Soluble Mn²⁺-dependent Adenylate Cyclase Activity in the Testis of the Blue Fox (Alopex lagopus)

Abstract

Soluble Mn²⁺ -dependent adenylate cyclase (MnAC) activity was found in testicular cytosol from blue foxes castrated during the breeding season. The rate of MnAC activity was approximately constant for 30 min at 35°C and for 2 hr after storage at 25°C. Activity was directly proportional to cytosol protein concentration and was optimal in the physiological pH range. Enzyme activity declined in the presence of an alkylating agent (N-ethyl maleimide, NEM) and was eliminated at a concentration of 1 mM NEM. Low concentrations (0.1—10 mM) of a reducing agent (β-mercapto ethanol, βME) did not increase MnAC activity, whereas a high concentration (100 mM) led to a significant reduction (p< 0.01) in activity. Substitution of Mn^2- in the assay medium with Mg^2- led to a total loss of enzyme activity, which could not be regained by adding hormones or by preincubation of cytosol for 60 min. The K_m for Mn²⁺ was estimated to be 3.5 mM. The affinity of the enzyme for Mn²⁺ was not altered by varying the concentration of ATP. In contrast, increasing concentrations of Mn²⁺ appeared to increase the affinity of the enzyme for MnATP^2-. The K_m for MnATP^2- thus varied from 6 to 18 mM.

Key Words:

• Testis
• Adenylate cyclase
• Germ cells