Abstract:
A phosphoprotein phosphatase (PPase M-I) thatdephosphorylates serine and threonine residues of histones was isolated fromthe goat cauda-epididymal sperm plasma membrane and partially characterized.The PPase was solubilized from the sperm membrane by treating it with 0.1 NNaOH at pH 11.4 and the solubilized enzyme was partially purified byconcanavalin A-sepharose affinity chromatography and high-performance liquidchromatography (HPLC), revealing it to be a 520-kDa protein. The PPase gave asingle protein band in native polyacrylamide gel electrophoresis (PAGE), butin the presence of SDS it resolved into multiple proteins (35-170 kDa)showing that the isolated enzyme contained a few contaminating proteins. Theenzyme is a glycoprotein because it binds with high affinity to concanavalinA. It was maximally active at pH 8.0 and its activity was not dependent onbivalent metal ions. The enzyme is a specific phosphatase as it displayedhigher affinity for dephosphorylation of large molecular weight phosphateesters. The PPase showed broad substrate specificity for the dephosphorylationof a variety of proteins. The membrane-associated PPase was strongly(70-80%) inhibited by detergents (0.5%) such as NonidetP-40, Lubrol PX, Triton X-100 and Tween-20. Pyrophosphate (5 mM) andorthovanadate (400 M) had no significant effect on the activity of theisolated PPase whereas polyamines such as spermine (10 mM) nd spermidine (10mM) slightly inhibited (20%) the enzymatic activity. Inorganicphosphate (10 mM) and NaF (10 mM), the well-known inhibitors of the cytosolicPPases, had no appreciable effect on the activity of PPase M-I, indicatingthat the membrane-bound PPase is distinct from the cytosolic PPases. Theenzyme was radiolabelled when the intact spermatozoa were subjected tolactoperoxidase-mediated radioiodination reaction. The results show that thePPase M-I is an ecto-enzyme that may play an important role in spermphysiology by causing the dephosphorylation of the sperm outer surfacephosphoproteins.