Abstract
Several methods for the iodination of recombinant v-H-ras protein were compared. The Iodobead method gave greates incorporation of radioactivity with minimal modification of theras protein. Upon treatment of theras protein with [125I] Nal and an Iodobead, radioactivity was initially incorporated into a 22 kDa species with a pl of 5.2, then predominantly into a 23 kDa species with a pl of 5.4. The specific activity of [125I]ras was 6×106 cpm/pmol totalras protein. Iondination did not alter the biological activity of theras protein as judged by its ability to bind GTPγS and induced maturation ofXenopus laevis oocytes. It is concluded that while iodination alters the apparent molecular weight and pI ofras, presumably by the oxidation of one or more classes of amino acids, this does not affect the biological function of the protein. Theras protein, radioactively-labelled with iodine using the Iodobead method, should be suitable for studies of protein-protein interactions involvingras. Treatment of iodinatedras with the chemical cross-linking agent disuccinimidyl suberate revealed the presence of several minor high molecular weight protein species. This result shows that, in a dilute solution of purifiedras protein, the monomeric form is in equilibrium with small amounts of polymeric forms.
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Abbreviations
- DSS:
-
Disuccinimidyl Suberate
- GTPγS:
-
Guanosine 5′-[γ-thio] triphosphate
- ATPγS:
-
Adenosine 5′[γ-thio] Triphosphate
References
Lowy DR, Willumsen BM: Function and regulation ofras. Ann Rev Biochem 62: 851–891, 1993
Egan SE, and Weinberg RA: The pathway to signal achievement. Nature 365: 781–783, 1993
Feig LA: The many roads that lead toras. Science 260: 767–768, 1993
Boguski MS, McCormick F: Proteins regulatingras and its relatives. Nature 366: 643–654, 1993
Moodie SA, Willumsen BM, Weber MJ and Wolfman A: Complexes ofras-GTP withraf-1 and mitogen-activated protein kinase kinase. Science 260: 1658–1661, 1993
Nur-E-Karmal MSA, Varga M, Maruta H: The GTPase-activating NF1 fragment of 91 amino acids reverses v-Ha-ras-induced malignant phenotype. J Biol Chem 268: 22331–22337, 1993
Downward J, Riehl R, Wu L, Weinberg RA: Identification of a nucleotide exchange-promoting activity for p21ras. Proc Natl Acad Sci USA 87: 5998–6002, 1990
Stagg BH, Temperley JW, Rochman H, Morley JS: Iodination and the biological activity of gastrin. Nature 228: 58–59, 1970
Heward CB, Yang YCS, Ormberg JF, Hadley ME, Hruby VJ: Effects of chloramine T and iodination on the biological activity of melanotropin. Hoppe-Seyler's Physiol Chem 360: 1851–1859, 1979
Santos E, Nebreda AR, Bryan T, Kempner ES: Oligomeric structure of p21ras proteins as determined by radiation inactivation. J Biol Chem 263: 9853–9858, 1988
Markwell MAK: A new solid-state reagent to iodinate proteins. Anal Biochem 125: 427–432, 1982
Tucker J, Sczakiel G, Feuerstein J, John J, Goody RS, Wittinghofer A: Expression of p21 proteins inEscherichia coli and stereochemistry of the nucleotide-binding site. EMBO J 5: 1351–1358, 1986
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the folin phenol reagent. J Biol Chem 193: 265–275, 1951
Peterson GL: A simplification of the protein assay method of Lowryet al. which is more generally applicable. Analytical Biochem 83: 346–356, 1977
Janknecht R, de Martynoff G, Lou J, Hipskind RA, Nordheim A, Stunnenberg HG: Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc Natl Acad Sci 88: 8972–8976, 1991
Shibuya EK, Polverino AJ, Chang E, Ruderman JV: Oncogenicras triggers the activation of 42-kDa mitogen-activated protein kinase in extracts of quiescentXenopus oocytes. Proc Natl Acad Sci 89: 9831–9835, 1992
Hunter WM, Greenwood FC: Preparation of iodine-132 labelled human growth hormone of high specific activity. Nature 194: 495–496, 1962
Harlow E, Lane D: Antibodies, a Laboratory Manual. Published by Cold Spring Harbour Laboratory, New York, pp 319–358, 1988
Fraker PJ, Speck JC: Protein and cell membrane iodinations with a sparingly soluble chloramide, 1,3,4,6-tetrachloro-3a, 6a-diphenylglycoluril. Biochem Biophys Res Commun 80: 849–857, 1978
Marchalonis JJ: An enzymic method for the trace iodinate proteins. Biochem J 113: 299–305, 1969
Morrison M, Bayes GS: Catalysis of iodination by lactoperoxidase. Biochemistry 9: 2995–3000, 1970
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680–685, 1970
Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354, 1979
Görg A, Postel W, Güther S: The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 9: 531–546, 1988
Switzer RC, Merril CR, Shifrin S: A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Analytical Biochem 98: 231–237, 1979
Satoh T, Nakafuku M, Kaziro Y: Studies onras proteins. Catalytic properties of normal and activatedras proteins purified in the absence of protein denaturants. Biochim Biophys Acta 949: 97–109, 1988
Wu M, Kim R, Kim S: The use ofXenopus oocytes for the bioassay ofras. Methods: A companion to Methods in Enzymology 1: 315–318, 1990
McConahey PJ, Dixon FJ: Radioiodination of proteins by the use of the chloramine-T method. Methods Enzymol 70: 210–213, 1980
Morrison M, Schonbaum GR: Peroxidase-catalyzed halogenation. Ann Rev Biochem 45: 861–888, 1976
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Chataway, T.K., Barritt, G.J. Studies on the iodination of aras protein and the detection ofras polymers. Mol Cell Biochem 137, 75–83 (1994). https://doi.org/10.1007/BF00926042
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DOI: https://doi.org/10.1007/BF00926042