Purification and biological characterization of N-acetyl beta-D glucosaminidase from Bufo arenarum Spermatozoa

Fertilization in Bufo arenarum requires the sperm to penetrate the egg enve lopes. The incubation of isolated vitelline envelopes with sperm induces th e acrosome reaction, releasing proteases and glycosidases to the media. In the present work N-acetyl-beta-D-glucosaminidase, beta-D-galactosidase, bet a-D-glucosidase, alpha-D-mannosidase, alpha-L-fucosidase, and alpha-D-gluco sidase activities are measured in spermatozoa. N-acetyl-beta-D-glucosaminid ase is the major sperm glycosidase activity assayed. However, N-acetyl-beta -D-galactosamine show competitive inhibitory effect. The glycosidase pH opt imum is 3.5 being inhibited at pHs higher than 7.5. in our study, N-acetyl- beta-D-glucosaminidase is the only glycosidase that in vitro binds to vitel line envelopes in conditions that resemble natural fertilization media. The isolation of the active enzyme will allow studies of its role in fertiliza tion. The enzyme has been purified in a two-step procedure. After native ge l electrophoresis, the activity-stained band was cut out and the eluted enz yme was finally subjected to ConA-sepharose chromatography. In SDS-PAGE, th e denatured enzyme migrates as a single band with a molecular mass of 45 kD a. Furthermore, analysis by size-exclusion on HPLC showed a peak of activit y at around 45 kDa. Preliminary localization studies showed higher relative activity in the acrosomal content. In addition, 10% of the N-acetyl-beta-D -glucosaminidase activity was associated with the reacted sperm. By in vitr o fertilization assay, it was observed that the inhibition of the enzyme re sults in the inhibition of fertilization. This last study shows that N-acet yl-beta-D-glucosaminidase plays an important role in toad fertilization. Mo l. Reprod. Dev. 57:194-203, 2000. (C) 2000 Wiley-Liss, inc.