Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1992 Oct 15;287(Pt 2):443–446. doi: 10.1042/bj2870443

Characterization of a Gi-protein from Trypanosoma cruzi epimastigote membranes.

O A Coso 1, A Díaz Añel 1, H Martinetto 1, J P Muschietti 1, M Kazanietz 1, D Fraidenraich 1, H N Torres 1, M M Flawia 1
PMCID: PMC1133185  PMID: 1445203

Abstract

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.

Full text

PDF
443

Images in this article

444Fig. 1.
on p.444
445Fig. 3.
on p.445
445Fig. 4.
on p.445

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Birnbaumer L., Abramowitz J., Brown A. M. Receptor-effector coupling by G proteins. Biochim Biophys Acta. 1990 May 7;1031(2):163–224. doi: 10.1016/0304-4157(90)90007-y. [DOI] [PubMed] [Google Scholar]
  2. Burstein E., Macara I. G. The ras-like protein p25rab3A is partially cytosolic and is expressed only in neural tissue. Mol Cell Biol. 1989 Nov;9(11):4807–4811. doi: 10.1128/mcb.9.11.4807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Eisenschlos C. D., Paladini A. A., Molina y Vedia L., Torres H. N., Flawiá M. M. Evidence for the existence of an Ns-type regulatory protein in Trypanosoma cruzi membranes. Biochem J. 1986 Aug 1;237(3):913–917. doi: 10.1042/bj2370913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gilman A. G. G proteins and dual control of adenylate cyclase. Cell. 1984 Mar;36(3):577–579. doi: 10.1016/0092-8674(84)90336-2. [DOI] [PubMed] [Google Scholar]
  5. Goldsmith P., Backlund P. S., Jr, Rossiter K., Carter A., Milligan G., Unson C. G., Spiegel A. Purification of heterotrimeric GTP-binding proteins from brain: identification of a novel form of Go. Biochemistry. 1988 Sep 6;27(18):7085–7090. doi: 10.1021/bi00418a062. [DOI] [PubMed] [Google Scholar]
  6. Holz G. G., 4th, Rane S. G., Dunlap K. GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels. Nature. 1986 Feb 20;319(6055):670–672. doi: 10.1038/319670a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Jakobs K. H., Saur W., Schultz G. Reduction of adenylate cyclase activity in lysates of human platelets by the alpha-adrenergic component of epinephrine. J Cyclic Nucleotide Res. 1976 Nov-Dec;2(6):381–392. [PubMed] [Google Scholar]
  8. Kornblihtt A. R., Flawia M. M., Torres H. N. Manganese ion dependent adenylate cyclase activity in rat testes: purification and properties. Biochemistry. 1981 Mar 3;20(5):1262–1267. doi: 10.1021/bi00508a033. [DOI] [PubMed] [Google Scholar]
  9. Kurachi Y., Nakajima T., Sugimoto T. On the mechanism of activation of muscarinic K+ channels by adenosine in isolated atrial cells: involvement of GTP-binding proteins. Pflugers Arch. 1986 Sep;407(3):264–274. doi: 10.1007/BF00585301. [DOI] [PubMed] [Google Scholar]
  10. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984 Dec;10(3-4):203–209. doi: 10.1016/0165-022x(84)90040-x. [DOI] [PubMed] [Google Scholar]
  11. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  12. Mumby S. M., Kahn R. A., Manning D. R., Gilman A. G. Antisera of designed specificity for subunits of guanine nucleotide-binding regulatory proteins. Proc Natl Acad Sci U S A. 1986 Jan;83(2):265–269. doi: 10.1073/pnas.83.2.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Muschietti J. P., Bianchini G. M., Martinetto H. E., Carricarte V. C., Giusto N., Farber D. B., Torres H. N., Flawia M. M. Reconstitution of a light-stimulated adenylate cyclase from retina and Neurospora crassa preparations. Characterization of the heterologous systems using normal and degenerative retinas. Eur J Biochem. 1989 Oct 20;185(1):205–210. doi: 10.1111/j.1432-1033.1989.tb15103.x. [DOI] [PubMed] [Google Scholar]
  14. Neville D. M., Jr Isolation of an organ specific protein antigen from cell-surface membrane of rat liver. Biochim Biophys Acta. 1968 Apr 9;154(3):540–552. doi: 10.1016/0005-2795(68)90014-7. [DOI] [PubMed] [Google Scholar]
  15. Northup J. K., Smigel M. D., Gilman A. G. The guanine nucleotide activating site of the regulatory component of adenylate cyclase. Identification by ligand binding. J Biol Chem. 1982 Oct 10;257(19):11416–11423. [PubMed] [Google Scholar]
  16. Simonds W. F., Goldsmith P. K., Codina J., Unson C. G., Spiegel A. M. Gi2 mediates alpha 2-adrenergic inhibition of adenylyl cyclase in platelet membranes: in situ identification with G alpha C-terminal antibodies. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7809–7813. doi: 10.1073/pnas.86.20.7809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Stryer L. Cyclic GMP cascade of vision. Annu Rev Neurosci. 1986;9:87–119. doi: 10.1146/annurev.ne.09.030186.000511. [DOI] [PubMed] [Google Scholar]
  18. Torruella M., Flawiá M. M., Eisenschlos C., Molina y Vedia L., Rubinstein C. P., Torres H. N. Trypanosoma cruzi adenylate cyclase activity. Purification and characterization. Biochem J. 1986 Feb 15;234(1):145–150. doi: 10.1042/bj2340145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Waldo G. L., Evans T., Fraser E. D., Northup J. K., Martin M. W., Harden T. K. Identification and purification from bovine brain of a guanine-nucleotide-binding protein distinct from Gs, Gi and Go. Biochem J. 1987 Sep 1;246(2):431–439. doi: 10.1042/bj2460431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Yatani A., Codina J., Imoto Y., Reeves J. P., Birnbaumer L., Brown A. M. A G protein directly regulates mammalian cardiac calcium channels. Science. 1987 Nov 27;238(4831):1288–1292. doi: 10.1126/science.2446390. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES