PURIFICATION AND PROPERTIES OF THE CGMP-INHIBITED CAMP PHOSPHODIESTERASE FROM BOVINE AORTIC SMOOTH-MUSCLE

Pure cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) in mu-g quantities was isolated from bovine aortic smooth muscle after more than 500-fold purification using DEAE ion-exchange and affinity chromatography with a derivative of the specific cGI-PDE inhibitor cilostamide conjugated as a ligand to aminoethyl agarose (CIT-agarose). The cGI-PDE, which constituted about half of the high affinity cAMP-PDE activity of a tissue homogenate, was identified with a 105-kDa protein on SDS-PAGE through use of antibodies towards the human platelet, bovine cardiac and bovine adipose tissue cGI-PDE in Western blot and immunoprecipitation/immunoinactivation analysis. As observed during purification of the enzyme from other tissues the enzyme protein was exquisitely sensitive to proteolytic nicking during purification, resulting in several 30-77-kDa polypeptide fragments. Rapid immunoprecipitation from fresh tissue extracts was the only way found to partially prevent the proteolysis. The native enzyme had apparent molecular sizes of approx. 100 000 or, mainly approx. 220 000 by gel chromatography, presumably indicating the presence of monomeric and dimeric forms. The enzyme hydrolyzed cAMP and cGMP with normal Michaelis-Menten kinetics with K(m) of 0.16 and 0.09-mu-M, respectively, with V(max) for hydrolysis of cGMP of 0.3 compared to 3.1-mu-mol/min per mg protein for cAMP. The enzyme was potently and selectively inhibited by cGMP (IC50 almost-equal-to 0.25-mu-M) and the cardiotonic/vasodilatory drugs OPC-3911 (a cilostamide derivative), milrinone and CI-930 (IC50 almost-equal-to 0.05, 0.40 and 0.25-mu-M, respectively). The cGI-PDE was phosphorylated by cAMP-dependent protein kinase as has been reported for the analogous enzymes in heart, adipose tissue and platelets. The identification of a cGI-PDE in the aortic smooth muscle and its inhibitor specificity is consistent with the hypothesis that inhibition of this enzyme is important in the mechanism through which these drugs produce vasorelaxation.