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 × g ) migrates as a single peak in a linear sucrose gradient (5–20%) with a sedimentation coefficient (S_{20, w}) of 4,1. The enzyme exhibits an Einstein-Stokes radius (r_s) of 30.6 Å as assessed by gel filtration (K_av= 0.50) on a calibrated Sephadex G-200 column. The molecular shape is close to spherical (f/f_o= 1.25). Isoelectric focussing of the AC peak (K_av= 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 α-D-manno- or α-D-glucopyranosyl terminal groups. (NH₄)₂SO₄ precipitates the AC activity (4°C) between 30–60% saturation. The yield approximates 85% of total AC activity with the specific activity reaching a plateau (110 pinoles 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.