Preferences help
enabled [disable] Abstract
Number of results
2001 | 48 | 2 | 551-562
Article title

Purification and functional reconstitution of intact ral-binding GTPase activating protein, RLIP76, in artificial liposomes.

Title variants
Languages of publication
We have recently shown that RLIP76, a ral-binding GTPase activating protein, mediates ATP-dependent transport of glutathione-conjugates (GS-E) and doxorubicin (DOX) (S. Awasthi et al., Biochemistry 39, 9327, 2000). Transport function of RLIP76 was found to be intact despite considerable proteolytic fragmentation in preparations used for those studies, suggesting either that the residual intact RLIP76 was responsible for transport activity, or that the transport activity could be reconstituted by fragments of RLIP76. If the former were true, intact RLIP76 would have a much higher specific activity for ATP-hydrolysis than the fragmented protein. We have addressed this question by comparing transport properties of recombinant RLIP76 and human erythrocyte membrane RLIP76 purified in buffers treated with either 100 or 500 μM serine protease inhibitor, PMSF. The purity and identity of recombinant and human erythrocyte RLIP76 was established by SDS/PAGE and Western-blot analysis. These studies confirmed the origin of the 38 kDa protein, previously referred to as DNP-SG ATPase, from RLIP76. Higher PMSF concentration resulted in lower yield of the 38 kDa band and higher yield of intact RLIP76 from both human and recombinant source. In contrast, the substrate-stimulated ATPase activity in presence of DNP-SG, doxorubicin, daunorubicin, or colchicine were unaffected by increased PMSF; similarly, ATP-dependent transport of doxorubicin in proteoliposomes reconstituted with RLIP76 was unaffected by higher PMSF. These results indicated that limited proteolysis by serine proteases does not abrogate the transport function of RLIP76. Comparison of transport kinetics for daunorubicin between recombinant vs human erythrocyte RLIP76 revealed higher specific activity of transport for tissue purified RLIP76, indicating that additional factors present in tissue purified RLIP76 can modulate its transport activity.
Physical description
  • Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX, U.S.A.
  • Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX, U.S.A.
  • Department of Human Biological Chemistry and Genetics, UTMB, Galveston, TX, U.S.A.
  • Department of Cellular Biochemistry, M. Nencki Institute of Experimental Biology, Warszawa, Poland
  • Department of Human Biological Chemistry and Genetics, UTMB, Galveston, TX, U.S.A.
  • Departments of Internal Medicine and Biochemistry & Molecular Biology, University of Arkansas for Medical Sciences and McClellan VA Hospital, Little Rock, AR, U.S.A.
  • Department of Human Biological Chemistry and Genetics, UTMB, Galveston, TX, U.S.A.
  • Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX, U.S.A.
  • 1. Awasthi, S., Cheng, J., Singhal, S.S., Pandya, U., Pikula, S., Pikula, J., Singh, S.V, Zimniak, P. & Awasthi, Y.C. (2000) Novel function of human RLIP76: ATP-dependent transport of glutathione conjugates and doxorubicin. Biochemistry 39, 9327-9334.
  • 2. Awasthi, S., Singhal, S.S., Srivastava, S.K., Zimniak, P., Bajpai, K.K., Saxena, M., Sharma, R., Ziller, S.A. III, Frenkel, E.P., Singh, S.V., He, N.G. & Awasthi, Y.C. (1994) Adenosine triphosphate-dependent transport of doxorubicin, daunomycin, and vinblastine in human tissues by a mechanism distinct from the P-glycoprotein. J. Clin. Invest. 93, 958-965.
  • 3. Awasthi, S., Singhal, S.S., He, N.G., Chaubey, M., Zimniak, P., Srivastava, S.K., Singh, S.V. & Awasthi, Y.C. (1996) Modulation of doxorubicin cytotoxicity by ethacrynic acid. Int. J. Cancer 68, 333-339.
  • 4. Awasthi, S., Singhal, S.S., Srivastava, S.K., Torman, R.T., Zimniak, P., Bandorowicz- Pikula, J., Singh, S.V., Piper, J.T., Awasthi, Y.C. & Pikula, S. (1998) ATP-dependent human erythrocyte glutathione-conjugate transporter. I. Purification, photoaffinity labeling and kinetic characteristics of ATPase activity. Biochemistry 37, 5231-5238.
  • 5. Awasthi, S., Singhal, S.S., Pikula, S., Piper, J.T., Srivastava, S.K., Torman, R.T., Bandorowicz-Pikula, J., Lin, J.T., Singh, S.V., Zimniak, P. & Awasthi, Y.C. (1998) ATP-dependent human erythrocyte glutathione-conjugate transporter. II. Functional reconstitution of transport activity. Biochemistry 37, 5239-5248.
  • 6. Awasthi, S., Singhal, S.S., Pandya, U., Gopal, S., Zimniak, P., Singh, S.V. & Awasthi, Y.C. (1999) ATP-dependent colchicine transport by human erythrocyte glutathione conjugate transporter. Toxicol. Appl. Pharmacol. 155, 215-226.
  • 7. Sharma, R., Awasthi, S., Zimniak, P. & Awasthi, Y.C. (2000) Transport of glutathione- conjugates in human erythrocytes. Acta Biochim. Polon. 47, 751-762.
  • 8. Jullien-Flores, V., Dorseuil, O., Romero, F., Letourneur, F., Saragosti, S., Berger, R., Tavitian, A, Gacon, G. & Camonis, J.H. (1995) Bridging Ral GTPase to Rho pathways. RLIP76, a Ral effector with CDC42/Rac GTPase-activating protein activity. J. Biol. Chem. 270, 22473-22477.
  • 9. Bauer, B., Mirey, G., Vetter, I.R., Garcia-Ranea, J.A., Valencia, A, Wittinghofer, A., Camonis, J.H. & Cool, R.H. (1999) Effector recognition by the small GTP-binding proteins Ras and Ral. J. Biol. Chem. 274, 17763-17770.
  • 10. Feig, L.A., Urano, T. & Cantor, S. (1996) Evidence for a Ras/Ral signaling cascade. Trends Biochem. Sci. 21, 438-441.
  • 11. Bos, J.L. (1998) All in the family? New insights and questions regarding interconnectivity of Ras, Rap1 and Ral. EMBO J. 17, 6776-6782.
  • 12. Chardin, P. (1988) The ras superfamily proteins. Biochimie 70, 865-868.
  • 13. Valencia, A., Chardin, P., Wittinghofer, A. & Sander, C. (1991) The ras protein family: Evolutionary tree and role of conserved amino acids. Biochemistry 30, 4637-4648.
  • 14. Nakashima, S., Morinaka, K., Koyama, S., Ikeda, M., Kishida, M., Okawa, K., Iwamatsu, A., Kishida, S. & Kikuchi, A. (1999) Small G protein Ral and its downstream molecules regulate endocytosis of EGF and insulin receptors. EMBO J. 18, 3629-3642.
  • 15. Quaroni, A. & Paul, E.C. (1999) Cytocentrin is a Ral-binding protein involved in the assembly and function of the mitotic apparatus. J. Cell Sci. 112, 707-718.
  • 16. Monk, T.J., Lau, S.S., Highet, R.J. & Gillette, J.R. (1985) Glutathione conjugates of 2-bromohydroquinone are nephrotoxic. Drug Metab. Dispos. 13, 553-559.
  • 17. Mertens, J.J., Gibson, N.W., Lau, S.S. & Monk, T.J. (1995) Reactive oxygen species and DNA damage in 2-bromo-(glutathion-S-yl) hydroquinone-mediated cytotoxicity. Arch. Biochem. Biophys. 320, 51-58.
  • 18. Bilzer, M., Krauth-Siegel, R.L., Schirmer, R.H., Akerboom, T.P., Sies, H. & Schulz, G.E. (1984) Interaction of a glutathione S-conjugate with glutathione reductase. Kinetic and x-ray crystallographic studies. Eur. J. Biochem. 138, 373-378.
  • 19. Awasthi, S., Srivastava, S.K., Ahmad, F., Ahmad, H. & Ansari, G.A.S. (1993) Interactions of glutathione S-transferase pi with ethacrynic acid and its glutathione conjugate. Biochim. Biophys. Acta 1164, 173-178.
  • 20. Park, S.H. & Weinberg, R.A. (1995) A putative effector of Ral has homology to Rho/Rac GTPase activating proteins. Oncogene 11, 2349-2355.
  • 21. Cantor, S.B., Yrano, T. & Feig, L.A. (1995) Identification and characterization of Ral-binding protein 1 and a potential downstream target of Ral GTPases. Mol. Cell. Biol. 15, 4578-4584.
  • 22. Jullien-Flores, V., Mahe, Y., Mirey, G., Leprince, C., Meunier-Bisceuil, B., Sorkin, A. & Camonis, J.H. (2000) RLIP76, an effector of the GTPase Ral, interacts with the AP2 complex: Involvement of the Ral pathway in receptor endocytosis. J. Cell Sci. 113, 2837-2844.
  • 23. Awasthi, S., Cheng, J., Singhal, S.S., Pandya, U., Sharma, R., Singh, S.V., Zimniak, P. & Awasthi, Y.C. (2001) Functional reassembly of ATP-dependent xenobiotic transport by the N- and C-terminal domains of RLIP76 and identification of ATP binding sequences. Biochemistry 40, 4159-4168.
  • 24. Minamide, L.S. & Bamburg, J.R. (1990) A filter paper dye-binding assay for quantitative determination of protein without interference from reducing agents or detergents. Anal. Biochem. 190, 66-70.
  • 25. Towbin, H., Staehelin, T. & Gordon, J. (1979) Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4353.
  • 26. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.
  • 27. Pikula, S., Hayden, J.B., Awasthi, S., Awasthi, Y.C. & Zimniak, P. (1994) Organic anion-transporting ATPase of rat liver. II. Functional reconstitution of active transport and regulation by phosphorylation. J. Biol. Chem. 269, 27574-27579.
  • 28. Sharma, R., Gupta, S., Singh, S.V., Medh, R.D., Ahmad, H., LaBelle, E.F. & Awasthi, Y.C. (1990) Purification and characterization of dinitrophenyl glutathione ATPase of human erythrocytes and its expression in other tissues. Biochem. Biophys. Res. Commun. 171, 155-161.
  • 29. Awasthi, Y.C., Singhal, S.S., Gupta, S., Ahmad, H., Zimniak, P., Radominska, A., Lester, R. & Sharma, R. (1991) Purification and characterization of an ATPase from human liver which catalyzes ATP hydrolysis in the presence of the conjugates of bilirubin bile acids and glutathione. Biochem. Biophys. Res. Commun. 175, 1090-1096.
  • 30. Singhal, S.S., Sharma, R., Gupta, S., Ahmad, H., Zimniak, P., Radominska, A., Lester, R. & Awasthi, Y.C. (1991) The anionic conjugates of bilirubin and bile acids stimulate ATP hydrolysis by S-(dinitrophenyl) glutathione ATPase of human erythrocyte. FEBS Lett. 281, 255-257.
  • 31. Saxena, M., Singhal, S.S., Awasthi, S., Singh, S.V., LaBelle, E.F., Zimniak, P. & Awasthi, Y.C. (1992) Dinitrophenyl S-glutathione ATPase purified from human muscle catalyzes ATP hydrolysis in the presence of leukotrienes. Arch. Biochem. Biophys. 298, 231-237.
Document Type
Publication order reference
YADDA identifier
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.