Purification and kinetic properties of the soluble Mn2+-dependent adenylyl cyclase of the rat testis.
The soluble Mn2+-dependent adenylyl cyclase (AC) of the rat testis was purified 1500-fold with some 19% yield of the initial activity. These results were accomplished by conventional separation techniques including (NH4)2SO4 precipitation of testis cytosol (106 000 x g), gel filtration (Sephadex G-200) and ion-exchange chromatography (Sephadex DEAE-A50) followed by Sephadex G-100 gel filtration and isoelectric focusing. Analysis by polyacrylamide-gel electrophoresis (PAGE) of aliquots from each purification step revealed the following. (a) The Mn2+-dependent AC migrated with a Rf value of 0.40 irrespective of the degree of purification. (2) The AC peak from the isoelectric focusing column separated into 2 major protein bands; however, only one band (Rf 0.40) had AC activity. The molecular weight emerging from the position of migration on the Sephadex G-200 and G-100 columns appeared to be consistent at 47 000-48 000 D, as estimated from the relationship log MW versus elution volume. The purified enzyme fulfilled the requirements for a simple Michaelis-Menten kinetics with an apparent Km for Mn2+ and MnATP2- of 6.7 and 2.5 mM, respectively. Varying the concentrations of ATP or Mn2+ separately did not alter the apparent affinity (Kmapp) for the other parameter. These and previous data from our laboratory show that the physico-chemical and kinetic properties (molecular weight and Kmapp for Mn2+ and MnATP2-) do not alter during purification. Furthermore, the additional step of affinity chromatography seems obligatory if a homogeneous AC preparation is to be obtained.
D000262:Adenylyl Cyclases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008345:Manganese / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D013737:Testis / Q000201:enzymology*
D008345:Manganese; D000262:Adenylyl Cyclases