A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Partial characterization of the purified enzyme.
A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Partial characterization of the purified enzyme.
复制标题
一种 Ca2 激活的蛋白酶，可能参与肌原纤维蛋白周转。
作者：
William R Dayton;W. Reville;D. E. Goll;M. Stromer
从猪骨骼肌中分离的纯化的Ca 2+激活的蛋白酶（CAF）能够从完整的肌原纤维中去除Z盘，在pH 7.5和1 mM Ca 2+和至少2 mM 2-巯基乙醇的存在下，对肌原纤维或酪蛋白底物具有最佳活性。当单独加入1 mM Mg 2+、Mn 2+、Ba 2+、Co 2+、Ni 2+和Fe 2+时，未检测到CAF活性。当加入1 mM Ca ~（2+）时，Co ~（2+）、Cu ~（2+）、Ni ~（2+）和Fe ~（2+）对CAF活性有抑制作用，而Mg ~（2+）、Mn ~（2+）和Ba ~（2+）对CAF活性无影响。CAF被碘乙酸不可逆地抑制，但不受大豆胰蛋白酶抑制剂的影响。S 0/20，W=5.90 S，沉淀平衡分子量为112 000。由于纯化的CAF在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中以分子量为80 000和30 000的两条多肽链迁移，因此CAF分子必须由这两条多肽链中的每一条组成。可以从校准的凝胶渗透柱数据或从S 0/20、W和分子量计算CAF的近似分子尺寸为38 X 220 A。纯化CAF的氨基酸组成和物理性质将其与已知的组织酶和血液或粒细胞中发现的其他蛋白酶区分开来。纯化的CAF去除了与I带中肌钙蛋白相关的400-A周期性的Z盘，并部分降解了M线，但与肌原纤维孵育时不会引起其他超微结构可检测的影响。这些结果与早期的发现一致，即纯化的CAF降解肌钙蛋白、原肌球蛋白和C蛋白，但对肌球蛋白、肌动蛋白或α-辅肌动蛋白没有影响，并表明CAF可能在肌原纤维蛋白代谢周转过程中对完整肌原纤维的分解具有生理作用。
The purified Ca2+-activated protease (CAF) isolated from porcine skeletal muscle and capable of removing Z-disks from intact myofibrils is optimally active on either myofibril or casein substrates at pH 7.5 and in the presence of 1 mM Ca2+ and at least 2 mM 2-mercaptoethanol. No CAF activity is detected when 1 mM Mg2+, Mn2+, Ba2+, Co2+, Ni2+, and Fe2+ are added singly. When added with 1 mM Ca2+, Co2+, Cu2+, Ni2+, and Fe2+ inhibit, whereas Mg2+, Mn2+, and Ba2+ have no effect on CAF activity. CAF is irreversibly inhibited by iodoacetate but is unaffected by soybean trypsin inhibitor. S0/20,W=5.90 S, and sedimentation equilibrium molecular weight - 112 000 for purified CAF. Because purified CAF migrates as two polypeptide chains with molecular weights of 80 000 and 30 000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the CAF molecule must consist of one each of these two polypeptide chains. Approximate molecular dimensions of 38 X 220 A can be calculated for CAF from calibrated gel permeation column data or from S0/20,W and the molecular weight. Amino acid composition and physical properties of purified CAF distinguish it from the known catheptic enzymes and from other proteases found in blood or in granulocytes. Purified CAF removes Z-disks the 400-A periodicity associated with troponin in the I band and partly degrades M lines but causes no other ultrastructurally detectable effects when incubated with myofibrils. These results agree with the earlier finding that purified CAF degrades troponin, tropomyosin, and C-protein but has no effect on myosin, actin, or alpha-actinin, and suggest that CAF may have a physiological role in disassembly of intact myofibrils during metabolic turnover of myofibrillar proteins.