Azanza J L, Raymond J, Robin J M, Cottin P, Ducastaing A
Biochem J. 1979 Nov 1;183(2):339-47. doi: 10.1042/bj1830339.
Ca(2+)-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial-Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulphate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only one polypeptide chain of mol.wt. 73000. The purification procedure used allowed us to eliminate a contaminant containing two components of mol.wt. about 30000 each. Whole casein or alpha(1)-casein were hydrolysed with a maximum rate at 30 degrees C, pH7.5, and with 5mm-CaCl(2), but myofibrils were found to be a very susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30000-dalton component as well as in various other higher- and lower-molecular-weight peptide fragments. Troponin T, troponin I, alpha-tropomyosin, some high-molecular-weight proteins (M protein, heavy chain of myosin) and three unidentified proteins are degraded. Thus the number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton, Goll, Zeece, Robson & Reville [(1976) Biochemistry15, 2150-2158]. The Ca(2+)-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. Thus the proteinase was proved to have an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca(2+)-activated neutral proteinase.
通过一种包括DEAE-葡聚糖凝胶层析、有机汞琼脂糖亲和层析以及Sephacryl S-200和Sephadex G-150凝胶过滤的方法，从兔骨骼肌中纯化出钙激活中性蛋白酶。十二烷基硫酸钠/聚丙烯酰胺凝胶电泳数据表明，纯化后的酶仅包含一条分子量为73000的多肽链。所采用的纯化程序使我们能够去除一种含有两种分子量约为30000的组分的污染物。全酪蛋白或α-酪蛋白在30℃、pH7.5以及5mM氯化钙存在的条件下被以最大速率水解，但发现肌原纤维是这种蛋白酶非常敏感的底物。这种活性与Z盘的破坏有关，而Z盘的破坏是由Z线蛋白的溶解引起的。该蛋白酶在体外的活性并不局限于去除Z线。在更大的平板上进行的十二烷基硫酸钠/聚丙烯酰胺凝胶电泳显示，该蛋白酶降解肌原纤维的能力比先前认为的更强。这种蛋白水解作用产生了一个分子量为30000的组分以及各种其他分子量更高和更低的肽片段。肌钙蛋白T、肌钙蛋白I、α-原肌球蛋白、一些高分子量蛋白质（M蛋白、肌球蛋白重链）以及三种未鉴定的蛋白质被降解。因此，肌原纤维中对蛋白酶敏感的区域数量比代顿、戈尔、泽斯、罗布森和雷维尔先前报道的[(1976)《生物化学》15, 2150 - 2158]更多。钙激活中性蛋白酶不是一种类胰凝乳蛋白酶或类胰蛋白酶，但它与所有用于组织蛋白酶B、木瓜蛋白酶、菠萝蛋白酶和无花果蛋白酶的经典巯基蛋白酶抑制剂发生反应。因此，证明该蛋白酶含有一个必需的巯基。抗蛋白酶和亮抑蛋白酶肽也是钙激活中性蛋白酶的抑制剂。
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