Phosphoprotein Phosphatase Activity of Sea Urchin Spermatozoa

Abstract

Spermatozoa of the sea urchin S. purpuratus contain phosphoprotein phosphatases capable of dephosphorylating phosphohistones and phosphorylase .alpha.. The phosphohistone phosphatase was purified .apprx. 400-fold by DEAE Sephacel, histone-Sepharose and high-pressure liquid-gel permeation chromatography to a specific activity of 0.3 .mu.mol phosphate formed per min per mg of protein. The apparent MW of this form was in excess of 300,000, but it could be converted to a 35,000 MW form by treatment with organic solvents. The enzyme did not resemble alkaline phosphatases from liver or intestine since phospho-Ser-histones were more effective substrates than phospho-Tyr-histones. When the supernatant fluid from the sperm homogenate was first treated with acetone to extract phosphatase actvity, subsequent chromatography resulted in a nearly homogeneous, 35,000 MW protein phosphatase and none of the larger MW form. The specific activity of the highly purified, low MW phosphate was 4.0 .mu.mol phosphate formed per min per mg of protein. It was strongly inhibited (> 85%) by fluoride (50 mM), Zn (0.2 mM) and pyrophosphate (2 mM). A fucose sulfate rich factor obtained from the jelly coat of S. purpuratus eggs (F-SP) caused .apprx. 3-fold increases in protein phosphatase activity in intact spermatozoa; the amount of released activity represented .apprx. 4% of the total cell phosphatase activity. Extracellular Ca2+ was required for the F-SP effect with half maximal responses at .apprx. 4 mM. Nigericin, an ionophore known to induce the sperm acrosome reaction, also caused apparent release of, or exposure of, phosphoprotein phosphatase activity in intact spermatozoa; speract, a peptide known to stimulate the respiration of spermatozoa, failed to increase protein phosphatase activity. Sea urchin spermatozoa probably contain phosphoprotein phosphatase activity similar to that found in many vertebrate cells and at least a part of this activity may reside in the sperm acrosomal region.