A cAMP-dependent phosphorylated motility protein in bovine epididymal sperm.

A putative cAMP-dependent protein kinase substrate associated with the cAMP stimulation of bovine sperm motility has been identified. Optimum conditions for a linear, concentration-dependent incorporation of [32P]ATP into phosphoproteins of an epididymal sperm sonicate by cAMP-dependent protein kinase are described. The motility state of the sperm was reduced by incubation at 37 degrees C and reactivated with theophylline. Endogenous levels of cAMP correlated with the motility state of the sperm. The phosphorylation state of phosphoproteins was frozen by addition of NaF (100 mM final concentration). After sonication and removal of endogenous nucleotides, 32P incorporation into phosphoprotein varied inversely with motility. The inverse relationship results from the procedure monitoring the capacity for incorporation of 32P into dephosphorylated cAMP-dependent protein kinase substrates. Over 70% of the cAMP-dependent label was in the soluble fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one cAMP-dependent phosphoprotein from the soluble fraction which had an inverse correlation with motility. This 55,000-dalton protein did not bind [3H]cholchicine (tubulin) or [3H]cAMP (REGULATORY SUBUNIT OF PROTEIN KINASE). These data indicate the existence of a cytosolic phosphorylated motility protein.