Purification of lysophospholipase of human spermatozoa and its implication in the acrosome reaction.
The lysophospholipase of human spermatozoa was purified to homogeneity by sequential ion-exchange, gel filtration, and hydrophobic chromatography. The final preparation exhibited a single protein band on SDS-PAGE. The molecular mass of the enzyme was estimated to be 51 kDa by SDS-PAGE and 52 kDa by gel filtration. The optimal pH of this enzyme is 8.0. Polyclonal antibodies against lysophospholipase were prepared by placing the enzyme adsorbed on nitrocellulose directly into the spleen of rabbits. These antibodies were purified by protein A-agarose and by affigel-lysophospholipase chromatography. The purified antibodies and enzyme were used to study the possible role of lysophospholipase in the acrosome reaction. The addition of these antibodies led to an increase in the acrosome reaction, thus suggesting that inhibition of lysophospholipase produces a higher lysophosphatidylcholine concentration and results in an acrosome reaction level similar to that obtained by the calcium ionophore A23187. Immunofluorescence localization of the enzyme indicated that the enzyme is located on the head of spermatozoa. The purified sperm lysophospholipase and its specific antibodies represent important tools for the study of the regulation of this enzyme in reproductive processes. Furthermore, the study of this enzyme will allow evaluation of the mechanisms underlying the acrosome reaction.
Lepage
N
N
Department of Biochemistry, University of Montreal, Québec, Canada.
Roberts
K D
KD
D000177:Acrosome / Q000737:chemistry*; D015153:Blotting, Western; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008244:Lysophosphatidylcholines / Q000378:metabolism; D008245:Lysophospholipase / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D013094:Spermatozoa / Q000201:enzymology*
D008244:Lysophosphatidylcholines; D008245:Lysophospholipase