Studies on the iodination of a ras protein and the detection of ras 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 the ras protein. Upon treatment of the ras 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 total ras protein. Iondination did not alter the biological activity of the ras protein as judged by its ability to bind GTPγS and induced maturation of Xenopus laevis oocytes. It is concluded that while iodination alters the apparent molecular weight and pI of ras, presumably by the oxidation of one or more classes of amino acids, this does not affect the biological function of the protein. The ras protein, radioactively-labelled with iodine using the Iodobead method, should be suitable for studies of protein-protein interactions involving ras. Treatment of iodinated ras 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 purified ras protein, the monomeric form is in equilibrium with small amounts of polymeric forms.

    Original languageEnglish
    Pages (from-to)75-83
    Number of pages9
    JournalMOLECULAR AND CELLULAR BIOCHEMISTRY
    Volume137
    Issue number1
    DOIs
    Publication statusPublished - 1 Aug 1994

    Keywords

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

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