Identification of guanine nucleotide binding proteins from Trypanosoma cruzi.
Guanine nucleotide binding proteins (GTP-binding proteins) function as transducers of signals in different cellular processes. We have identified several GTP-binding proteins in Trypanosoma cruzi by Western blot analyses. Six polypeptide bands, p20, p25, p28, p31, p37 and p38, were specifically detected in epimastigote crude extracts, using polyclonal antibodies directed against transducin (T) or the alpha-subunit of transducin (T alpha). Four of these bands, p28, p31, p37 and p38, were found in both the soluble and the particulate epimastigote fractions. On the other hand, two of the polypeptides, p20 and p25, were observed only in the particulate fraction, and were not solubilized using 0.2% Triton X-100 and 0.2% Nonidet P-40. A rat monoclonal antibody directed against the ras oncogene, immunorecognized a band with molecular mass of 20,000 daltons, in epimastigote homogenates. In view of their identical apparent molecular weight and solubilization properties, p20, recognized by anti-T or anti-T alpha antibodies, and the 20 KDa band, recognized by anti-ras antibodies, seem to correspond to the same polypeptide. [3H] GDP and [3H] GMP-PNP binding experiments revealed the presence of guanine nucleotide binding proteins in total epimastigote crude extracts, as well as, in the soluble, detergent soluble, and particulate fractions. A primary screening of a T. cruzi cDNA library with anti-T alpha antibodies, followed by secondary and tertiary screenings with anti-ras antibodies yielded six positive clones. One of these clones (Tc-ras1) contains a 600 bp insert which we believe encodes for the ras protein from T. cruzi. On a Northern blot, this cDNA hybridizes to a unique mRNA band of 2.0 Kilobases in epimastigotes.
Bubis
J
J
Universidad Simón Bolívar, Departamento de Biología Celular, Caracas, Venezuela.
Millan
E J
EJ
Martinez
R
R
D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002642:Chick Embryo; D005260:Female; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000276:immunology / Q000502:physiology; D015723:Gene Library; D011905:Genes, ras / Q000276:immunology; D006801:Humans; D051379:Mice; D015800:Protozoan Proteins / Q000032:analysis* / Q000276:immunology; D011817:Rabbits; D051381:Rats; D015398:Signal Transduction; D015293:Transducin / Q000276:immunology; D014349:Trypanosoma cruzi / Q000737:chemistry* / Q000502:physiology; D014404:Tubulin / Q000276:immunology
D000911:Antibodies, Monoclonal; D015800:Protozoan Proteins; D014404:Tubulin; D019204:GTP-Binding Proteins; D015293:Transducin