Studies of kidney plasma membrane adenosine-3′,5′-monophosphate-dependent protein kinase

Abstract

Porcine kidney cortex was utilized for the preparation of plasma-membrane-enriched and soluble cytoplasmic (cytosol) fractions for the purpose of examining the relative properties of cyclic [ 3H]AMP receptor and cyclic AMP-dependent protein kinase activities of these preparations. The affinity, specificity and reversibility of cyclic [ 3H]AMP interaction with renal membrane and cytosol binding sites were indicative of physiological receptors. Binding sites of cytosol and deoxycholate-solubilized membranes were half-saturated at approx. 50nM and 100 nM cyclic [ 3H]AMP. Native plasma membranes exhibited multiple binding sites which were not saturated up to 1 mM cyclic [ 3H]AMP. Modification of the cyclic phosphate configuration or 2′-hydroxyl of the ribose moiety of cyclic AMP produced a marked reduction in the effectiveness of the cyclic AMP analogue as a competitor with cyclic [ 3H]AMP for renal receptors. The cyclic [ 3H]AMP interaction with membrane and cytosol fractions was reversible and the rate and extent of dissociation of bound cyclic [ 3H]AMP was temperature dependent. With the plasma-membrane preparation, dissociation of cyclic [ 3H]AMP was enhanced by ATP or AMP. Assay of both kidney subcellular fractions for protein kinase activity revealed that cyclic AMP enhanced the phosphorylation of protamine, lysine-rich and arginine-rich histones but not casein. The potency and efficacy of activation of renal membrane and cytosol protein kinase by cyclic AMP analogues such as N 6-butyryl-adenosine cyclic 3′,5′-monophosphate or N 6, O 2-dibutyryl-adenosine cyclic 3′,5′-monophosphate supported the observations on the effectiveness of cyclic AMP analogues as competitors with cyclic [ 3H]AMP in competitive binding assays. This study suggested that the membrane cyclic [ 3H]AMP receptors may be closely associated with the membrane-bound catalytic moiety of the cyclic AMP-dependent protein kinase system of porcine kidney.