Roth D A, Rehemtulla A, Kaufman R J, Walsh C T, Furie B, Furie B C
Center for Hemostasis and Thrombosis Research, New England Medical Center, Boston, MA.
Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8372-6. doi: 10.1073/pnas.90.18.8372.
A vitamin K-dependent carboxylase has recently been purified from bovine liver microsomes and candidate cDNA clones have been isolated. Definitive identification of the carboxylase remains circumstantial since expression of candidate carboxylase cDNAs in mammalian cells is confounded by the presence of endogenous carboxylase activity. To overcome this problem, a recombinant strain of baculovirus (Autographa california nuclear polyhedrosis virus, AcMNPV) encoding a putative carboxylase (vbCbx/AcMNPV) was used to infect Sf9 insect cells, which we demonstrate have no endogenous carboxylase activity. Infection with vbCbx/AcMNPV conferred vitamin K-dependent carboxylase activity to Sf9 insect cells. Carboxylase activity was demonstrated to peak 2-3 days after infection with vbCbx/AcMNPV. Metabolic radiolabeling with L-[35S]methionine revealed that the 90-kDa recombinant protein is the major protein synthesized at the time of peak activity after infection. An anti-peptide antibody directed against residues 86-99 reacted with bovine liver carboxylase on Western blot analysis and immunoprecipitated recombinant carboxylase from infected Sf9 microsomal protein preparations. Since Sf9 insect cells lack endogenous vitamin K-dependent carboxylase activity, expression of carboxylase activity in Sf9 insect cells with recombinant baculovirus demonstrates that the protein encoded by this cDNA is a vitamin K-dependent gamma-glutamyl carboxylase.
一种维生素K依赖性羧化酶最近已从牛肝微粒体中纯化出来，并且已分离出候选cDNA克隆。由于哺乳动物细胞中内源性羧化酶活性的存在，使得候选羧化酶cDNA的表达受到干扰，因此对该羧化酶的最终鉴定仍属间接证据。为克服这一问题，使用一种编码假定羧化酶的杆状病毒重组株（苜蓿银纹夜蛾核型多角体病毒，AcMNPV）感染Sf9昆虫细胞，我们证明这些细胞没有内源性羧化酶活性。用vbCbx/AcMNPV感染赋予Sf9昆虫细胞维生素K依赖性羧化酶活性。已证明羧化酶活性在vbCbx/AcMNPV感染后2至3天达到峰值。用L-[35S]甲硫氨酸进行代谢放射性标记显示感染后活性峰值时合成的主要蛋白质是90 kDa的重组蛋白。一种针对86 - 99位残基的抗肽抗体在蛋白质印迹分析中与牛肝羧化酶发生反应，并从感染的Sf9微粒体蛋白制剂中免疫沉淀重组羧化酶。由于Sf9昆虫细胞缺乏内源性维生素K依赖性羧化酶活性，用重组杆状病毒在Sf9昆虫细胞中表达羧化酶活性表明该cDNA编码的蛋白质是一种维生素K依赖性γ-谷氨酰羧化酶。
