Purification of the proteolytically solubilized, active catalytic subunit of adenylate cyclase from ram sperm

Abstract

Ram spermatozoa adenylate cyclase is insensitive to all usual regulatory processes. The purification of its active catalytic subunit was accomplished after proteolytic solubilization of a particulate fraction by .alpha.-chymotrypsin. The purification (26000-fold from the particulate fraction or 125000-fold from the whole-sperm proteins) was achieved by conventional procedures (DEAE-Trisacryl, Ultrogel AcA 34, DEAE-Sephacel hydroxyapatite), in the absence of detergent, and with a yield of 5-10% and a final specific activity of 19 .mu.mol cyclic AMP formed mg protein-1 min-1 at 30.degree. C in the presence of manganase as cosubstrate. The solubilized enzyme, stable at the beginning of the purification procedure, beme unstable at the later stages. After the last step (chromatography on hydroxyapaptite) half-lives of 27 min, 50 min and 160 min were obtained at 30.degree. C, 20.degree. C and 4.degree. C respectively. The enzyme was stabilized by addition of bovine serum albumin and Lubrol PX, 80% of the activity remaining after 24 h at 4.degree. C. The purified enzyme exhibited a Km value similar to that of the native enzyme (Km = 1.4 mM). Unlike the native enzyme, the purified enzyme has an absolute requirement for MnATP; no significant activity was recovered in the presence of MgATP. Adenosine inhibited the activity of both the native and purified forms of the enyzme to the same extent and in a non-competitive manner. This indicates that (a) adenosine acts on the catalytic components itself and (b) the inhibition site and the catalytic site are different. Data obtained with adenosine analogs indicate that adenosine interacts with the cyclase catalytic subunit with a "P-sit" specificity. The purified adenylate cyclase, which had an apparent molecular mass of 38 kDa on a high-performance liquid chromatography column [Stengel, D., Guenet, L. and Hanoune, J. (1982) J. Biol. Chem. 257, 10818-10826], gave a doublet of 36 kDa and 34 kDa on sodium dodecyl sulfate gel electrophoresis. This represents the smallest protein entity associated with adenylate cyclase activity so far reported.