Benders A G, van Kuppevelt T H, Oosterhof A, Wevers R A, Veerkamp J H
Department of Biochemistry, University of Nijmegen, Netherlands.
Biochim Biophys Acta. 1992 Nov 23;1112(1):89-98. doi: 10.1016/0005-2736(92)90258-n.
Na+/K(+)-ATPase, Mg(2+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase are examined in cultured human skeletal muscle cells of different maturation grade and in human skeletal muscle. Na+/K(+)-ATPase is investigated by measuring ouabain binding and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase). SR Ca(2+)-ATPase is examined by ELISA, Ca(2+)-dependent phosphorylation and its activities on ATP and 3-O-methylfluorescein phosphate. Na+/K(+)-ATPase and SR Ca(2+)-ATPase are localized by immunocytochemistry. The activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase show a good correlation with the other assayed parameters of these ion pumps. All ATPase parameters investigated increase with the maturation grade of the cultured muscle cells. The number of ouabain-binding sites and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-MFPase are significantly higher in cultured muscle cells than in muscle. The Mg(2+)-ATPase activity, the content of SR Ca(2+)-ATPase and the activities of SR Ca(2+)-ATPase and Ca(2+)-dependent 3-O-MFPase remain significantly lower in cultured cells than in muscle. The ouabain-binding constant and the molecular activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase are equal in muscle and cultured cells. During ageing of human muscle the activity as well as the concentration of SR Ca(2+)-ATPase decrease. Thus the changes of the activities of the ATPases are caused by variations of the number of their molecules. Na+/K(+)-ATPase is localized in the periphery of fast- and slow-twitch muscle fibers and at the sarcomeric I-band. SR Ca(2+)-ATPase is predominantly confined to the I-band, whereas fast-twitch fibers are much more immunoreactive than slow-twitch fibers. The presence of cross-striation for Na+/K(+)-ATPase and SR Ca(2+)-ATPase in highly matured cultured muscle cells indicate the development and subcellular organization of a transverse tubular system and SR, respectively, which resembles the in vivo situation.
在不同成熟度的培养人骨骼肌细胞和人骨骼肌中检测钠钾ATP酶、镁ATP酶和肌浆网（SR）钙ATP酶。通过测量哇巴因结合以及钠钾ATP酶和钾依赖性3 - O - 甲基荧光素磷酸酶（3 - O - MFPase）的活性来研究钠钾ATP酶。通过酶联免疫吸附测定（ELISA）、钙依赖性磷酸化及其对ATP和3 - O - 甲基荧光素磷酸的活性来检测SR钙ATP酶。通过免疫细胞化学对钠钾ATP酶和SR钙ATP酶进行定位。钠钾ATP酶和SR钙ATP酶的活性与这些离子泵的其他检测参数具有良好的相关性。所研究的所有ATP酶参数均随着培养肌细胞的成熟度而增加。培养肌细胞中哇巴因结合位点的数量以及钠钾ATP酶和钾依赖性3 - O - MFPase的活性显著高于肌肉中的。培养细胞中的镁ATP酶活性、SR钙ATP酶含量以及SR钙ATP酶和钙依赖性3 - O - MFPase的活性仍显著低于肌肉中的。肌肉和培养细胞中钠钾ATP酶和SR钙ATP酶的哇巴因结合常数和分子活性相等。在人类肌肉衰老过程中，SR钙ATP酶的活性以及浓度均降低。因此，ATP酶活性的变化是由其分子数量的变化引起的。钠钾ATP酶定位于快肌和慢肌纤维的周边以及肌节I带。SR钙ATP酶主要局限于I带，而快肌纤维的免疫反应性比慢肌纤维强得多。高度成熟的培养肌细胞中钠钾ATP酶和SR钙ATP酶横纹的存在分别表明了横管系统和SR的发育及亚细胞组织，这类似于体内情况。
