Characterization of Sea Urchin Sperm Adenylate Cyclase1

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Abstract
Sea urchin (Lytechinus pictus) sperm adenylate cyclase was inhibited by the methylxanthines, 1-methyl-3-isobutylxanthine (MIX), caffeine and theophylline. MIX was the most potent inhibitor (I5 0 = 1.4mM) while caffeine and theophylline were about equal in potency (I5 0 = 15 mM). A more detailed kinetic analysis with theophylline indicated that it was a linear competitive inhibitor with respect to MnATP. The sperm adenylate cyclase was inactivated by temperatures as low as 30°, and Mn2+, but not Mg2+ or Ca2+ accelerated this rate of denaturation. Double reciprocal plots were linear as a function of either free Mn2+ or MnATP, and slope and intercept replots of these data also were linear. Thus, the sperm adenylate cyclase conforms to an enzyme model in which both a MnATP substrate site and a Mn2+ regulatory site exist. Furthermore, these data suggest that free ATP is not a potent inhibitor of the enzyme. When the concentration of total Mn2+ was less than the concentration of total ATP, Ca2+ and Mg2+ were capable of activating the enzyme, but when the concentration of total Mn2+ exceeded that of ATP, both Ca2+ and Mg2+ acted as inhibitors. Various phosphorylated nucleosides (GDP, ADP, GTP, dGTP) inhibited the enzyme, but adenosine and guanosine were without effect. A number of carbohydrates (acetate, pyruvate, glucose), fatty acids, and fluoride also had little, if any, effect. Two polyamines, cadaverine (2mM) and putrescine (2 mM) caused a slight stimulation (∼30 percent) of enzyme activity. Under a variety of conditions, solutions containing factors released from sea urchin eggs either had no effect or inhibited the sperm adenylate cyclase.