Determination of cyclic AMP-dependent protein kinase subunits by an immunoassay reveals a different subcellular distribution of the enzyme in rat parotid than does determination of the enzyme activity.

G Schwoch, S M Lohmann, U Walter, U Jung
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Abstract

The distribution of cyclic cAMP-dependent protein kinase in subcellular fractions of rat parotid was determined by two independent biochemical methods, measurement of kinase catalytic activity or by quantitation of the catalytic and regulatory subunits in an enzyme-linked immunosorbent assay using monospecific antibodies. The major amount (85%) of the catalytic activity was found associated with the 100,000 g soluble fraction, whereas only 1/3 of the total catalytic subunit was demonstrated in the soluble fraction by the immunoassay. The immunoassay results furthermore indicated that approximately 50% of the total cellular protein kinase was associated with the extranuclear particulate fraction and that the predominant form of the kinase in the particulate fractions was the type II isoenzyme. The reasons for the differences in the distribution of the protein kinase demonstrated by the two methods were examined. Incomplete extraction of membrane-bound protein kinase and the influence of membrane localized ATPases on activity measurements were, at least in part, responsible for the low percentage of kinase activity measured in the particulate fractions. These results emphasize that the precise quantitation of protein kinase subunits merits investigation by more than one method. For the parotid, the finding that approximately 2/3 of the total catalytic subunit may be particulate associated provides additional evidence that cyclic AMP could be involved in membrane mechanisms of hormone-regulated secretion.

通过免疫测定法测定环amp依赖性蛋白激酶亚基,揭示了该酶在大鼠腮腺中的亚细胞分布与测定酶活性不同。
环camp依赖性蛋白激酶在大鼠腮腺亚细胞部分的分布是通过两种独立的生化方法来确定的,一种是测量激酶的催化活性,另一种是用单特异性抗体在酶联免疫吸附法中定量测定催化和调节亚基。大部分(85%)的催化活性与100,000 g可溶性部分有关,而免疫分析显示可溶性部分仅占总催化亚基的1/3。免疫分析结果进一步表明,大约50%的细胞蛋白激酶与核外颗粒部分有关,颗粒部分中激酶的主要形式是II型同工酶。研究了两种方法所证明的蛋白激酶分布差异的原因。膜结合蛋白激酶的不完全提取和膜定位atp酶对活性测量的影响,至少在一定程度上,是颗粒馏分中测量的激酶活性百分比低的原因。这些结果强调蛋白激酶亚基的精确定量值得采用多种方法进行研究。对于腮腺,发现大约2/3的总催化亚基可能与颗粒相关,这为环状AMP可能参与激素调节分泌的膜机制提供了额外的证据。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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