Mn2+-dependent adenylyl cyclase (AC) in rat testis: kinetic properties and optimalization of assay conditions.
The aim of the present study was to investigate some kinetic properties of the soluble Mn2+-dependent adenylyl cyclase (AC) in rat testis and to examine the viability of this enzyme under various assay conditions. 1. Constant AC activity was found during the first 35 min of incubation, and AC activity was proportional to protein concentrations in the range tested (10-45 micrograms/tube). 2. The enzyme activity was constant for at least 24 h at 4 degrees C and for at least 2 h at 35 degrees C. At higher temperatures (above 45 degrees C) the AC activity rapidly declines. 3. The enzyme displays a pH optimum in the physiological range (7.0-7.4) and increasing ionic strength (0-0.4 m NaCl/KCl) caused only a marginal decrease in enzyme activity. 5. The soluble Mn2+-dependent AC in rat testis could not be stimulated by hormones (hCG, FSH, prostaglandins), guanyl-nucleotides (GMP-P(NH)P, 2-4 x 10(-5) M) or F-. Ca2+ (0.1-5 mM) caused a dose-dependent stimulation of AC activity; the most pronounced effect was seen at low Mn2+ concentrations. 6. The Km for MnATP2- was estimated to 3.0 mM and the Km for Mn2+ to 7.2 mM. Increasing concentrations of Mn2+ did not alter the affinity for MnATP2- and neither did varying levels of ATP affect the affinity for Mn2+.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008345:Manganese / Q000494:pharmacology*; D051381:Rats; D013696:Temperature; D013737:Testis / Q000187:drug effects / Q000201:enzymology*
D008345:Manganese; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases; D002118:Calcium