Properties of the cyclic GMP phosphodiesterase from rat lung: Inhibition by unsaturated fatty acids

Abstract

Catalytic and regulatory properties of the major form of cyclic GMP phophodiesterase (3′:5′-cyclic-GMP 5′-nucleotidohydrolase, EC 3.1.4.35) from rat lung were studied. The enzyme partially purified by a DEAE-Sepharose chromatography displayed a much higher affinity toward cyclic GMP than toward cyclic AMP, the apparent K m values being 5.7 μM and 482 μM for the guanylic and the adenylic cyclic nucleotide, respectively. In contrast, the V value for cyclic AMP was about 3-times higher than the V value for cyclic GMP. Linear double reciprocal plots of initial velocity were observed with each cyclic nucleotide. From 10 −8 to 3.3·10 −6 M, cyclic GMP did not change the hydrolysis of 1 or 10 μM cyclic [ 3H]AMP, while it became inhibitory at higher concentrations. In contrast with a calmodulin-sensitive phosphodiesterase prepared from rat brain, the lung enzyme was not stimulated by a heat-stable Ca 2+-dependent factor from rat lung or by rat brain calmodulin or by lipids including fatty acids and lysophosphatidylcholine. Various unsaturated 18- and 20-carbon fatty acids inhibited at varying degrees the cyclic GMP phosphodiesterase from rat lung. The inhibitory potency increased with the number of double bonds in the hydrocarbon chain. In contrast, the methyl esters of the unsaturated fatty acids and the saturated fatty acids of variable hydrocarbon chain lengths had no appreciable effects. A linear Hill plot of phosphodiesterase inhibition with a slope of unity was obtained with arachidonic acid up to 30 μM, suggesting only one type of inhibitory site. In this range of concentrations the inhibition was entirely reversible. Kinetics analysis demonstrated that up to 30 μM arachidonic acid was a purely competitive inhibitor with an apparent K i of 20 μM. Over 30 μM, the Hill coefficient increased progressively, indicating the binding to other inhibitory sites, while the reversibility disappared.