Sonnenburg W K, Rybalkin S D, Bornfeldt K E, Kwak K S, Rybalkina I G, Beavo J A
Department of Pharmacology, University of Washington, Seattle, Washington 98195, USA.
Methods. 1998 Jan;14(1):3-19. doi: 10.1006/meth.1997.0561.
The calmodulin-stimulated cyclic nucleotide phosphodiesterases (PDE1s) constitute a large gene family and are found in a wide variety of tissues and cells. Because of the functional diversity of PDE1 genes and the observation that these isozymes often make up a major component of the total cyclic nucleotide hydrolytic activity in certain cell types, PDE1s are of growing interest as targets for therapeutic intervention. Here we describe a series of methodologies to identify, quantitate, and determine the cellular expression of PDE1 isozymes. We describe first the resolution of different PDEs using high-performance anion-exchange chromatography and then a Western blotting methodology for identifying or authenticating PDE1 activities. Next we present an immunoprecipitation method that can be used for quantitating specific PDE1 isoforms and describe the use of RNase protection analysis for further identification of PDE1 subtypes. Finally, we provide a simple, immunocytochemical method for determining the cellular expression of PDE1 isozymes. Combined, the above methodologies should allow an investigator to identify, quantitate, and determine the cellular localization of PDE1 isozymes in any tissue with little ambiguity.
钙调蛋白刺激的环核苷酸磷酸二酯酶（PDE1s）构成一个庞大的基因家族，存在于多种组织和细胞中。由于PDE1基因的功能多样性，以及观察到这些同工酶在某些细胞类型中常构成总环核苷酸水解活性的主要成分，PDE1s作为治疗干预靶点越来越受到关注。在此，我们描述了一系列用于鉴定、定量和确定PDE1同工酶细胞表达的方法。我们首先描述了使用高效阴离子交换色谱法分离不同的磷酸二酯酶，然后介绍了一种用于鉴定或验证PDE1活性的蛋白质印迹法。接下来，我们介绍一种可用于定量特定PDE1同工型的免疫沉淀方法，并描述使用核糖核酸酶保护分析进一步鉴定PDE1亚型。最后，我们提供一种简单的免疫细胞化学方法来确定PDE1同工酶的细胞表达。综合起来，上述方法应能使研究人员在几乎没有歧义的情况下，鉴定、定量并确定任何组织中PDE1同工酶的细胞定位。
