HEXOKINASE PRESENT IN HUMAN SPERM IS NOT TYROSINE-PHOSPHORYLATED BUT ITS ANTIBODIES AFFECT FERTILIZING-CAPACITY

The present study was conducted to investigate the presence of hexokin ase in human sperm cell, its subcellular localization, its modulation and role in capacitation/acrosome reaction, and tyrosine kinase activi ty, if any. These studies were conducted using antibodies (Ab) raised against rat brain hexokinase (type I isozyme) that have significant cr oss-reaction with various human tissue hexokinases and neutralize the catalytic activity of the enzyme. Hexokinase Ab reacted with acrosomal , mid-piece and tail regions of methanol-fixed (similar to 70-80%) and live (similar to 35-52%) sperm in the indirect immunofluorescence tec hnique (IFT) and the immunobead binding technique (IBT), respectively. Hexokinase Ab specifically recognized a band of 116 kDa on the Wester n blot of detergent-solubilized human sperm preparation that was diffe rent from the 95-kDa phosphotyrosine protein. Hexokinase Ab caused a s ignificant (P < 0.01 to < 0.001) and concentration-dependent inhibitio n of human sperm penetration of zona-free hamster oocytes in the sperm penetration assay (SPA). These data indicate that the hexokinase of 1 16-kDa molecular weight is present in acrosomal, mid-piece, and tail r egions of human sperm. The sperm hexokinase is a glycoprotein that is different from the 95-kDa phosphotyrosine protein and is not phosphory lated at tyrosine residues; however, its antibodies cause agglutinatio n and a concentration-dependent inhibition of fertilizing capacity of human sperm.