VESICLES ISOLATED FROM EPIDIDYMAL FLUID: AFFINITY SITES FOR N-ACETYL GLICOSAMINIDASE AND 6-GALACTOSIDASE

FORNES MW.; GRIMALT P.

San Francisco, California

Congreso; 6th International Congress on Cell Biology. 36th American Society for Cell Biology Annual Meeting; 1996

American Society for Cell Biology

The sperm maturation during the epididymal transit depends on the epididymal environment. This environment is maintained by the secretion of epididymal epithelium. Some components of the secretion is constituted by hydrolases. It was described that membrane bound vesicles that arise from principal cells contained some of these enzymes (Fornes et al., 1994). In this work, we tested if two of these hydrolases, N acetyl glicosaminidase (Nag) and 6 galactosidase (6Gal), posses high affinity sites in the vesicles isolated from rat epididymal fluid. Vesicles were isolated by differential centrifugation and washed with PBS containing 0.6M KCl to release endogenous enzymes. Then, vesicles were incubated with increasing concentration of NAG and 6Gal and the free and bound enzyme were assayed by a fluorometric methodology. In others experiments, the binding assay were performed with colloidal gold particles (20 nm) coated with 6Gal and observed by EM. The binding assay shows a saturation pattern and the electron micrograph of gold-6Gal shows the dark particles located in patches on the membrane. The results indicate that NAG and 6Gal have high affinity sites on the membrane of the vesicles.