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The Effect of pH and Various Cations on the GTP Hydrolysis of Rice Heterotrimeric G-protein α Subunit Expressed in Escherichia Coli

  • Seo, Hak-Soo (Division of Applied Life Sciences, Graduate School of Gyeongsang National University) ;
  • Jeong, Jin-Yong (Division of Infectious Disease, Samsung Biomedical Research Institute) ;
  • Nahm, Min-Yeop (Division of Applied Life Sciences, Graduate School of Gyeongsang National University) ;
  • Kim, Sam-Woong (Division of Applied Life Sciences, Graduate School of Gyeongsang National University) ;
  • Lee, Sang-Yeol (Division of Applied Life Sciences, Graduate School of Gyeongsang National University) ;
  • Bahk, Jeong-Dong (Division of Applied Life Sciences, Graduate School of Gyeongsang National University)
  • Published : 2003.03.31
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Abstract

Previously, we reported the biochemical properties of RGA1 that is expressed in Escherichia coli (Seo et al., 1997). The activities of RGA1 that hydrolyzes and binds guanine nucleotide were dependent on the $MgCl_2$ concentration. The steady state rate constant ($k_{cat}$) for GTP hydrolysis of RGA1 at 2 mM $MgCl_2$ was $0.0075{\pm}0.0001\;min^{-1}$. Here, we examined the effects of pH and cations on the GTPase activity. The optimum pH at 2 mM $MgCl_2$ was approximately 6.0; whereas, the pH at 2 mM $NH_4Cl$ was approximately 4.0. The result from the cation dependence on the GTPase (guanosine 5'-triphosphatase) activity of RGA1 under the same condition showed that the GTP hydrolysis rate ($k_{cat}=0.0353\;min^{-1}$) under the condition of 2mM $NH_4Cl$ at pH 4.0 was the highest. It corresponded to about 3.24-fold of the $k_{cat}$ value of $0.0109\;min^{-1}$ in the presence of 2 mM $MgCl_2$ at pH 6.0.

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References

  1. Bimbaumer, L., Abramowitz, J. and Brown, A. M. (1990) Receptor-effector coupling by G proteins. Biochim. Biophys. Acta 1031, 163-224. https://doi.org/10.1016/0304-4157(90)90007-Y
  2. Bourne, H. R., Sanders, D. A. and McCormick, F. (1990) The GTPase superfamily: a conserved switch for diverse cell functions. Nature 348, 125-132. https://doi.org/10.1038/348125a0
  3. Brandt, D. R. and Ross, E. M. (1985) GTPase activity of the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Accumulation and turnover of enzyme-nucleotide intermediates. J. Biol. Chem. 260, 266-272.
  4. Coleman, D. E. and Sprang, S. R. (1999) Structure of Gialpha1.GppNHp, autoinhibition in a galpha protein-substrate complex. J. BioI. Chem. 274, 16669-16672. https://doi.org/10.1074/jbc.274.24.16669
  5. Freissmuth, M., Casey, P. J. and Gilman, A. G. (1989) G proteins control diverse pathways of transmembrane signaling. FASEB J. 3, 2125-2131.
  6. Hanzal-Bayer, M., Renault, L., Roversi, P., Wittinghofer, A. and Hillig, R. C. (2002) The complex of ArI2-GTP and PDE delta: from structure to function. EMBO J. 21, 2095-2106. https://doi.org/10.1093/emboj/21.9.2095
  7. Im, M.-J. (2001) Regulation of a novel guanine nucleotide binding protein tissue transglutaminase (Gah). J. Biochem. Mol. Biol. 34, 95-101.
  8. Isida, S., Takahashi, Y. and Nagata, T. (1993) Isolation of cDNA of an auxin-regulated gene encoding a G protein beta subunit- like protein from tobacco BY-2 cells. Proc. Natl. Acad. Sci. USA 90, 11152-11156 https://doi.org/10.1073/pnas.90.23.11152
  9. Ishikawa, A., Iwasaki, Y. and Asahi, T. (1996) Molecular cloning and characterization of a cDNA for the beta subunit of a G protein from rice. Plant Cell Physiol. 37, 223-228. https://doi.org/10.1093/oxfordjournals.pcp.a028935
  10. Kalbitzer, H. J., Feuerstein, J., Goody, R. S. and Wittinghofer, A. (1990) Stereochemistry and lifetime of the GTP hydrolysis intermediate at the active site of elongation factor Tu from Bacillus stearothermophilus as inferred from the 17O-55Mn superhyperfine interaction. Eur. J. Biochem. 188, 355-359. https://doi.org/10.1111/j.1432-1033.1990.tb15411.x
  11. Krab, I. M. and Parmeggiani, A. (2002) Mechanisms of EF-Tu, a pioneer GTPase. Prog. Nucleic Acid Res. Mol. BioI. 71, 513-551. https://doi.org/10.1016/S0079-6603(02)71050-7
  12. Kim, W. Y., Cheong, N. E., Lee, D. C., Je, D. Y., Bahk, J. D., Cho, M. J. and Lee, S. Y. (1995) Cloning and sequencing analysis of a full-length cDNA encoding a G protein alpha subunit, SGA1, from soybean. Plant Physiol. 108, 1315-1316. https://doi.org/10.1104/pp.108.3.1315
  13. Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E. and Sigler, P. B. (1996) The 2.0 A crystal structure of a heterotrimeric G protein. Nature 379, 311-319. https://doi.org/10.1038/379311a0
  14. Lee, H. J., Tucker, E. B., Crain, R. C. and Lee, Y. S. (1993) Stomatal opening is induced in epidermal peels of Commelina communis L. by GTP analogs or pertussis toxin. Plant Physiol. 102, 95-100. https://doi.org/10.1104/pp.102.1.95
  15. Legendre, L., Heistein, P. F. and Low, P. S. (1992) Evidence for participation of GTP-binding proteins in elicitation of the rapid oxidative burst in cultured soybean cells. J. Biol. Chem. 267, 20140-20147.
  16. Legendre, L., Yueh, Y. G., Crain, R., Haddock, N., Heinstein, P. F. and Low, P. S. (1993) Phospholipase C activation during elicitation of the oxidative burst in cultured plant cells. J. Biol. Chem. 268, 24599-24563.
  17. Li, W. and Assmann, S. M. (1993) Characterization of a G-protein-regulated outward potassium current in mesophyll cells of Vicia faba. Proc. Natl. Acad. Sci. USA 91, 262-266.
  18. Ma, H., Yanofsky, M. F. and Meyerowitz, E. M. (1990) Molecular cloning and characterization of GPA1, a G protein alpha subunit gene from Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 87, 3821-3825. https://doi.org/10.1073/pnas.87.10.3821
  19. Ma, H., Yanofsky, M. F. and Huang, H. (1991) Isolation and sequence analysis of TGA1 cDNAs encoding a tomato G protein alpha subunit. Gene 107, 189-195. https://doi.org/10.1016/0378-1119(91)90318-6
  20. Millner, P. A. and Causier, B. A. (1996) G-protein-coupled receptors in plant cells. J. Exp. Bot. 47, 983-992. https://doi.org/10.1093/jxb/47.8.983
  21. Mixon, M. B., Lee, E., Colemal, D. E., Berghuis, A. M., Gilman, A. G. and Sprang, S. R. (1995) Tertiary and quaternary structural changes in Gi alpha 1 induced by GTP hydrolysis. Science 270, 954-960. https://doi.org/10.1126/science.270.5238.954
  22. Muegge, I., Schweins, T. and Warshel, A. (1998) Electrostatic contributions to protein-protein binding affinities: application to Rap/Raf interaction. Proteins 30, 407-423. https://doi.org/10.1002/(SICI)1097-0134(19980301)30:4<407::AID-PROT8>3.0.CO;2-F
  23. Neuhaus, G., Bowler, C., Kerm, R. and Chua, N. H. (1993) Calcium/calmodulin-dependent and -independent phytochrome signal transduction pathways. Cell 73, 937-952. https://doi.org/10.1016/0092-8674(93)90272-R
  24. Northup, J. K., Smigel, M. D. and Gilman, A. G. (1982) The guanine nucleotide activating site of the regulatory component of adenylate cyclase. Identification by ligand binding. J. BioI. Chem. 257, 11416-11423.
  25. Park, S., Nelson, T. J., Alkon, D. L. and Kim, J. (2000) Functional characterization of the squid calexcitin-2, a calcium and GTP-binding protein. J. Biochem. Mol. Biol. 33, 391-395.
  26. Poulsen. C., Mai, X. M. and Borg, S. (1994) A Lotus japonicus cDNA encoding an alpha subunit of a heterotrimeric G-protein. Plant Physiol. 105, 1453-1454. https://doi.org/10.1104/pp.105.4.1453
  27. Schweins, T., Scheffzek, K., Aiiheuer. R. and Wtlinghofer, A. (1997) The role of the metal ion in the p21ras catalysed GTP-hydrolysis: $Mn^{2+}$ versus $Mg^{2+}$. J. Mol. BioI. 266, 847-856. https://doi.org/10.1006/jmbi.1996.0814
  28. Seo, H. S., Choi, C. H., Lee, S. Y., Cho, M. J. and Bahk, J. D. (1997) Biochemical characteristics of a rice (Oryza sativa L., IR36) G-protein alpha-subunit expressed in Escherichia coli. Biochem. J. 324, 273-281.
  29. Seo, H. S., Kim, H. Y., Jeong, J. Y., Lee, S. Y., Cho, M. J. and Bahk, J. D. (1995) Molecular cloning and characterization of RGA1 encoding a G protein alpha subunit from rice (Oryza sativa L. IR-36). Plant Mol. Biol. 27, 1119-1131. https://doi.org/10.1007/BF00020885
  30. Sondek, J., Lambright, D. G., Noel, J. P., Hamm, H. E. and Sigler, P. B. (1994) GTPase mechanism of Gproteins from the 1.7-A crystal structure of transducin alpha-GDP-AIF-4. Nature 372. 276-279. https://doi.org/10.1038/372276a0
  31. Wagner, P., Molenaar, C. M. T., Rauh, A. G. J., Brokel, R., Smith, H. D. and Gallwitz, D. (1987) Biochemical properties of the ras-related YPT protein in yeast: a mutational analysis. EMBO J. 6, 2373-2379.
  32. Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A, Posner, B. A., Gilman, A. G. and Sprang, S. R. (1995) The structure of the G protein heterotrimer Gi alpha 1 beta 1 gamma 2. Cell 83, 1047-1058. https://doi.org/10.1016/0092-8674(95)90220-1
  33. Warpeha, K. M., Hamm, H. E., Rasenick, M. M. and Kaufman, L. S. (1991) A blue-light-activated GTP-binding protein in the plasma membranes of etiolated peas. Proc. Natl. Acad. Sci. USA 88, 8925-8929. https://doi.org/10.1073/pnas.88.20.8925
  34. Weiss, C. A., Garnaat, C. W., Mukai, K., Hu, Y. and Ma. H. (1994) Isolation of cDNAs encoding guanine nucleotide- binding protein beta-subunit homologues from maize (ZGB1) and Arabidopsis (AGB1). Proc. Natl. Acad. Sci. USA 91, 9554-9558. https://doi.org/10.1073/pnas.91.20.9554
  35. Weiss, C. A, Huang, H. and Ma, H. (1993) Immunolocalization of the G protein alpha subunit encoded by the GPA1 gene in Arabidopsis. The Plant Cell 5, 1513-1528. https://doi.org/10.2307/3869735
  36. Wieden, H. J., Gromadski, K., Rodnin, D. and Rodnina, M. V. (2002) Mechanism of elongation factor (EF)-Ts-catalyzed nucleotide exchange in EF-Tu. Contribution of contacts at the guanine base. J. BioI. Chem. 277, 6032-6036. https://doi.org/10.1074/jbc.M110888200
  37. Wise, A. and Millner, P. A. (1992) Evidence for the presence of GTP-binding proteins in tobacco leaf and maize hypocotyl plasmalemma. Biochem. Soc. Trans. 20, 7.
  38. Wise, A., Thomas. P. G., Murphy, G. A. and Millner, P. A. (1997) Expression of the Arabidopsis G-protein GP alpha1 : purification and characterization of the recombinant protein. Plant Mol. Biol. 33, 723-728. https://doi.org/10.1023/A:1005732423622
  39. Wise, A, White, I. R. and Millner, P. A. (1993) Stimulation of guanosine 5' -O-(3-thio)triphosphate binding to higher plant plasma membranes by the mastoparan-analogue, Mas 7. Biochem. Soc. Trans. 21, 228.

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