对肌钙蛋白抑制亚基具有活性的骨骼肌磷酸酶的鉴定、特性及其与其他磷蛋白磷酸酶的关系。

The identification and properties of phosphatases in skeletal muscle with activity towards the inhibitory subunit of troponin, and their relationship to other phosphoprotein phosphatases.

作者信息

Ray K P, England P J

出版信息

Biochem J. 1976 Aug 1;157(2):369-80. doi: 10.1042/bj1570369.

DOI:10.1042/bj1570369

PMID:183748

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1163862/

Abstract

Phosphoprotein phosphatases with activity towards the inhibitory subunit of troponin (troponin I), phosphorylase a and lysine-rich histone (fraction F1) have been fractionated from rat skeletal muscle by chromatography on Sephadex G-200 and polylysine-Sepharose. Six separate fractions were identified on the basis of substrate specificity and behaviour during chromatography. 2. All fractions showed similar Km values for any given protein substrate. The Km for troponin I (5 muM) was significantly lower than that previously reported. 3. Phosphatase activities towards troponin I and hosphorylase a did not show a requirement for bivalent-metal ions. Two of the fractions with only minor activity towards histone were activated by Mn2+. 4. Discontinuous polyacrylamide-gel-electrophoresis studies indicated that several of the fractions contained more than one phosphatase activity, and additionally showed that several of the activities could exist in different aggregation states. On the basis of these studies at least two phosphatases with activity only towards troponin I were identified. In addition, phosphorylase phosphatase (which has considerable activity towards troponin I) and a general phosphatase with activity towards all three substrates were found. 5. A fraction with mol.wt. of 150000 could be activated by freezing with 2-mercaptoethanol or by heating to 55 degrees C. This activation was accompanied by a decrease in mol.wt. to 25000. 6. The total amount of phosphatase with activity towards troponin I which was extracted would be sufficient to dephosphorylate all the troponin I present in skeletal muscle in approximately 10s.

摘要

利用葡聚糖凝胶G - 200和聚赖氨酸 - 琼脂糖柱色谱法，从大鼠骨骼肌中分离出了对肌钙蛋白（肌钙蛋白I）抑制亚基、磷酸化酶a和富含赖氨酸组蛋白（F1组分）具有活性的磷蛋白磷酸酶。根据底物特异性和色谱过程中的行为，鉴定出了六个不同的组分。2. 对于任何给定的蛋白质底物，所有组分都显示出相似的米氏常数（Km）值。肌钙蛋白I的Km值（5μM）显著低于先前报道的值。3. 对肌钙蛋白I和磷酸化酶a的磷酸酶活性不显示对二价金属离子的需求。对组蛋白只有轻微活性的两个组分被Mn2 +激活。4. 不连续聚丙烯酰胺凝胶电泳研究表明，几个组分含有不止一种磷酸酶活性，此外还表明几种活性可以以不同的聚集状态存在。基于这些研究，至少鉴定出了两种仅对肌钙蛋白I有活性的磷酸酶。此外，还发现了对肌钙蛋白I有相当活性的磷酸化酶磷酸酶和对所有三种底物都有活性的一般磷酸酶。5. 分子量为150000的一个组分可以通过用2 - 巯基乙醇冷冻或加热到55℃来激活。这种激活伴随着分子量降至25000。6. 提取的对肌钙蛋白I有活性的磷酸酶总量足以在大约10秒内使骨骼肌中所有的肌钙蛋白I去磷酸化。