The role of β-N-acetylglucosaminidase (β-NAGase) which existed in several species sperm acrosome, was investigated by many years. This enzyme, together with acrosine play an important role in mammals, amphibians and ascidians toward the sperm penetration of oocyte. Teleosts fish possess spermatozoa that lack an acrosome and our earlier experiments revealed that did not contain arylsulfatase, one of acrosomal enzymes. Interestingly we found the β-NAGase activity in Rainbow trout milt plasma and spermatozoa. The characteristics of this enzyme might revealed the role of β-NAGase in not-acrosomal sperm.

Milt obtained from Rainbow trout was centrifuged to obtain milt plasma and spermatozoa, and its was used to isolation β-NAGase from each sources separately. After ion exchange chromatography (Q Sepharose) of milt plasma we obtained 2 protein peaks possessing β-NAGase activity in milt plasma, and one protein peak in sperm extracts. This step of purification enabled us to obtain about 5-7 fold purification. But after gel filtration (Superdex 200) of Rainbow trout milt plasma peaks I and II we obtained 165 and 242 fold purification respectively. The molecular mass established during gel filtration was 127 kDa for Rainbow trout spermatozoal β-NAGase, and 74 kDa for each forms from milt plasma. SDS-PAGE electrophoresis revealed that after Rainbow trout sperm extract chromatography we observed 3 protein bands. The electrophoresis of peaks of Rainbow trout milt plasma revealed 2 and 6 bands in the 1^st and 2^nd peaks respectively.

The kinetic parameters were determined for not-purified enzymes: milt plasma and spermatozoa extracts. The optimum pH for Rainbow trout milt plasma and sperm extract β-NAGase was at the range 4.4 - 4.8. We also calculated the K_m value, it was 7.79x10^-4M for Rainbow trout milt plasma β-NAGase and 5.80x10^-4M for Rainbow trout sperm extract β-NAGase. The incubation at 56^oC by 20 minutes of all sources of enzymes revealed that at this temperature 100% of β-NAGase activity was observed in spermatozoa extracts, but in Rainbow trout milt plasma only 2% of β-NAGase activity was observed. Form of enzyme observed in spermatozoa extracts was thermal stable.

The preliminary results indicated that in Rainbow trout milt might exist two or three forms of β-NAGase, however further study (hydrophobic chromatography and/or Concavalin A Sepharose) are necessarily for obtaining a detailed characteristics of all those enzymes forms.

This study is supported by Ministry of Science and Higher Education Grant No N 308 018 31.

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