Omar A. Coso1, A Díaz Añel1, Horacio Martinetto1, Jorge Muschietti1, Marcelo G. Kazanietz1, Diego Fraidenraich1, Héctor N. Torres1, Mirtha M. Flawiá1
1Instituto de Investigaciones en Ingeniería Genética y Biología Molecular and Facultad de Ciencias Exactas y Naturales. Obligado 2490, 1428 Buenos Aires, Argentina
Tóm tắt
A guanosine 5′-[gamma-[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. 43,000. 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.
