Phosphoprotein phosphatase activity of sea urchin spermatozoa

Abstract

Spermatozoa of the sea urchin Strongylocentrotus purpuratus were shown to contain phosphoprotein phosphatases capable of dephosphorylating phosphohistones and phosphorylase a. The phosphohistone phosphatase was purified approximately 400-fold by DEAE Sephacel, histone-Sepharose and high-pressure liquid-gel permeation chromatography to a specific activity of 0.3 μ moI phosphate formed per mm per mg of protein. The apparent molecular weight of this form was in excess of 300,000, but it could be converted to a 35,000 M_r 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 activity, subsequent chromatography resulted in a nearly homogeneous, 35,000 M_r protein phosphatase and none of the larger molecular weight form. The specific activity of the highly purified, low molecular weight phosphatase was 4.0 μ mol phosphate formed per mm per mg of protein. It was strongly inhibited (>85%) by fluoride (50 mM), zinc (0.2 mM) and pyrophosphate (2 mM). A fucose sulfate rich factor obtained from the jelly coat of S. purpuratus eggs (F-SP) caused approximately 3-fold increases in protein phosphatase activity in intact spermatozoa; the amount of released activity represented about 4% of the total cell phosphatase activity. Extracellular Ca²⁺ was required for the F-SP effect with half maximal responses at approximately 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, while speract, a peptide known to stimulate the respiration of spermatozoa, failed to increase protein phosphatase activity. These results suggest that sea urchin spermatozoa 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.