Characterization of a G(i)-protein from trypanosoma cruzi epimastigote membranes

COSO, O.A.; DIAZ ANEL, A.; MARTINETTO, H.; MUSCHIETTI, J.P.; KAZANIETZ, M.; RAIDENRAICH, D.; TORRES, H.N.; FLAWIA, M.M.

BIOCHEMICAL JOURNAL

PORTLAND PRESS LTD

Año: 1992 vol. 287 p. 443 - 446

0264-6021

A guanosine 5´-[γ-[35S]thio]triphosphate-binding activity was detergent-extracted from Trypanosoma cruzi membranes. This binding activity was co-eluted from gel-filtration columns with a factor which, in a heterologous reconstitution system, blocks glucagon stimulation of adenylate cyclase activity in liver membranes. ADP-ribosylation of these membranes by pertussis toxin eliminated this blocking capacity. Incubation of T. cruzi membranes with activated pertussis toxin and [adenylate-32P]NAD+ led to the incorporation of radioactivity into a labelled product with an apparent M(r) of approx. 43000. Crude membranes were electrophoresed on SDS/polyacrylamide gels and analysed, by Western blotting, with GA/1 anti-α(common), AS/7 anti-α(i1), anti-α(i2) and anti-α12 polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 anti-β antibody, of about 30 kDa, was also detected in the membrane fraction.