P95, THE MAJOR PHOSPHOTYROSINE-CONTAINING PROTEIN IN MOUSE SPERMATOZOA, IS A HEXOKINASE WITH UNIQUE PROPERTIES

Mouse sperm contain a major phosphotyrosine-containing protein of M(r) 95,000 (nonreducing conditions) which has been implicated as a sperm membrane receptor for the egg zona pellucida glycoprotein, ZP3 (Leyton , L., and Saling, P. (1989) Cell 57, 1123-1130; Leyton, L., LeGuen, P. , Bunch, D., and Saling, P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11692-11695). This protein was purified and subjected to limited trypt ic digestion and subsequent amino acid analysis. Three sequenced pepti des revealed 100% amino acid identity to a mouse hepatoma hexokinase ( Arora, K. K., Fanciulli, M., and Pederson, P. L. (1990) J. Biol. Chem. 265, 6481-6488). The purified protein, which migrated at M(r) 116,000 under reducing conditions (p95/116), reacted with an antiserum to the purified rat brain hexokinase, type 1, and comigrated on sodium dodec yl sulfate-polyacrylamide gel electrophoresis with the purified rat br ain enzyme under both nonreducing and reducing conditions. Unlike p95/ 116, the rat brain enzyme was not a phosphotyrosine-containing protein . The p95/116 protein could be immunoprecipitated with the hexokinase antiserum or an O-phosphotyrosine antibody. Limited tryptic digestion of the purified p95/116 and the rat brain enzyme generated subsets of identical peptides which reacted with the hexokinase antiserum. Howeve r, p95/116 also contained phosphotyrosine-containing peptides that wer e not present in the rat brain hexokinase. When different mouse tissue s were probed with the hexokinase antiserum all tissues, with the exce ption of liver, contained immunoreactive protein. In contrast, only sp erm and testis possessed a phosphotyrosine-containing form of hexokina se. These data suggest that the germ cell component of the testis poss esses a unique tyrosine-phosphorylated form of hexokinase.