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 envelopes. The incubation of isolated vitelline envelopes with sperm induces the acrosome reaction, releasing proteases and glycosidases to the media. In the present work N-acetyl-beta-D-glucosaminidase, beta-D-galactosidase, beta-D-glucosidase, alpha-D-mannosidase, alpha-L-fucosidase, and alpha-D-glucosidase activities are measured in spermatozoa. N-acetyl-beta-D-glucosaminidase is the major sperm glycosidase activity assayed. However, N-acetyl-beta-D-galactosamine show competitive inhibitory effect. The glycosidase pH optimum 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 vitelline envelopes in conditions that resemble natural fertilization media. The isolation of the active enzyme will allow studies of its role in fertilization. The enzyme has been purified in a two-step procedure. After native gel electrophoresis, the activity-stained band was cut out and the eluted enzyme was finally subjected to ConA-sepharose chromatography. In SDS-PAGE, the denatured enzyme migrates as a single band with a molecular mass of 45 kDa. Furthermore, analysis by size-exclusion on HPLC showed a peak of activity 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 vitro fertilization assay, it was observed that the inhibition of the enzyme results in the inhibition of fertilization. This last study shows that N-acetyl-beta-D-glucosaminidase plays an important role in toad fertilization.