Adrenal medullary cyclic nucleotide phosphodiesterase: Subcellular distribution, partial purification and regulation of enzyme activity

Abstract

The major cyclic nucleotide phosphodiesterase (3′:5′-cyclic-AMP 5′-nucleotidohydrolase, EC 3.1.4.17) of bovine adrenal medulla has been purified 360-fold and characterized with respect to subcellular distribution, substrate specificity and kinetic properties. When assayed at millimolar concentrations of cyclic nucleotides, only one major activity was found in this tissue, and it was localized in the 100 000 × g supernatant. The enzyme has activity directed against both cyclic AMP ( K m = 83.8 μ M ; V = 126 nmol/min per mg protein) and cyclic GMP ( K m = 69.4 μ M ; V = 131 nmol/min per mg protein). Cyclic AMP and cyclic GMP appear to be hydrolyzed at the same catalytic site. In addition to the observations that the cyclic AMP and cyclic GMP hydrolyzing activities copurified during protein fractionation, and had identical electrophoretic behavior, each cyclic nucleotide was determined to be a competitive inhibitor of the hydrolysis of the other, with K i values being roughly similar to their respective K m values as substrates. The enzyme displays positive cooperative kinetic behavior when either cyclic nucleotide is the substrate; however, the cooperativity is more marked with cyclic AMP than with cyclic GMP. Low concentrations of cyclic GMP (1 μM) activate cyclic AMP hydrolysis approximately six fold. This activation is due to a loss of cooperativity accompanied by a two fold decrease in the apparent K m value for cyclic AMP. Cyclic IMP is a less effective activator of the enzyme. Low concentrations of para-chloromercuribenzoate activate cyclic AMP hydrolysis in the absence of cyclic GMP, but do not potentiate the cyclic GMP activation of the enzyme. The enzyme is competitively inhibited by aminophylline and noncompetitively inhibited by desipramine. The finding that the major cyclic nucleotide phosphodiesterase of adrenal medulla is a cyclic GMP-activated cyclic nucleotide phosphodiesterase suggests that this enzyme may be important in regulating changes in the cyclic AMP: cyclic GMP ratio following cholinergic stimulation in this tissue.