CHARACTERIZATION OF A G(I)-PROTEIN FROM TRYPANOSOMA-CRUZI EPIMASTIGOTE MEMBRANES

A guanosine 5'-[gamma-[S-35]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-P-32]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-alpha(common), AS/7 anti-alpha(t), anti-alpha(i1) and anti-alpha(i2) 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-beta antibody, of about 30 kDa, was also detected in the membrane fraction.