High-affinity sites for beta-D-galactosidase on membrane-bound vesicles isolated from rat epididymal fluid

Glycosidases in rat epididymal fluid are secreted under androgen stimulatio n and possess receptors on the sperm surface. One of these enzymes, beta-D- galactosidase (gal), was found in the epididymal fluid as a soluble enzyme and also in a heterogeneous population of membrane bound vesicles (mbv). be ta-D-Galactosidase was specifically localized to a subpopulation of larger, electron-dense mbv. The aim of this study was to analyze the high-affinity sites for gal on the membrane of mbv using two different methods: classica l fluorometric assay (used in previous papers) and colloidal gold (20 nm) c onjugated to gal as a marker in ultrastructural studies. beta-D-Galactosida se bound to mbv with high-affinity (K-d in a nanomolar range) are in a satu rable form. Furthermore, 25 mM fructose-1,6-diphosphate (f-1,6-dip), a suga r that competes for the binding site, showed 50% inhibition of the binding. The gold conjugates were mostly observed on the surface of the large, elec tron-dense mbv but not on the small, electron-lucent mbv. Gold particles we re also observed on the larger vesicles, but less frequently in the presenc e of f-1,6-dip. Larger mbv possesses high-affinity sites for gal on their m embrane.