PH-SENSITIVE DISSOCIATION AND ASSOCIATION OF BETA-N-ACETYLHEXOSAMINIDASE FROM BOAR SPERM ACROSOME

Beta-N-acetylhexosaminidase (beta-Hex, EC. 3.2.1.52) was released from cauda epididymal boar sperm by treatment with ionophore A23187, indic ating that this enzyme is localized in the acrosome. Beta-hex was extr acted on a large scale, with 2% acetic acid containing 0.2% Brij 35, f rom washed ejaculated sperm. By gel filtration chromatography, beta-He x was separated into a high-molecular-weight fraction (beta-Hex I) and a low-molecular-weight fraction (beta-Hex II). Beta-hex I, which is p redominant under acidic conditions (pH 6.5), dissociated into beta-Hex II under alkaline conditions (pH 7.4). Beta-hex 11, converted from be ta-Hex I, associated again to form beta-Hex I under acidic conditions. By sequential chromatography on ion-exchange, lectin, gel filtration, and ion-exchange HPLC columns, beta-Hex I and II were purified 1200-f old and 4000-fold, respectively, with a combined recovery of 23% as me asured with synthetic substrate. An inhibitor of beta-Hex, O-(2-acetam ido-2-deoxy-D-glucopyranosylidene) amino N-phenyl-carbamate (PUGNAC), reduced the in vitro fertilization rate in porcine cumulus-enclosed eg gs, but barely changed the rate when cumulus-free eggs were used. Beta -hex I was shown to possess cumulus dispersion activity, suggesting th at beta-Hex plays a role in the passing by sperm through cumulus cells before they bind to the zona pellucida.