Establishment of an efficient purification method and further characterization of 32K calmodulin-binding protein in testis.
A heat-stable 32K calmodulin-binding protein has been purified approximately 3,670-fold from porcine testis to apparent homogeneity as judged by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and polyacrylamide gel electrophoresis under native conditions. The purification employed calmodulin-Sepharose 4B affinity chromatography; elution was performed with a free Ca2+ gradient. This provided a simple and efficient procedure, and approximately 1.62 mg of pure heat-stable calmodulin-binding protein was obtained from 390 g of porcine testis with a yield of 47% in activity. The purified protein was asymmetric (f/fo = 1.89) and consisted of a single polypeptide of Mr = 32,000. It is a highly acidic protein (pI = 3.9) with a diffusion coefficient of 5.4 X 10(-7) cm2/s, a sedimentation coefficient of 1.43 S, and a Stokes radius of 39.5 A in its free form and 41.3 A in its complex form with calmodulin. The extent of inhibition of phosphodiesterase by the calmodulin-binding protein was affected by the order of addition of the agents to the reaction mixture. The extent of inhibition was maximal when phosphodiesterase was added last, while it was minimal when the calmodulin-binding protein was added last. This protein was indistinguishable from a heat-stable calmodulin-binding protein in rat testis (Ono, T., Koide, Y., Arai, Y., & Yamashita, K. (1984) J. Biol. Chem. 259, 9011-9016).
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D008297:Male; D011487:Protein Conformation; D051381:Rats; D013552:Swine; D013737:Testis / Q000032:analysis*
D002148:Calmodulin-Binding Proteins; C051666:calspermin; D015105:3',5'-Cyclic-AMP Phosphodiesterases