Evidence for convertible forms of soluble uterine cyclic nucleotide phosphodiesterase

Abstract

The cyclic nucleotide phosphodiesterase (3′ : 5′-cyclic nucleotide 5′-nucleotidohydrolase, EC 3.1.4.17) systems of many tissues show multiple physical and kinetic forms. In contrast, the soluble rat uterine phosphodiesterase exists as a single enzyme form with non-linear Lineweaver-Burk kinetics for cyclic AMP (app. K_m of approx. 3 and 20 μM) and linear kinetics for cyclic GMP (app. K_m of approx. 3 μM) since the two hydrolytic activities are not separated by a variety of techniques. In uterine cytosolic fractions, cyclic AMP is a non-competitive inhibitor of cyclic GMP hydrolysis (K_i approx. 32 μM). Also, cyclic GMP is a non-competitive inhibitor of cyclic AMP hydrolysis (K_i approx 16 μM) at low cyclic GMP/cyclic AMP substrate ratios. However, cyclic GMP acts as a competitive inhibitor of cyclic AMP phosphodiesterase (K_i approx 34 μM) at high cyclic GMP/cyclic AMP substrate ratios. When a single hydrolytic form of uterine phosphodiesterase, separated initially by DEAE anion-exchange chromatography, is treated with trypsin (0.5 μg/ml for 2 min) and rechromatographed on DEAE-Sephacel, two major forms of phosphodiesterase are revealed. One form elutes at 0.3 M NaOAc⁻ and displays anomalous kinetics for cyclic AMP hydrolysis (app. K_m of 2 and 20 μM) and linear kinetics for cyclic GMP (app. K_m approx. 5 μM), kinetic profiles which are similar to those of the uterine cytosolic preparations. A second form of phosphodiesterase elutes at 0.6 M NaOAc⁻ and displays a higher apparent affinity for cyclic AMP (app. K_m approx. 1.5 μ) without appreciable cyclic GMP hydrolytic activity. These data provide kinetic and structural evidence that uterine phosphodiesterase contains distinct catalytic sites for cyclic AMP and cyclic GMP. Moreover, they provide further documentation that the multiple forms of cyclic nucleotide phosphodiesterase in mammalian tissue smay be conversions from a single enzyme species.