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 chromatog raphy. The final preparation exhibited a single protein band on SDS-PA GE. 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 p lacing 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 t he acrosome reaction. The addition of these antibodies led to an incre ase in the acrosome reaction, thus suggesting that inhibition of lysop hospholipase produces a higher lysophosphatidylcholine concentration a nd results in an acrosome reaction level similar to that obtained by t he calcium ionophore A23187. Immunofluorescence localization of the en zyme indicated that the enzyme is located on the head of spermatozoa. The purified sperm lysophospholipase and its specific antibodies repre sent important tools for the study of the regulation of this enzyme in reproductive professes. Furthermore, the study of this enzyme will al low evaluation of the mechanisms underlying the acrosome reaction.