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Studies on the iodination of aras protein and the detection ofras polymers

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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

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Correspondence to Greg J. Barritt.

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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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Key words

  • ras
  • iodination
  • GTPγS binding
  • oocyte maturation
  • polymeric forms