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首页> 外文期刊>Molecular and Cellular Biology >Fine-structure mapping of charge-shift mutations in regulatory subunit of type I cyclic AMP-dependent protein kinase.
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Fine-structure mapping of charge-shift mutations in regulatory subunit of type I cyclic AMP-dependent protein kinase.

机译：I型环AMP依赖性蛋白激酶调节亚基中电荷转移突变的精细结构图。

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摘要

A variety of structural mutations that alter functional properties of regulatory subunit (R) of type I cyclic AMP-dependent protein kinase are available in the cultured S49 mouse lymphoma cell system. Many of these mutations also alter the electrostatic charge of R by about 1 or 2 units. By a novel peptide mapping procedure, a number of these "charge-shift" structural mutations were localized to small regions within the R polypeptide. The procedure employed two-dimensional polyacrylamide gel electrophoresis to separate large overlapping fragments generated from denatured, affinity-purified R by limited digestion with papain. Mutations were mapped to intervals between the endpoints of these fragments. The position of one mutation was confirmed by mapping a new site for cleavage by Staphylococcus aureus V8 protease. Six different Ka mutations, which increase the concentrations of cyclic AMP required for kinase activation, mapped to three clusters in the carboxy-terminal half of R. Second-site mutations that cause phenotypic reversion of a single Ka mutant strain mapped to either side of the original mutation. By using charge-shift mutations for calibration, a map of charge density distribution was constructed for the R polypeptide. This map allowed tentative assignment of mutational lesions to portions of the R amino acid sequence implicated in cyclic AMP binding.
机译：在培养的S49小鼠淋巴瘤细胞系统中可获得多种改变I型环状AMP依赖性蛋白激酶调节亚基（R）功能特性的结构突变。这些突变中的许多也将R的静电荷改变了约1或2个单位。通过新颖的肽作图程序，许多这些“电荷转移”结构突变被定位在R多肽内的小区域。该方法采用二维聚丙烯酰胺凝胶电泳，通过木瓜蛋白酶的有限消化来分离从变性的，亲和纯化的R产生的大的重叠片段。突变被映射到这些片段的端点之间的间隔。通过定位新的金黄色葡萄球菌V8蛋白酶切割位点来确认一个突变的位置。六个不同的Ka突变（增加了激酶激活所需的环AMP的浓度）映射到R的羧基末端一半的三个簇中。第二个位点的突变导致单个Ka突变株的表型回复，该单个Ka突变株映射到R的任一侧。原始突变。通过使用电荷转移突变进行校准，构建了R多肽的电荷密度分布图。该图允许将突变性损伤暂定为与环状AMP结合有关的R氨基酸序列的一部分。

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