Byus C V, Fletcher W H
J Cyclic Nucleotide Protein Phosphor Res. 1985;10(1):9-22.
A direct cytochemical procedure that specifically locates free catalytic subunits (C) from cAMP-dependent protein kinase has been used to follow the kinetics of kinase dissociation in parental Chinese hamster ovary cells (CHO 10001) and 4 mutant CHO cell lines variously deficient in this enzyme (CHO 10215, 10248, 10260, 10265) (1-5). When cultures of wild-type cells were stimulated with 8BrcAMP a time- and dose-dependent dissociation of kinase was observed. The catalytic unit appeared first in the cytoplasm and nucleolus and with time in the nucleoplasm as well. At peak protein kinase activation (30 min) more than 80% of the cells possessed abundant C in all of these subcellular compartments. The data indicate that both the type I and type II isozymes of the cAMP-dependent protein kinase are localized in similar areas of the cell. Stimulation of the mutant cell lines with 8-BrcAMP revealed that they each contained activatable kinase, determined cytochemically, that paralleled in amount the total assayable protein kinase determined biochemically (1-6). There were differences in the basal (unstimulated) levels of free C in the mutants relative to each other and to wild-type cells. For example, wild-type and mutants 10248, and 10260 had barely detectable cytoplasmic catalytic-subunit in unstimulated cultures whereas mutant 10215 possessed a significant amount of free C. Upon stimulation with 8-BrcAMP, the subcellular distribution of C was in all cases similar; although there were significant quantitative disparities between the wild type and various mutant cell lines. All cell lines examined had roughly equivalent amounts of nucleolar C but differed predominantly in the quantity of cytoplasmic kinase. Specifically, two of the mutant lines (10260 and 10248) contained barely detectable amounts of nucleoplasmic C whereas the other mutants had moderate (10265) to near normal (10215) amounts of nucleoplasmic enzyme. Mutants having only type I isozyme (10248) and those with only type II isozyme (10265) gave similar patterns of free C localization after 8BrcAMP stimulation.
一种直接的细胞化学方法可特异性定位环磷酸腺苷（cAMP）依赖性蛋白激酶的游离催化亚基（C），已被用于追踪亲代中国仓鼠卵巢细胞（CHO 10001）和4种该酶不同程度缺陷的突变型CHO细胞系（CHO 10215、10248、10260、10265）中激酶解离的动力学过程（1 - 5）。当用8 - 溴环磷酸腺苷（8BrcAMP）刺激野生型细胞培养物时，观察到激酶呈时间和剂量依赖性解离。催化亚基首先出现在细胞质和核仁中，随着时间推移也出现在核质中。在蛋白激酶激活的峰值（30分钟）时，超过80%的细胞在所有这些亚细胞区室中都含有丰富的C。数据表明，cAMP依赖性蛋白激酶的I型和II型同工酶都定位于细胞的相似区域。用8 - BrcAMP刺激突变细胞系表明，通过细胞化学测定，它们各自都含有可激活的激酶，其含量与通过生化方法测定的可检测的总蛋白激酶量相当（1 - 6）。相对于彼此以及野生型细胞，突变体中游离C的基础（未刺激）水平存在差异。例如，野生型以及突变体10248和10260在未刺激的培养物中几乎检测不到细胞质催化亚基，而突变体10215则含有大量的游离C。在用8 - BrcAMP刺激后，所有情况下C的亚细胞分布都相似；尽管野生型和各种突变细胞系之间存在显著的数量差异。所有检测的细胞系核仁C的量大致相当，但主要在细胞质激酶的量上有所不同。具体而言，其中两个突变系（10260和10248）几乎检测不到核质C，而其他突变体的核质酶量中等（10265）至接近正常（10215）。仅具有I型同工酶的突变体（10248）和仅具有II型同工酶的突变体（10265）在8BrcAMP刺激后呈现出相似的游离C定位模式。
