ATP HYDROLYSIS-DEPENDENT PROTEASE ACTIVITY OF THE ION (CAPR) PROTEIN OF ESCHERICHIA-COLI K-12
ATP HYDROLYSIS-DEPENDENT PROTEASE ACTIVITY OF THE ION (CAPR) PROTEIN OF ESCHERICHIA-COLI K-12
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DOI：
10.1073/pnas.78.8.4728
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发表时间：
1981-01-01
期刊：
影响因子：
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通讯作者：
MARKOVITZ, A
中科院分区：
文献类型：
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作者：
CHARETTE, MF;HENDERSON, GW;MARKOVITZ, A
大肠杆菌CAPR基因突变导致多种表型，其中之一是不能降解异常和正常蛋白质(Deg-)。先前对部分纯化制剂的研究表明，Lon(CAPR)基因产物是一种94,000道尔顿多肽，与核酸有亲和力。Lon(CAPR)蛋白现已得到高度纯化，并被证明具有依赖于ATP的蛋白酶活性。该酶在含镁离子和三磷酸腺苷的Tris缓冲液中将标记的α-酪蛋白水解成三氯乙酸可溶的形式。该反应的最适pH为8.5，最适核苷酸为三磷酸腺苷。CTP和UTP可替代ATP(分别为75%和67%)，而GTP、ADP、AMP、cAMP和PPI不能替代。离子蛋白(CAPR)的蛋白分解需要三磷酸腺苷(ATP)的水解。ATP和CTP的非水解性类似物不能促进酪蛋白的切割。当使用低浓度的三磷酸腺苷时，蛋白质分解停止，因为三磷酸腺苷池耗尽。酪蛋白刺激离子(CAPR)ATPase活性，其产物为等摩尔数的ADP和PI。未检测到蛋白激酶活性。部分纯品中存在的DNA结合活性保留在纯化蛋白中。从一个具有Deg表型(CapR9)的Lon无义突变体中提纯的基因产物，虽然保留了DNA结合活性，但缺乏ATP依赖的蛋白酶和ATPase活性。缺乏依赖于ATP的蛋白酶活性可以解释在Lon突变体中观察到的许多多效性效应。
Mutations in the E. coli lon (capR) gene result in multiple phenotypes, one of which is the failure to degrade abnormal and normal proteins (Deg-). Previous work with partially purified preparations showed that the lon (capR) gene product is a 94,000-dalton polypeptide with an affinity for nucleic acids. The lon (capR) protein has now been highly purified and is demonstrated to have an ATP-dependent protease activity. The enzyme hydrolyzed 3H-labeled .alpha.-casein into trichloroacetic acid-soluble forms in Tris buffer containing Mg2+ and ATP. The reaction had a pH optimum of 8.5 and ATP was the preferred nucleotide. CTP and UTP could substitute for ATP (75% and 67%, respectively) but GTP, ADP, AMP, cAMP and PPi could not. Proteolysis by the lon (capR) protein required ATP hydrolysis. Nonhydrolyzable analogs of ATP and CTP did not promote casein cleavage. When low concentrations of ATP were used, proteolysis stopped as the ATP pool was depleted. Casein stimulated lon (capR) ATPase activity, and the products were ADP and Pi in equimolar amounts. No protein kinase activity was detected. The DNA-binding activity, present in partially pure preparations, was retained in the purified protein. The gene product purified from a lon nonsense mutant that exhibits the Deg- phenotype (capR9), lacked both the ATP-dependent protease and ATPase activities, though it retained DNA-binding activity. Absence of an ATP-dependent protease activity could account for many of the pleiotropic effects observed in lon mutants.