Caracterização parcial de uma fração enriquecida em atividade ATP/GTPase obtida a partir de testículo de coelho

Abstract

In this work we isolated and partially characterized a fraction of rabbit testis containing the ATPase/GTPase activity by using a phosphocellulose column. Testis was homogenated in 50mM Imidazole pH 8.5 with 10 mM EDTA/EGTA, 1.0 mM DTT, 0.3 mM PMSF, 1.0 mM benzamidine, and then centrifuged by 15.000g for 40 min. The supematant was loaded in DEAE-Sepharose column. Bound proteins were eluted with 50 mM Imidazole pH 6.5 and the eluate was directly loaded in a phosphocellulose column. The fraction containing ATPase activity was then eluated with 50 mM Imidazole pH 8.0. No proteins were retained in GTP-agarose column. The isolated fraction presented the following properties. 1) At high concentrations (mM) of ATP or GTP, GTPase larger than ATPase activity. At low substrate concentrations both nucleotides are indistinctly hidrolyzed. 2) Both activities were dependent on the presence of Mg++ or Ca++. In both cases, the Ca++-nucleotidase was larger of than Mg++-nucleotidase activity. 3) Ehfferenttly from the corresponding fraction isolated from rat brain (Coelho, 1988), Mg++ATPase activity presented no stimulation by Ca++-CaM. 4) ATPase activity is well preserved when stored at 4 to 10 «C by one week, but not at 75 »C by 5 minutes or in a freezer at -20 °C. 5) (K+-EDTA)ATPase activity was not detected. 6) Mg-ATPase activity was not sensitive to high salt concentrations (0.3 M KC1 or NaCl), 0.2 % Triton X-100, 1.0 mM azide w 250 nM NaVO3. However, it was 80% inhibited by A1F3 (2.4 mM NaF/ 0.1 mM AlClj) and 35 % in presence ofthe 1 mM NaVO3 or 5 mM NaF. . 1 4. tn GTP-agarose column were analyzed in Some polypeptides not bound to Utr agaiu , O0 J60 100 74 e 48 kDa bands. The column SDS-PAGE and caractenzed as 160, . ^tained just two polypeptides, with 32 and 16 kDa, that didn’t e i ite SMÍ.Í ilial ” lhe vesicle transport. The proteins here described may have a participation in mechanotransduction and signalization events, for what they still need more investigation.