A novel receptor mediated ATP transport system regulated by tyrosine and serine/threonine phosphokinases in Trypanosoma cruzi trypomastigotes.
Trypanosoma cruzi has a plasma membrane ATP transport system that may consist of an exterior receptor domain (ATP-R) and an interior domain regulated by tyrosine and serine/threonine kinases. The addition of exogenous ATP to freely swimming trypomastigotes resulted in a receptor-mediated inward movement of the nucleotide, and the system obeyed mass action law (Km, 9.42 microM and Vmax, 77.7 nmol x min(-1) x 4 x 10(6) trypomastigotes(-1)). Preloaded [3H]ADPi was not exchanged for ATP following the addition of increasing concentrations of exogenous ATPo to swimming trypomastigotes. Trypomastigote [ATP]o <--> ATP-R --> [ATP]i transport was [ATP]o-dependent and saturable at 100 microM. [ATP]o <--> ATP-R --> [ATP]i transport was abrogated by the tyrosine kinase inhibitors, genistein and lavendustin A. [ATP]o <--> ATP-R --> [ATP]i transport was also inhibited by the serine/threonine kinase inhibitor, staurosporin. Suramin, the antagonist of P2x and P2y purinergic receptors and the inhibitor of tyrosine kinase growth factor receptors, was also a very effective competitive inhibitor of the trypomastigote ATP transport system. The action of exogenous [gamma32P]ATPo resulted in the initial and simultaneous phosphorylation of a 63-kDa polypeptide (p63) and of a 92.4-kDa polypeptide (p92.4), which was followed by the abrupt phosphorylation of many other substrate proteins. The trypomastigote p63/p92.4 polypeptides may represent substrate proteins of a putative ATP-R-related tyrosine phosphokinase, and ATP receptors may transmit their signals by phosphorylation of specific substrate proteins.
Sadigursky
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M
The Department of Pathology, Faculty of Medicine, Federal University of Bahia, Brazil.
Santos-Buch
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CA
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D014349:Trypanosoma cruzi / Q000378:metabolism*
D004791:Enzyme Inhibitors; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases