Partial purification and characterization of a phosphoprotein phosphatase from sperm plasma membrane

A phosphoprotein phosphatase (PPase M-I) that dephosphorylates serine and t hreonine residues of histones was isolated from the goat cauda-epididymal s perm plasma membrane and partially characterized. The PPase was solubilized from the sperm membrane by treating it with 0.1 N NaOH at pH 11.4 and the solubilized enzyme was partially purified by concanavalin A-sepharose affin ity chromatography and high-performance liquid chromatography (HPLC), revea ling it to be a 520-kDa protein. The PPase gave a single protein band in na tive polyacrylamide gel electrophoresis (PAGE), but in the presence of SDS it resolved into multiple proteins (35-170 kDa) showing that the isolated e nzyme contained a few contaminating proteins. The enzyme is a glycoprotein because it binds with high affinity to concanavalin A. It was maximally act ive at pH 8.0 and its activity was not dependent on bivalent metal ions. Th e enzyme is a specific phosphatase as it displayed higher affinity for deph osphorylation of large molecular weight phosphate esters. The PPase showed broad substrate specificity for the dephosphorylation of a variety of prote ins. The membrane-associated PPase was strongly (70-80%) inhibited by deter gents (0.5%) such as Nonidet P-40, Lubrol PX, Triton X-100 and Tween-20. Py rophosphate (5 mM) and orthovanadate (400 mu M) had no significant effect o n the activity of the isolated PPase whereas polyamines such as spermine(10 mM) and spermidine(10 mM) slightly inhibited (20%) the enzymatic activity. Inorganic phosphate (10 mM) and NaF (10 mM), the well-known inhibitors of the cytosolic PPases, had no appreciable effect on the activity of PPase M- I, indicating that the membrane-bound PPase is distinct from the cytosolic PPases. The enzyme was radiolabelled when the intact spermatozoa were subje cted to lactoperoxidase-mediated radioiodination reaction. The results show that the PPase M-I is an ecto-enzyme that may play an important role in sp erm physiology by causing the dephosphorylation of the sperm outer surface phosphoproteins.