Amino-terminal alanine functions in a calcium-specific process essential for membrane binding by prothrombin fragment 1.
Amino-terminal alanine functions in a calcium-specific process essential for membrane binding by prothrombin fragment 1.
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DOI：
10.1021/bi00413a052
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发表时间：
1988-06
期刊：
影响因子：
2.9
通讯作者：
D. Welsch;G. Nelsestuen
中科院分区：
文献类型：
--
作者：
D. Welsch;G. Nelsestuen
凝血酶原片段1(牛凝血酶原的156个氨基酸残基)上的两个乙酰化位点对于蛋白质的紧密钙和膜结合功能是必不可少的；钙保护这两个位点不受乙酰化的影响[Welsch，D.J.，Pletcher，C.H.，&NELLESTUEN，G.L.(1988)生物化学(本期三篇论文中的第一篇)]。赖氨酸残基(第3、11、44、57和97位)的epsilon-氨基对蛋白质功能不是关键的，在钙保护的蛋白质中是乙酰化的。两个必需的乙酰化位点中活性最强的是氨基末端丙氨酸。为了确定这个位点，片段1首先在有钙存在的情况下乙酰化，以衍生化非必需的位点。去除钙并用放射性试剂进行部分乙酰化可得到单一的主要放射性多肽。该多肽的分离和鉴定表明，其放射性活性与氨基端丙氨酸有关。此外，钙保护蛋白的序列分析表明，存在未衍生化的氨基端丙氨酸。令人惊讶的是，三硝基苯基的共价修饰并没有改变膜的结合活性。因此，氨基末端的正电荷似乎不是其功能的关键。氨基末端丙氨酸的乙酰化而不是第二必需位点的乙酰化，产生了对钙需求很高的片段1衍生物，它已经失去了大部分的膜结合功能。然而，该蛋白对镁离子的亲和力只有轻微的变化。与这种金属离子选择性一致，氨基端丙氨酸的保护是钙专一性的，而镁离子不能保护这个位点不受乙酰化的影响。
Two acetylation sites on prothrombin fragment 1 (amino-terminal 156 amino acid residues of bovine prothrombin) are essential for the tight calcium and membrane binding functions of the protein; calcium protects both of these sites from acetylation [Welsch, D. J., Pletcher, C. H., & Nelsestuen, G. L. (1988) Biochemistry (first of three papers in this issue)]. The epsilon-amino groups of the lysine residues (positions 3, 11, 44, 57, and 97) were not critical to protein function and were acetylated in the calcium-protected protein. The most reactive of the two essential acetylation sites was identified as amino-terminal alanine. To identify this site, fragment 1 was first acetylated in the presence of calcium to derivatize the nonessential sites. Removal of calcium and partial acetylation with radioactive reagent produced a single major radioactive peptide. Isolation and characterization of this peptide showed that the radioactivity was associated with amino-terminal alanine. In addition, sequence analysis of calcium-protected protein showed the presence of underivatized amino-terminal alanine. Surprisingly, covalent modification with a trinitrophenyl group did not alter membrane binding activity. Thus, the positive charge on the amino terminus did not appear critical to its function. Acetylation of amino-terminal alanine without acetylation of the second essential site produced a fragment 1 derivative which had a high requirement for calcium and which had lost most membrane binding function. However, this protein had only slightly altered affinity for magnesium ion. In agreement with this metal ion selectivity, protection of amino-terminal alanine was calcium specific, and magnesium ion did not protect this site from acetylation.(ABSTRACT TRUNCATED AT 250 WORDS)