大鼠心肌肌膜钙依赖性ATP酶的纯化与特性分析

Purification and characterization of a Ca2+-dependent ATPase from rat heart sarcolemma.

作者信息

Tuana B S, Dhalla N S

出版信息

J Biol Chem. 1982 Dec 10;257(23):14440-5.

Abstract

The Ca2+-dependent ATPase was solubilized from rat heart sarcolemmal membranes upon digestion with trypsin and was found to be different from Ca2+-stimulated Mg2+-dependent ATPase (Dhalla, N. S., Anand-Srivastava, M. B., Tuana, B. S., and Khandelwal, R. L. (1981) J. Mol. Cell. Cardiol. 13, 413-423). The enzyme was purified by high speed centrifugation, ammonium sulfate fractionation, and column chromatography and was seen as a single protein band in nondenaturing polyacrylamide gel electrophoresis. In sodium dodecyl sulfate-acrylamide gels, the enzyme dissociated into two subunits or fragments with molecular weights of about 55,000 and 12,000. The molecular weight of the enzyme, estimated by gel filtration on a Sephadex G-100 column, was found to be about 67,000. The enzyme utilized ATP with a Km of 0.20-0.26 mM but was also able to utilize ITP, CTP, GTP, and ADP as substrates at much lower rates. It was activated by Ca2+ with a Ka of 0.13-0.21 mM; it was also activated by other cations in the order Ca2+ greater than Mn2+ greater than Sr2+ greater than Ba2+ greater than Mg2+. Divalent cations like Cu2+, Ni2+, and Mg2+ were potent inhibitors. The enzyme was insensitive to ouabain, verapamil, oligomycin, cyanide, and vanadate but was markedly inhibited by N-ethylmaleimide. Calmodulin failed to stimulate Ca2+-dependent ATPase and instead inhibited slightly. Unlike K+, Na+ produced a marked inhibition of the Ca2+-dependent ATPase activity, and this inhibition was associated with an 8- 10-fold decrease in the affinity of the enzyme for Ca2+. The competitive action of Na+ indicates that the Ca2+-dependent ATPase may be a site of Na+-Ca2+ antagonism in the cell membrane.

摘要

用胰蛋白酶消化大鼠心肌肌膜后，可溶解出钙依赖性ATP酶，发现它与钙刺激的镁依赖性ATP酶不同（达拉，N.S.，阿南德 - 斯里瓦斯塔瓦，M.B.，图阿纳，B.S.，和坎德瓦尔，R.L.（1981年）《分子与细胞心脏病学杂志》13卷，413 - 423页）。该酶通过高速离心、硫酸铵分级分离和柱色谱法进行纯化，在非变性聚丙烯酰胺凝胶电泳中呈现为单一蛋白条带。在十二烷基硫酸钠 - 丙烯酰胺凝胶中，该酶解离为两个亚基或片段，分子量分别约为55,000和12,000。通过在葡聚糖G - 100柱上进行凝胶过滤估计，该酶的分子量约为67,000。该酶利用ATP的米氏常数为0.20 - 0.26 mM，但也能够以低得多的速率利用ITP、CTP、GTP和ADP作为底物。它被Ca²⁺激活，激活常数为0.13 - 0.21 mM；它也被其他阳离子以Ca²⁺＞Mn²⁺＞Sr²⁺＞Ba²⁺＞Mg²⁺的顺序激活。二价阳离子如Cu²⁺、Ni²⁺和Mg²⁺是强效抑制剂。该酶对哇巴因、维拉帕米、寡霉素、氰化物和钒酸盐不敏感，但被N - 乙基马来酰亚胺显著抑制。钙调蛋白未能刺激钙依赖性ATP酶，反而略有抑制作用。与K⁺不同，Na⁺对钙依赖性ATP酶活性产生显著抑制，这种抑制与该酶对Ca²⁺的亲和力下降8 - 10倍有关。Na⁺的竞争性作用表明钙依赖性ATP酶可能是细胞膜中Na⁺ - Ca²⁺拮抗作用的一个位点。