Partial purification and characterization of a trypsin-like serine protease from bovine sperm.
Sperm proteolytic activities are relevant in the enzymatic mechanism of fertilization. Several authors have suggested the presence of serine proteases other than acrosin in mice and human spermatozoa. In this work we describe the characterization of a partially purified bovine sperm serine protease BSp66 and its dimmer, BSp120. Partial purification of the monomer was performed from fresh spermatozoa, while the dimer form of the protease was obtained from cryopreserved spermatozoa. The Mr of BSp120 and BSp66 estimated by zymography and gel filtration chromatography were 120 and 66 kDa, respectively. They were positively stained by Schiff-PAS reagent for glycoproteins and they both digested synthetic peptides with basic amino acids in the P1 site. Polyclonal antibodies against acrosin or proacrosin did not cross-react neither with BSp120, nor BSp66. In addition, antibodies raised in our laboratory against BSp120 and BSp66 did not recognize acrosin or proacrosin suggesting that they are not antigenically related proteins. Also, no cross- reactivity was detected with proteins in the range of 120-66 kDa when antibodies against the proteasome were used. The cellular localization of this protease by optical immunocytochemistry using specific antibodies revealed a positive signal in the apical portion of the sperm head suggesting acrosomal or membrane localization. The evidences presented here characterize BSp66 as a trypsin-like serine protease, a putative new member of this highly redundant proteolytic system of the sperm acrosome.
Cesari
Andreina
A
Instituto de Investigaciones Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, CC:1245 (7600) Mar del Plata, Argentina. acesari@mdp.edu.ar
Cacciato
Claudio Santiago
CS
De Castro
Rosana Esther
RE
Sánchez
Jorge Julián
JJ
D000818:Animals; D002417:Cattle; D015925:Cryopreservation; D019281:Dimerization; D008297:Male; D012697:Serine Endopeptidases / Q000378:metabolism; D013094:Spermatozoa / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism*
D012697:Serine Endopeptidases; D014357:Trypsin