Iodophenylarsine oxide and arsenical affinity chromatography: new probes for dithiol proteins. Application to tubulins and to components of the insulin receptor-glucose transporter signal transduction pathway.
In our studies of the effects of the trivalent arsenical phenylarsine oxide on insulin-dependent hexose uptake in 3T3-L1 adipocytes, we needed direct methods to study arsenical-protein interactions. In this report, we describe two such new tools. The first is the radiolabeled covalent affinity reagent 4-[125I]iodophenylarsine oxide. This compound has effects on 3T3-L1 adipocytes similar to those of phenylarsine oxide both with respect to effects of hexose uptake and the accumulation of pp15, a phosphotyrosine-containing putative mediator of insulin action. Iodophenylarsine oxide labels numerous proteins in intact cells in a concentration-dependent, but apparently insulin-independent fashion. The second tool is trivalent arsenical affinity chromatography, which we use to show novel direct interactions between trivalent arsenicals and several proteins from 3T3-L1 adipocytes including the insulin-responsive glucose transporter GLUT4, the insulin proreceptor, and both the alpha and beta subunits of tubulin. The non-insulin-dependent glucose transporter GLUT1, the mature insulin receptor, and the fatty acid-binding protein 422(aP2) do not show strong interactions with arsenical resin. These results provide a new chemical approach to the study of both insulin-dependent hexose transport and tubulin function.
Hoffman
R D
RD
Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205.
Lane
M D
MD
D016475:3T3 Cells; D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001152:Arsenicals / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D002846:Chromatography, Affinity / Q000379:methods*; D004220:Disulfides / Q000032:analysis*; D007328:Insulin / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D011336:Probability; D011487:Protein Conformation; D011972:Receptor, Insulin / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D015398:Signal Transduction / Q000187:drug effects*; D014404:Tubulin / Q000378:metabolism*
D000345:Affinity Labels; D001152:Arsenicals; D004220:Disulfides; D007328:Insulin; D007457:Iodine Radioisotopes; D009004:Monosaccharide Transport Proteins; D014404:Tubulin; C029341:oxophenylarsine; C075599:4-iodophenylarsine oxide; D011972:Receptor, Insulin