Characterization of a Gi-protein from Trypanosoma cruzi epimastigote membranes.
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.
Coso
O A
OA
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Buenos Aires, Argentina.
Díaz Añel
A
A
Martinetto
H
H
Muschietti
J P
JP
Kazanietz
M
M
Fraidenraich
D
D
Torres
H N
HN
Flawia
M M
MM
D000244:Adenosine Diphosphate / Q000378:metabolism; D037361:Adenylate Cyclase Toxin; D000595:Amino Acid Sequence; D000818:Animals; D019204:GTP-Binding Proteins / Q000032:analysis / Q000276:immunology / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000032:analysis / Q000276:immunology / Q000502:physiology; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D015800:Protozoan Proteins / Q000032:analysis* / Q000276:immunology; D013462:Sulfur Radioisotopes; D014349:Trypanosoma cruzi / Q000737:chemistry* / Q000502:physiology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D046911:Macromolecular Substances; D008565:Membrane Proteins; D015800:Protozoan Proteins; D013462:Sulfur Radioisotopes; D010566:Virulence Factors, Bordetella; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins