Studies on the iodination of a ras protein and the detection of ras polymers.
Several methods for the iodination of recombinant v-H-ras protein were compared. The Iodobead method gave greatest 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 x 10(6) cpm/pmol total ras protein. Iondination did not alter the biological activity of the ras protein as judged by its ability to bind GTP gamma S and induce 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.
Chataway
T K
TK
Department of Medical Biochemistry, School of Medicine, Flinders University, Adelaide, Australia.
Barritt
G J
GJ
D000818:Animals; D015153:Blotting, Western; D002700:Chloramines; D003432:Cross-Linking Reagents / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007457:Iodine Radioisotopes / Q000378:metabolism*; D007526:Isoelectric Point; D007553:Isotope Labeling; D007784:Lactoperoxidase; D008722:Methods; D008863:Microspheres; D008970:Molecular Weight; D015689:Oncogene Protein p21(ras) / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D009866:Oogenesis / Q000187:drug effects; D011108:Polymers; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012973:Sodium Hypochlorite; D013388:Succinimides / Q000494:pharmacology; D014105:Tosyl Compounds; D014508:Urea / Q000031:analogs & derivatives; D014982:Xenopus laevis; D000096926:Benzenesulfonamides
D002700:Chloramines; D003432:Cross-Linking Reagents; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D007457:Iodine Radioisotopes; D007784:Lactoperoxidase; D015689:Oncogene Protein p21(ras); D011108:Polymers; D011993:Recombinant Fusion Proteins; D012973:Sodium Hypochlorite; D013388:Succinimides; D014105:Tosyl Compounds; D014508:Urea; C016300:chloramine-T; C030263:chloramine-B; D000096926:Benzenesulfonamides; C012385:1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril; C019358:disuccinimidyl suberate