Preliminary physico‐chemical characterization of the soluble Mn²⁺‐dependent adenylate cyclase in the rat testis

This paper reports some physico-chemical properties of the Mn+-dependent adenylate cyclase (AC) of the rat testis. The AC activity in the crude cytosol (106 000 x g) migrates as a single peak in a linear sucrose gradient (5--20%) with a sedimentation coefficient (S20,w) of 4,1. The enzyme exhibits an Einstein-Stokes radius (rs) of 30.6 A as assessed by gel filtration (Kav = 0.50) on a calibrated Sephadex G-200 column. The molecular shape is close to spherical (f/f0 = 1.25). Isoelectric focussing of the AC peak (Kav = 0.50) from the Sephadex G-200 column revealed a pI of 5.7. Ion exchange chromatography of the crude cytosol on a Whatman DE-52 column gave a single peak eluting between 0.13--0.17 M NaCl. Electrophoresis on polyacrylamide gels resulted in a migrating AC peak with an apparent Rf of 0.40 relative to bromophenol blue. The Mn2+-dependent AC is not retained on a Con-A Sepharose 4B column indicating that the enzyme molecule does not contain sugars possessing alpha-D-manno- or alpha-D-glucopyranosyl terminal groups. (NH4)2SO4 precipitates the AC activity (4 degrees C) between 30--60% saturation. The yield approximates 85% of total AC activity with the specific activity reaching a plateau (110 pmoles cAMP/mg protein/min.) at 50% saturated (NH4)2SO4. The soluble Mn+-dependent AC of the rat testis thus appears to be relatively symmetrical with a size of approximately 52 000 D and a negative charge at physiological pH. Apparently it does not contain sugar moieties and it exhibits little molecular heterogeneity.