Characterization of a rat liver cyclic GMP-activated phosphodiesterase by chromatography on hexyl-agarose. Inhibition of phosphodiesterase activity by hexyl-agarose

Abstract

Chromatography on hexyl-agarose resolved a partially purified GMP-activated phosphodiesterase from rat liver into 2 peaks of activity: the 1st was eluted with 0.5 M KCl and was cAMP-specific. The 2nd was tightly bound to hexyl-agarose and was not eluted with KCl (0-2.0 M), which enhanced the hydrophobic interactions of this form with the matrix. It was eluted with 0.5 M-Tris, hydrolyzed cAMP and cGMP and was specifically activated by cGMP. The cGMP-activated phosphodiesterase was immobilized on hexyl-agarose. Enzyme activity, quantitatively bound to hexyl-agarose, was not released from the hydrophobic matrix in the presence of cAMP or cGMP, under assay conditions. The immobilized form of the enzyme retained catalytic activity, was inhibited by 0.1 mM cAMP and was activated by micromolar concentrations of cGMP to a lesser extent (7-fold) than the control, i.e., the enzyme mixed with unsubstituted agarose (15-fold). When the enzyme was immobilized, inhibition of cAMP phosphodiesterase activity was only observed in the presence of cGMP (at 3 .mu.M): in its absence, activity remained unchanged. The kinetic behavior of the immobilized enzyme is consistent with the hypothesis of a binding site distinct from the hydrolytic and activating sites.