Vitamin K-dependent carboxylation of the carboxylase
Vitamin K-dependent carboxylation of the carboxylase
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DOI：
10.1073/pnas.95.2.466
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发表时间：
1998-01-20
影响因子：
11.1
通讯作者：
Pudota, BN
中科院分区：
文献类型：
--
作者：
Berkner, KL;Pudota, BN
维生素K依赖(VKD)蛋白质需要用VKD-γ-谷氨酰羧基酶修饰，VKD-谷氨酰羧基酶是一种酶，在需要维生素K氢醌的反应中将谷氨酸簇转化为GLAS。对于它们的活性，我们发现羧基酶也在依赖维生素K的反应中使自己羧化。当纯人重组羧基酶与(CO2)-C-14体外孵育，然后经过SDS/PAGE分析后，观察到一条与羧基酶大小相对应的放射性标记条带，随后通过碱水解和高效液相色谱对体外修饰的羧基酶进行的GLE分析表明，所有的放射性活性都可以归因于GLA残基。对谷氨酸、天冬氨酸和谷氨酸残基的定量测定表明，3molGla/molCxylase。放射性标记的GLA是酸不稳定的，证实了它的特性，如果在体外反应中没有观察到维生素K，在杆状病毒(羧基酶)感染的昆虫细胞中也检测到羧化酶羧化，而在不表达内源VKD蛋白或羧基酶的模拟感染的昆虫细胞中不检测到。最后，我们证明了该羧化酶在体内是羧化的。从有或无维生素K培养的重组羧化酶BHK细胞中纯化了羧化酶并分析了GLA残基，只有在维生素K培养的BHK细胞中分离的羧化酶才能观察到羧化作用，并检测到3molGla/mol羧化酶，体外羧化酶和凝血因子IX的羧化分析表明羧化酶可能在凝血因子IX的转化中起作用，体内研究表明羧化酶在羧化酶稳定性中可能起作用，羧化酶的发现对VKD蛋白羧化的机制和基于华法林的抗凝治疗具有广泛的意义，需要进一步研究和回顾。
Vitamin K-dependent (VKD) proteins require modification by the VKD-gamma-glutamyl carboxylase, an enzyme that converts clusters of glus to glas in a reaction that requires vitamin K hydroquinone, for their activity, We have discovered that the carboxylase also carboxylates itself in a reaction dependent on vitamin K. When pure human recombinant carboxylase was incubated in vitro with (CO2)-C-14 and then analyzed after SDS/PAGE, a radiolabeled band corresponding to the size of the carboxylase was observed, Subsequent gla analysis of in vitro-modified carboxylase by base hydrolysis and HPLC showed that all of the radioactivity could be attributed to gla residues. Quantitation of gla, asp, and glu residues indicated 3 mol gla/mol carboxylase. Radiolabeled gla was acid-labile, confirming its identity, and was not observed if vitamin K was not included in the in vitro reaction, Carboxylase carboxylation also was detected in baculovirus-(carboxylase) -infected insect cells but not in mock-infected insect cells, which do not express endogenous VKD proteins or carboxylase. Finally, we showed that the carboxylase was carboxylated in vivo. Carboxylase was purified from recombinant carboxylase BHK cells cultured in the presence or absence of vitamin K and analyzed for gla residues, Carboxylation of the carboxylase only was observed with carboxylase isolated from BHK cells cultured in vitamin K, and 3 mol gla/mol carboxylase were detected, Analyses of carboxylase and factor IX carboxylation in vitro suggest a possible role for carboxylase carboxylation in factor IX turnover, and in vivo studies suggest a potential role in carboxylase stability, The discovery of carboxylase carboxylation has broad implications for the mechanism of VKD protein carboxylation and Warfarin-based anti-coagulant therapies that need to be considered both retrospectively and in the future.