Nucleophilic behavior of lysine-501 of the alpha-polypeptide of sodium and potassium ion activated adenosinetriphosphatase consistent with a role in binding adenosine triphosphate.
Nucleophilic behavior of lysine-501 of the alpha-polypeptide of sodium and potassium ion activated adenosinetriphosphatase consistent with a role in binding adenosine triphosphate.
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钠离子和钾离子激活的三磷酸腺苷酶的α-多肽的赖氨酸-501的亲核行为与结合三磷酸腺苷的作用一致。
DOI：
10.1021/bi00433a041
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Kyte,J
中科院分区：
文献类型：
--
作者：
Xu,KY;Kyte,J
加州大学圣迭戈分校化学系，拉霍亚，加利福尼亚州，92093收到1988年4月8日；修订稿件收到1988年12月5日摘要：一种针对羧基末端序列Gaper的免疫吸附剂，它由羊钠和钾离子激活的三磷酸腺苷酶[(Na++K+)-ATPase]的第502-506位残基组成，用于分离完整蛋白质中紧接在该序列之前的赖氨酸与[~3H]乙酸酐或荧光素5‘-异硫氰酸盐之间的反应产物。当ATP被稳定在E！中的完整的天然酶结合时，两种试剂都观察到了这种赖氨酸Lys50i的表观亲核性的变化。构象或当酶的结构从构象变为E_2-P构象时。对于这两种试剂，在前一次改变中，掺入产率下降了4倍以上，但在后一次改变中仅下降了2倍。由于在没有构象变化的情况下结合ATP时的降幅比在没有占据活性部位的情况下发生主要构象变化时的降幅要大得多，这些[~3H]乙酰掺入的变化表明a多肽中的Lys50i直接参与了(Na++K+)-ATPase活性部位内的ATP结合。本文还研究了抗Sequence-Gaper的特异性多克隆抗体与变性或酶活性(Na++K+)-ATPase之间的免疫化学反应。Western blotting和抗体引起的酶活性抑制表明，它可以分别与变性形式和天然形式的α-多肽结合。抗丙型免疫球蛋白与天然酶结合对荧光素S异硫氰酸酯掺入Lys50i的影响表明，它们与Lys501附近的天然酶结合。当免疫球蛋白被结合时，该酶就不再活跃。
Department of Chemistry, University of California, San Diego, La Jolla, California 92093 Received April 8, 1988; Revised Manuscript Received December 5, 1988 abstract: An immunoadsorbent specific for the carboxy-terminal sequence-GAPER, which comprises residues 502-506 of the a-polypeptide of ovine sodium and potassium ion activated adenosinetriphosphatase [(Na++ K+)-ATPase], was used toisolate the products of the reaction between the lysine immediately preceding this sequence in the intact protein and either [3H] acetic anhydride or fluorescein 5'-isothiocyanate. Changes in the apparent nucleophilicity of this lysine, Lys50i, were observed with both reagents when ATP was bound by the intact, native enzyme poised in the E! conformation or when the structure of the enzyme was changed from theE [conformation into the E2-P conformation. With both reagents, a decrease of more than 4-fold in the yield of incorporation occurred during the former change, but a decrease of only 2-fold occurred during the latter. Because a much larger decrease occurred when ATP was bound in the absence of a conformational change than occurred when a major conformational change took place in the absence of the occupation of the active site, these changes in the incorporation of [3H] acetyl suggest that Lys50i from the a polypeptide is directly involved in binding ATP within the active site of (Na++ K+)-ATPase. The immunochemical reactions between the specific polyclonal antibodiesraised against the sequence-GAPER and denatured or enzymically active (Na++ K+)-ATPase were also investigated. Western blots and the inhibition of enzymic activity caused by the antibody have shown that it can bind to both the denatured and the native form of the a-polypeptide, respectively. The effects of the binding of anti-GAPERimmu-noglobulins to the native enzyme on the incorporation of fluorescein S'-isothiocyanate at Lys50i demonstrate that they are binding to the native enzyme in the immediate vicinity of Lys501. When an immunoglobulin is bound, the enzyme is inactive.