PURIFICATION AND IDENTIFICATION OF BOVINE LIVER GAMMA-CARBOXYLASE
PURIFICATION AND IDENTIFICATION OF BOVINE LIVER GAMMA-CARBOXYLASE
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DOI：
10.1073/pnas.89.14.6242
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发表时间：
1992-07-15
影响因子：
11.1
通讯作者：
SUTTIE, JW
中科院分区：
文献类型：
--
作者：
BERKNER, KL;HARBECK, M;SUTTIE, JW
在依赖维生素K的反应中，微体伽马-羧基酶催化将一组有限的谷氨酰残基修饰为伽玛-羧基-谷氨酰残基，该反应也利用O2和CO2。我们报道了牛肝微粒体羧基酶的纯化。亲和层析利用羧基酶与凝血酶原前体的结合，以及羧基酶与前肽序列的结合，与离子交换层析和固定化凝集素分离相结合。纯化倍数为3.5×10(5)，是迄今报道的最高纯化倍数，为35倍。从这一分离中获得了单一的98 kDa蛋白。通过两个不同的标准，羧基酶活性与这种蛋白质有关。当用于蛋白质分析时，针对羧基酶制备的抗体检测到了98 kDa的蛋白质。此外，当在非变性凝胶体系中进行电泳时，显示出单一的98 kDa蛋白具有较高的活性。纯化的羧基酶制剂的可获得性将有助于加深对该蛋白质所执行的复杂生化反应的了解。
The microsomal gamma-carboxylase catalyzes modification of a limited set of glutamyl residues to gamma-carboxy-glutamyl 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.