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.
Takada
M
M
Department of Chemistry, Faculty of Science, Chiba University, Japan.
Yonezawa
N
N
Yoshizawa
M
M
Noguchi
S
S
Hatanaka
Y
Y
Nagai
T
T
Kikuchi
K
K
Aoki
H
H
Nakano
M
M
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D000177:Acrosome / Q000187:drug effects / Q000201:enzymology*; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D005260:Female; D005307:Fertilization in Vitro; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010091:Oximes / Q000494:pharmacology; D048448:Phenylcarbamates; D013084:Sperm-Ovum Interactions / Q000187:drug effects; D013552:Swine; D001619:beta-N-Acetylhexosaminidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D010091:Oximes; D048448:Phenylcarbamates; C068836:N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)oxime; D000001:Calcimycin; D001619:beta-N-Acetylhexosaminidases; D000117:Acetylglucosamine