Stringent requirement for Ca2+ in the removal of Z-lines and alpha-actinin from isolated myofibrils by Ca2+-activated neutral proteinase.
Stringent requirement for Ca2+ in the removal of Z-lines and alpha-actinin from isolated myofibrils by Ca2+-activated neutral proteinase.
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DOI：
10.1042/bj2090635
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发表时间：
1983-03
期刊：
影响因子：
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通讯作者：
M. Reddy;M. Rabinowitz;R. Zak
中科院分区：
文献类型：
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作者：
M. Reddy;M. Rabinowitz;R. Zak
用Ca 2+激活的中性蛋白酶（CANP）处理分离的肌原纤维导致Z线和α-辅肌动蛋白的特异性去除。为了研究这些过程的离子需求，我们测量Z线去除相差和干涉显微镜和α-辅肌动蛋白去除十二烷基硫酸钠/聚丙烯酰胺凝胶电泳分析肌原纤维蛋白。天然纯化蛋白质的蛋白水解消化直接在聚丙烯酰胺凝胶上和通过荧光胺技术测量。结果表明，CANP对Z线和α-辅肌动蛋白的去除以及蛋白降解产物的释放只在Ca ~（2+）存在时才发生，而Sr ~（2+）、Ba ~（2+）、Mn ~（2+）、Mg ~（2+）、Co ~（2+）和Zn ~（2+）均不起作用。与对Ca 2+的这种严格要求相反，用变性酪蛋白、天然和变性血红蛋白、天然肌动蛋白和原肌球蛋白测量的CANP的蛋白水解活性也在其他二价阳离子存在下发生，以以下顺序：Ca 2+大于Sr 2+大于Ba 2+。这些数据表明，只有Ca 2+可以产生构象变化的肌原纤维，使他们容易受到CANP的行动，而其蛋白水解活性是由几个二价离子刺激。
Treatment of isolated myofibrils with Ca2+-activated neutral proteinase (CANP) results in specific removal of Z-line and of alpha-actinin. To investigate the ionic requirement for these processes, we measured Z-line removal by phase-contrast and interference microscopy and alpha-actinin removal by sodium dodecyl sulphate/polyacrylamide-gel electrophoretic analysis of myofibrillar proteins. The proteolytic digestion of native purified proteins was measured directly on polyacrylamide gels and by the fluorescamine technique. We found that the removal of Z-line and alpha-actinin as well as the release of proteolytic degradation products from isolated myofibrils by CANP occur only in the presence of Ca2+; Sr2+, Ba2+, Mn2+, Mg2+, Co2+ and Zn2+ are all ineffective. In contrast with this stringent requirement for Ca2+, the proteolytic activity of CANP measured with denatured casein, native and denatured haemoglobin, native actin and tropomyosin also occurs in the presence of other bivalent cations, in the following order: Ca2+ greater than Sr2+ greater than Ba2+. These data suggest that only Ca2+ can produce the conformational change in myofibrils that renders them susceptible to the action of CANP, whereas its proteolytic activity is stimulated by several bivalent ions.