LOCATION OF GAMMA-CARBOXYGLUTAMYL RESIDUES IN PARTIALLY CARBOXYLATED PROTHROMBIN PREPARATIONS
LOCATION OF GAMMA-CARBOXYGLUTAMYL RESIDUES IN PARTIALLY CARBOXYLATED PROTHROMBIN PREPARATIONS
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DOI：
10.1021/bi00423a019
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发表时间：
1988-11-15
期刊：
影响因子：
2.9
通讯作者：
SUTTIE, JW
中科院分区：
文献类型：
--
作者：
LISKA, DJ;SUTTIE, JW
凝血酶原含有10。-羧基谷氨酰基（Gla）残基在蛋白质的n端（片段1）结构域。在使用抗凝剂后，低羧化的、生理上活性较低的凝血酶原会分泌到牛或人的血浆中。现在已经研究了这些凝血酶原的羧基化位点。血浆中含有部分羧基化形式的原凝血酶混合物，从双酚醇处理过的牛中获得，部分羧基化（4、6或8 Gla）的三个池通过吸附在柠檬酸钡和草酸钡上、硫酸铵分馏和层析纯化。从这些变体中得到的片段1用3H2O平衡，在干燥状态下加热，使Gla脱羧，并将3H掺入所得的Glu残基中。然后对该肽进行Edman降解测序，并对每个潜在的含glu位点测定PTH-Glu的比放射性。当PTH-Glu比活性的对数与循环数绘制时，从正常凝血酶原片段1获得的数据符合线性模型。对80%变异的分析表明，羧基化只在最后两个Gla残基中减少，而从60%变异中获得的数据表明，从氨基最多的Gla残基到羧基最多的Gla残基的羧基化普遍减少。在40%的Gla变体中，除了最氨基末端的Gla残基外，所有Gla残基似乎都是低羧化的。这些数据表明。凝血酶原中谷氨酰残基的羧化不是随机发生的，而是优先发生氨基末端Gla残基的羧化。当羧基化受到限制时，羧基末端残基越多，羧基化的损害就越严重。
Prothrombin contains 10 .gamma.-carboxyglutamyl (Gla) residues in the N-terminal (fragment 1) domain of the protein. Following anticoagulant administration, a spectrum of undercarboxylated, physiologically less active forms of prothrombin in secreted into bovine or human plasma. The sites of undercarboxylation in these prothrombin species have now been investigated. Plasma containing a mixture of partially carboxylated forms of prothombin was obtained from a dicoumarol-treated bovine, and three pools of partially carboxylated (four, six, or eight Gla) species were purified by adsorption onto barium citrate and barium oxalate, ammonium sulfate fractionation, and chromatography. Fragment 1 obtained from these variants was equilibrated with 3H2O and heated in a dry state to decarboxylated Gla and incorporate 3H into the resulting Glu residues. This peptide was then sequenced by Edman degradation, and the specific radioactivity of PTH-Glu was determined for each potential Gla-containing site. Data obtained from normal prothrombin fragment 1 fit a linear model when the log of specific activity of PTH-Glu was plotted against the cycle number. Analysis of the 80% variant showed a decrease in carboxylation only in the last two Gla residues, while data obtained from the 60% variant indicated a general decrease in carboxylation from the most amino-to the more carboxyl-terminal Gla residues. In the 40% Gla variant, all but the most amino-terminal of the Gla residues appeared to be undercarboxylated. These data indicate that the .gamma.-carboxylation of glutamyl residues in prothrombin does not occur randomly but instead with preferential carboxylation of the most amino-terminal Gla residues. When carboxylation is limited, the impairment of carboxylation is more severe at the more carboxyl-terminal residues.