PURIFICATION AND CHARACTERIZATION OF A PROTEIN-KINASE FROM GOAT SPERMPLASMA-MEMBRANE

A protein kinase that causes phosphorylation of serine and threonine r esidues of casein has been partially purified from goat cauda-epididym al sperm plasma membrane and characterized. The kinase, solubilized fr om the membrane with 1.0% Triton X-100, was purified to 480-fold by us ing DEAE-cellulose and casein-Sepharose affinity chromatographic techn iques. The kinase is a strongly basic protein with pI of 9.5. The enzy me has a molecular mass of 310 kilodaltons as estimated by Sephacryl S -300 gel exclusion. The kinase showed affinity for protein substrates in the order membrane proteins > casein > phosvitin > histone > protam ine. The apparent K-m values of the kinase for casein and membrane pro teins were 1 and 0.15 mg/mL, respectively. The synthetic peptides Kemp tide and poly(Glu(80)Tyr(20)) did not serve as substrates of the enzym e. ATP, rather than GTP or PPi, is the donor of phosphate for the phos phorylation reaction. Cyclic AMP and GMP, NaCl (0.25 M), KCl (0.25 M), Ca2+, calmodulin, phosphatidylserine, and muscle protein kinase inhib itor had no appreciable effect on the kinase activity. Heparin (0.5 mu g/mL) showed high affinity for inhibiting only 40% of the kinase acti vity, whereas polyamines at a relatively high concentration (5 mM) inh ibited 40-50% of the enzymic activity. The kinase appears to be distin ct from other protein kinases including casein kinases. The activity o f the kinase derived from the purified sperm plasma membrane was marke dly (similar to 90%) lost when the intact spermatozoa were pretreated with diazonium salt of sulfanilic acid, a membrane nonpenetrating surf ace probe. The data are consistent with the view that the isolated enz yme is an ecto-protein kinase whose catalytic site is oriented primari ly to the surface of viable sperm cell to cause phosphorylation of the endogenous outer cell-surface phosphoproteins.