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, indicating that this enzyme is localized in the acrosome. beta-Hex was extracted on a large scale, with 2% acetic acid containing 0.2% Brij 35, from washed ejaculated sperm. By gel filtration chromatography, beta-Hex was separated into a high-molecular-weight fraction (beta-Hex I) and a low-molecular-weight fraction (beta-Hex I). beta-Hex I, which is predominant under acidic conditions (pH 6.5), dissociated into beta-Hex II under alkaline conditions (pH 7.4). beta-Hex II, converted from beta-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-fold and 4000-fold, respectively, with a combined recovery of 23% as measured with synthetic substrate. An inhibitor of beta-Hex, O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl-carbamate (PUGNAC), reduced the in vitro fertilization rate in porcine cumulus-enclosed eggs, but barely changed the rate when cumulus-free eggs were used. beta-Hex I was shown to possess cumulus dispersion activity, suggesting that beta-Hex plays a role in the passing by sperm through cumulus cells before they bind to the zona pellucida.