Association of cyclic nucleotide phosphodiesterase of buffalo spermatozoa with sperm chromatin.

Buffalo sperm heads contain more than 50% of the total cyclic AMP-phosphodiesterase activity (EC 3.1.4.17) present in spermatozoa. Its distribution in sperm heads revealed no activity in acrosome and other membrane structures present in the head. All the cyclic AMP-phosphodiesterase activity was found firmly bound to sperm chromatin which could not be solubilized. In addition to cyclic AMP, cyclic GMP was also hydrolysed by chromatin preparation. The rate of hydrolysis was 2.5-times more rapid with cyclic AMP than with cyclic GMP at their optimum pH of 7.5 and 8.0, respectively. The pH and heat stability profiles, inhibition studies and the effect of divalent metal ions indicated that the two activities are not associated with the same protein. Mixed substrate analysis showed two sites at which the hydrolysis of cyclic AMP and cyclic GMP is catalysed. Chromatin cyclic nucleotide phosphodiesterases exhibited kinetics typical of one enzyme species both for cyclic AMP (K m = 100 microM; V = 1.0 nmol/min per mg protein) and cyclic GMP (Km = 23 microM; V = 0.4 nmol/min per mg protein). Each cyclic nucleotide was found to be a competitive inhibitor of the hydrolysis of the other with a Ki value of 30.18 microM for cyclic AMP hydrolysis and 256 microM for cyclic GMP hydrolysis. Hill coefficients of 1.0 obtained in the presence of cyclic AMP for cyclic GMP hydrolysis and vice-versa indicated no allosteric interactions. It is suggested that chromatin cyclic nucleotide phosphodiesterase may have a role post fertilization in cell growth and differentiation with no role in sperm motility which is regulated by similar enzymes present in sperm flagella.