Purification and characterization of guanosine 3':5'-monophosphate-specific phosphodiesterase from guinea pig lung.

A low K,,, guanosine 3’:5’-monophosphate (cyclic GMP)specific phosphodiesterase was purified about 250-fold from crude extracts of the guinea pig lung by steps of DEAEcellulose chromatography, hydroxylapatite treatment, and preparatory polyacrylamide gel electrophoresis. Analytical gel electrophoresis revealed two protein bands, indicating that the enzyme preparation was about 50% homogeneous. The relative rate of hydrolysis of cyclic GMP and adenosine 3’:5’-monophosphate (cyclic AMP), using 1 yM substrate concentrations, was at least 1,OOO:l. The apparent K,, for cyclic GMP (0.8 pM) was about 200 times lower than the apparent K,, for cyclic AMP (150 PM). No significant hydrolysis of inosine 3’:5’-monophosphate (cyclic IMP) and the Sbromo and 8-benzylamino derivatives of cyclic GMP, cyclic AMP, or cyclic IMP by the purified enzyme was noted. The specificity of the enzyme was unaltered by pH, most metal ions, temperature, or the endogenous protein activator and Ca2+. The purified cyclic GMP-specific enzyme was extremely labile and required bovine serum albumin for stabilization. The molecular weight of the enzyme was estimated by linear sucrose density gradient ultracentrifugation and Sephadex G-200 gel filtration to be 137,000 and 168,000, respectively. The enzyme required Mg2+, Mn2+, or Co2+ for its activity and was not stimulated by protein activator. In addition, maximal enzyme activity was observed at a pH range of 7.5 to 8.0 and at a temperature range of 37-42”, with an activation energy of 11.9 kcal/mol. Cyclic AMP and cyclic IMP were found to inhibit the cyclic GMP-specific enzyme in a competitive manner with apparent Ki values of 8.1 mM and 1.1 yM, respectively. Inhibition by cyclic IMP was highly specific for the cyclic GMPspecific enzyme when compared to a cyclic AMP phosphodiesterase purified from the same tissue.