Vilsen B, Andersen J P
Danish Biomembrane Research Centre, Institute of Physiology, University of Aarhus.
J Biol Chem. 1992 Dec 25;267(36):25739-43.
Use of the nonphosphorylating beta,gamma-bidentate chromium(III) complex of ATP to induce a stable Ca(2+)-occluded form of the sarcoplasmic reticulum Ca(2+)-ATPase was combined with molecular sieve high performance liquid chromatography of detergent-solubilized protein to examine the ability of the Ca(2+)-ATPase mutants Gly-233-->Glu, Gly-233-->Val, Glu-309-->Gln, Gly-310-->Pro, Pro-312-->Ala, Ile-315-->Arg, Leu-319-->Arg, Asp-703-->Ala, Gly-770-->Ala, Glu-771-->Gln, Asp-800-->Asn, and Gly-801-->Val to occlude Ca2+. This provided a new approach to identification of amino acid residues involved in Ca2+ binding and in the closure of the gates to the Ca2+ binding pocket of the Ca(2+)-ATPase. The "phosphorylation-negative" mutant Asp-703-->Ala and mutants of ADP-sensitive phosphoenzyme intermediate type were fully capable of occluding Ca2+, as was the mutant Gly-770-->Ala. Mutants in which carboxylic acid-containing residues in the putative transmembrane segments had been substituted ("Ca(2+)-site mutants") and mutant Gly-801-->Val were unable to occlude either of the two calcium ions. In addition, the mutant Gly-310-->Pro, previously classified as ADP-insensitive phosphoenzyme intermediate type (Andersen, J.P., Vilsen, B., and MacLennan, D.H. (1992). J. Biol. Chem. 267, 2767-2774), was unable to occlude Ca2+, even though Ca(2+)-activated phosphorylation from MgATP took place in this mutant.
将ATP的非磷酸化β,γ-双齿铬(III)配合物用于诱导肌浆网Ca(2+)-ATP酶形成稳定的Ca(2+)封闭形式，并与去污剂增溶蛋白的分子筛高效液相色谱相结合，以检测Ca(2+)-ATP酶突变体Gly-233→Glu、Gly-233→Val、Glu-309→Gln、Gly-310→Pro、Pro-312→Ala、Ile-315→Arg、Leu-319→Arg、Asp-703→Ala、Gly-770→Ala、Glu-771→Gln、Asp-800→Asn和Gly-801→Val封闭Ca2+的能力。这为鉴定参与Ca2+结合以及Ca(2+)-ATP酶Ca2+结合口袋门关闭的氨基酸残基提供了一种新方法。“磷酸化阴性”突变体Asp-703→Ala以及ADP敏感型磷酸酶中间体类型的突变体与突变体Gly-770→Ala一样，完全能够封闭Ca2+。在假定的跨膜片段中含有羧酸残基的取代突变体（“Ca(2+)位点突变体”）和突变体Gly-801→Val无法封闭两个钙离子中的任何一个。此外，先前被归类为ADP不敏感型磷酸酶中间体类型的突变体Gly-310→Pro（Andersen, J.P., Vilsen, B., and MacLennan, D.H. (1992). J. Biol. Chem. 267, 2767-2774）即使在该突变体中发生了由MgATP激活的Ca(2+)磷酸化，也无法封闭Ca2+。
