Physicochemical and Kinetic Properties of the Mn2†-Dependent Adenylyl Cyclase of the Human Testis*

The present study shows the presence of a soluble Mn2+-dependent adenylyl cyclase (AC) in the human testis and some physicochemical and kinetic properties have been examined. The results can be summarized as follows: 1) At 35 C the AC activity was constant as a function of incubation time for at least 60 min and proportional to protein concentration ranging from 10–150 jug protein per assay tube. 2) Enzyme activity was maintained for at least 24 h at 4 C; at 35 C, however, a marked loss of activity (approximately 40%) was observed after 2 h. At higher temperatures (above 45 C) the activity was destroyed completely after a few minutes. 3) The AC exhibited a pHoptimum around pH 7.5 and increasing ionic strength (0–0.4 M NaCl/KCl) apparently had no significant effect on enzyme activity. 4) Free cysteinyl thiol groups are required for enzyme activity: alkylating agents (N-ethylmaleimide, 0.8–6.0 mM) and reducing agents (β-mercaptoethanol, 1–5 mM) enhanced AC activity. 5) The Mn2+-dependent AC activity was insensitive to hormones (gonadotropins and prostaglandins), guanosine mono phosphate- P(NH)P, and fluoride. Ca2† (0.1–10 mM) enhanced the enzyme activity in a dose-dependent fashion. The stimulatory effect was most pronounced in the presence of subsaturating concentrations of Mn2+. 6) The Km for MnATP2− and Mn2+ were estimated to be 1.8 and 3.1 mM, respectively. The Km for MnATP2− was constant irrespective of the amount of free Mn2+ present, and the affinity for Mn2+ was unaffected by alterations in ATP levels. 7) The Mn2+-dependent AC activity was to a great extent (approximately 80%) present as high molecular weight aggregates when analyzed by both sucrose gradient centrifugation and gel filtration. A small molecular weight component, probably representing the “monomeric” form, displayed the following properties: Stoke's radius (estimated by Sephadex G-200 chromatography) 28.6 A, sedimentation coefficient (measured by sucrose gradient centrifugation) 4.1 S2o,w, frictional ratio 1.20, and isoelectric point (estimated from an LKB isoelectric focusing column) 5.6. (J. Clin Endocrinol Metab53: 465, 1981)