Association between the bovine sperm protease BSp120 and the sperm membranes“.

KATUNAR MR AND CESARI A,

Bariloche. Argentina

Congreso; XXXI Reunión Anual, Sociedad Argentina de Investigación. Bioquímica y Biología Molecular (SAIB).; 2003

BSp120 is a dimeric glycoprotein with trypsin-like activity located in the apical portion of cryopreserved sperm. The aim of this work was to investigate whether BSp120 was associated to sperm membranes. Different chaotropic agents were tested during the extraction of proteins from cryopreserved bovine sperm:1- 50mM Tris-HCl pH 7.4 (buffer A); 2- 0.6M KCl in buffer A; 3- 1% Triton X100 in Buffer A; 4- 1% SDS in Buffer A. rude extract was loaded onto a DEAE column. Fractions containing BSp120 were assayed by zymography, azocaseinolytic activity and Western blotting. The yield of proteolytic activity, protein concentration and specific activity for each condition were as follows: 1- 1.22x10- 4U, 0.625µg/µl, 1.95x10-4 U/µg; 2- 1.61 x10-4U, 0.625µg/µl, 2.57 x10-4 U/µg; 3- 1.08 x10-3U, 0.5µg/µl, 2.16 x10-3 U/µg; 4- Nd, 0.67µg/µl, Nd. Condition 3 showed the highest activity corresponding to the electrophoretic mobility of BSp120. The chromatographic profile of BSp120 extracted with Triton X100 on DEAE-Sephacel differed from that previously reported as it was not retained at pH 7.5. Protein obtained with mild chaotropic agents (KCl, acidic buffers, alkali) are superficially related to membranes, while proteins obtained with strong agents (detergents) are integrated to the lipidic bilayers. Therefore, we suggest that BSp120 is tightly associated to the plasmatic or acrosomal membrane of the bovine sperm. Besides, the net charge of BSp120 in sperm extracts changes depending on the condition of the extraction.