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

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 αchymotrypsin. The purification (2600-fold from the particulate fraction or 125000-fold from the whole-sperm proteins) was achieved by conventional procedures (DEAE-Trisacryl, Ultogel AcA 34, DEAE-Sephacel, hydroxyapatite), in the absence of detergent, and with a yield of 5-10% and a final specific activity of 19 µmol cyclic AMP formed mg protein-1 min -1 at 30°C in the presence of manganese as cosubstrate. The colubilized enzyme, stable at the beginning of the purification procedure, became unstable at the later stages. After the last step (chromatography on hydroxyapatite_ half-lives of 27 min min, 50 min, and 160 min were obtained at 30°C and 4°C respectively. The enzyme was stabilized by addition of bovine serum albumin and Lubrol PX, 80% of the activity remaining after 24 at 4°C. The purified enzyme 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 enzyme to the same extent and in a non-competitive manner. This indicated that (a) adenosine acts on the catalytic component itself and (b) the inhibition site and the catalytic site are different. Data obtained with adenosine analogs 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-site' specificity. The purified adenylate cyclase, which had an apparent molecular mass of 35 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. [ABSTRACT FROM AUTHOR]