CHARACTERIZATION OF PARTICULATE CYCLIC-NUCLEOTIDE PHOSPHODIESTERASES FROM BOVINE BRAIN - PURIFICATION OF A DISTINCT CGMP-STIMULATED ISOENZYME

In the absence of detergent, ≈80-85% of the total cGMP-stimulated phosphodiesterase (PDE) activity in bovine brain was associated with washed particulate fractions; ≈85-90% of the calmodulin-sensitive PDE was soluble. Particulate cGMP-stimulated PDE was higher in cerebral cortical gray matter than in other regions. Homogenization of the brain particulate fraction in 1% Lubrol increased cGMP-stimulated activity ≈100% and calmodulin-stimulated ≈400-500%. Although 1% Lubrol readily solubilized these PDE activities, ≈75% of the cAMP PDE activity (0.5 μM [3H]cAMP) that was not affected by cGMP was not solubilized. This cAMP PDE activity was very sensitive to inhibition by Rolipram but not cilostamide. Thus, three different PDE types, i.e., cGMP stimulated, calmodulin sensitive, and Rolipram inhibited, are associated in different ways with crude bovine brain particulate fractions. After solubilization and purification by chromtography on cGMP-agarose, heparin-agarose, and Superose 6, the brain particulate cGMP-stimulated PDE cross-reacted with antibody raised against a cGMP-stimulated PDE purified from calf liver supernatant. The brain enzyme exhibited a slightly greater subunit Mr than did soluble forms from calf liver or bovine brain, as evidenced by protein staining or immunoblotting after polyacrylamide gel electrophoresis under denaturing conditions. Incubation of brain particulate and liver soluble cGMP-stimulated PDEs with V8 protease produced several peptides of similar size, as well as at least two distinct fragments of ≈27 kDa from the brain and ≈23 kDa from the liver enzyme. After photolabeling in the presence of [32P]cGMP and digestion with V8 protease, [32P]cGMP in each PDE was predominantly recovered with a peptide of ≈14 kDa. All of these observations are consistent with the existence of at least two discrete forms (isoenzymes) of cGMP-stimulated PDE. © 1990, American Chemical Society. All rights reserved.