors.forename: C M
authors.initials: CM
authors.lastname: Sanders-Hinck
authors.forename: J W
authors.initials: JW
authors.lastname: Suttie
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019735:Carbon-Carbon Ligases; D002417:Cattle; D008025:Ligases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight
chemical_list: D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase