Calmodulin sensitive phosphodiesterase of porcine cerebral cortex: kinetic behavior, calmodulin activation, and stability.

The calmodulin sensitive phosphodiesterase of porcine cerebral cortex was characterized in terms of kinetic behavior, calmodulin activation, and stability. This enzyme displayed non-Michaelis-Menten kinetics in the presence or absence of calmodulin. The apparent affinity for cyclic GMP was higher than that for cyclic AMP but at saturating levels of substrate, this enzyme catalyzed the hydrolysis of cyclic AMP at a greater rate than it did cyclic GMP. The affinity of this enzyme for calmodulin was about 20-fold lower than usually reported. The apparent loss of phosphodiesterase activity after storage was found to be due to a strong association with container surfaces and could be prevented or reversed by the presence of 0.1% Triton X-100.

Irreversible inhibition of calmodulin-sensitive cyclic nucleotide phosphodiesterase.

Photolysis of solutions containing 4-azido-7-phenylpyrazolo-[1,5a]-1,3,5-triazine (APPT) and calmodulin-sensitive cyclic nucleotide phosphodiesterase resulted in reduction of both cyclic GMP and cyclic AMP hydrolytic activity. The inactivation was dependent upon both time of exposure to ultraviolet irradiation and the initial concentration of APPT. The photo-induced inactivation could be attenuated by the presence of cyclic GMP, 1-methyl-3-isobutylxanthine, and papaverine. alpha-Chymotrypsin treatment caused the enzyme to be fully active in the absence of calmodulin but this treatment did not alter the ability of APPT to inactivate the enzyme. Thus, inhibition of calmodulin-binding did not contribute to the photo-induced inactivation. These data indicate that APPT acts as a photoaffinity agent to covalently modify the APPT-binding site of calmodulin-sensitive phosphodiesterase.