CA-2+-ACTIVATED PROTEASE POSSIBLY INVOLVED IN MYOFIBRILLAR PROTEIN TURNOVER - PURIFICATION FROM PORCINE MUSCLE
CA-2+-ACTIVATED PROTEASE POSSIBLY INVOLVED IN MYOFIBRILLAR PROTEIN TURNOVER - PURIFICATION FROM PORCINE MUSCLE
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DOI：
10.1021/bi00655a019
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发表时间：
1976-01-01
期刊：
影响因子：
2.9
通讯作者：
REVILLE, WJ
中科院分区：
文献类型：
--
作者：
DAYTON, WR;GOLL, DE;REVILLE, WJ
Busch 等人描述了 P0-40 粗肌肉提取物中 Ca2+ 激活的 Z 盘去除活性。连续使用5柱层析程序从猪骨骼肌提取物中纯化：6%琼脂糖； DEAE-纤维素；葡聚糖凝胶 G-200； DEAE-梯度非常浅的纤维素；葡聚糖凝胶 G-150。所有 Z 盘去除活性均以单个峰从每根柱上洗脱下来。 Z 盘去除活性总是与 Ca2+ 激活的蛋白水解活性共洗脱，因此 P0-40 粗肌肉提取物中的 Z 盘去除活性归因于单一 Ca2+ 激活的蛋白酶 (CAF)。 5 柱色谱程序使 Ca2+ 激活的蛋白水解酶活性的比活性增加了 140 倍；因为在层析之前制备 P0-40 粗 CAF 级分使比活性增加了 127 倍，所以整个过程使 CAF 比活性增加了 17,800 倍。比活性的增加表明肌肉含有 3.4 μg CAF/g 肌肉鲜重；该含量与我们每克肌肉 0.25-0.76 微克纯化 CAF 的产量相当吻合。纯化的 CAF 在 pH 7.5 Tris-HCl 缓冲液中的聚丙烯酰胺凝胶电泳过程中迁移为单条带，但在十二烷基硫酸钠中的聚丙烯酰胺凝胶电泳过程中迁移为 MW 为 80,000 和 30,000 的 2 个条带。十二烷基硫酸钠-聚丙烯酰胺凝胶的光密度扫描显示，80,000 道尔顿和 30,000 道尔顿亚基构成纯化 CAF 制剂中蛋白质的 85-90%，并且这些亚基以等摩尔比存在。
Ca2+-activated Z-disk-removing activity in the P0-40 crude muscles extracts described by Busch et al. was purified from porcine skeletal muscle extracts by using 5 column chromatographic procedures in succession: 6% agarose; DEAE-cellulose; Sephadex G-200; DEAE-cellulose with a very shallow gradient; Sephadex G-150. All Z-disk-removing activity eluted in a single peak off each column. Z-disk-removing activity always coeluted with Ca2+-activated proteolytic activity, so Z-disk-removing activity in the P0-40 crude muscle extract is due to a single Ca2+-activated protease (CAF). The 5 column chromatographic procedures produced a 140-fold increase in specific activity of the Ca2+-activated proteolytic enzymic activity; because preparation of the P0-40 crude CAF fraction before chromatography produced a 127-fold increase in specific activity, the entire procedure produces a 17,800-fold increase in specific activity of CAF. This increase in specific activity suggests that muscle contains 3.4 .mu.g of CAF/g of muscle fresh weight; this content is in reasonably good agreement with our yields of 0.25-0.76 .mu.g of purified CAF/g of muscle. Purified CAF migrated as a single band during polyacrylamide gel electrophoresis in pH 7.5 Tris-HCl buffer but migrated as 2 bands with MW of 80,000 and 30,000 during polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Densitometric scans of sodium dodecyl sulfate-polyacrylamide gels show that the 80,000- and 30,000-dalton subunits make up 85-90% of the protein in purified CAF preparations and that these subunits are present in equimolar ratios.