Purification of labeled cyanogen bromide peptides of the alpha polypeptide from sodium ion and potassium ion activated adenosinetriphosphatase modified with N-[3H]ethylmaleimide.
Purification of labeled cyanogen bromide peptides of the alpha polypeptide from sodium ion and potassium ion activated adenosinetriphosphatase modified with N-[3H]ethylmaleimide.
复制标题
从用N-[3H]乙基马来酰亚胺修饰的钠离子和钾离子激活的腺苷三磷酸酶中纯化α多肽的标记溴化氰肽。
作者：
Le,DT
美国加州大学圣迭戈分校化学系，拉霍亚，加利福尼亚州，92093收到1985年7月18日；修订稿件收到1985年10月18日摘要：从狗肾脏分离的钠离子和钾离子激活的三磷酸腺苷酶在三种不同的构象下与N-[~3H]乙基马来酰亚胺反应，表面上分别是E_2-P、E_2和EB。这些指认是在考虑了各自烷基化混合物中配体的特定浓度后作出的。反应30分钟后，发现这三种不同的样品之间的剩余酶活性从未烷基化对照的90%到30%不等。在所有情况下，a多肽被提纯并用溴化氰消化，在每一次消化中，相同的两个不同的放射性多肽通过Sephadex LH-60凝胶过滤得到鉴定和纯化。N-[~3H]乙基马来酰亚胺掺入这两个肽中的一个与酶失活密切相关，而掺入另一个的最广泛的是当与ATP结合的活性部位未被占据时。然而，后一肽中残基的烷基化不会导致酶的失活。用高压液相色谱进一步纯化这两种多肽，并用手工Dansyl Edman或固相技术测定它们的氨基末端序列。含有被三磷酸腺苷保护的巯基的多肽，其氨基末端是与荧光素S‘-异硫氰酸酯专一反应的赖氨酸[Farley，R.A.，Tran，C.M.，Carilli，C.T.，Hawke，D.，&Shively，Je(1984)J.Biol]。化学。259,9532-9535]，
Dzung The Le Department of Chemistry, University of California, San Diego, La Jolla, California 92093 Received July 18, 1985; Revised Manuscript Received October 18, 1985 abstract: Sodium ion and potassium ion activated adenosinetriphosphatase, isolated from canine kidney, was reacted with N-[3H] ethylmaleimide while it was poised in three different conformations, ostensibly E2-P, E2, and Eb respectively. These assignments were made from a consideration of the particular concentrations of ligands in the respective alkylation mixtures. After a 30-min reaction, the remaining enzymatic activity was found to vary among these three different samples from 90 to 30% of that of unalkylated controls. In all cases, the a polypeptide was purified and subjected to digestion with cyanogen bromide, and in each digest the same two distinct radioactive peptides were identified and purified by gel filtration on a column of Sephadex LH-60. The incorporation of N-[3H] ethylmaleimideinto one of these two peptidescorrelated closely with enzymatic inactivation, while the incorporation into the other was most extensive when the portion of the active site to which ATP binds was unoccupied. Alkylation of the residue within the latter peptide, however, does notresult in inactivation of the enzyme. Both peptides were further purified by high-pressure liquid chromatography, and their amino-terminal sequences were determined by manual dansyl Edman or solid-phase techniques. The peptide containing the sulfhydryl protectedby ATP has, as its amino terminus, the lysine that reacts exclusively with fluoresceinyl S'-isothiocyanate [Farley, R. A., Tran, C. M., Carilli, C. T., Hawke, D., & Shively, JE (1984) J. Biol. Chem. 259, 9532-9535],