Uptake and binding of radiolabelled phenylarsine oxide in 3T3-L1 adipocytes.
Phenylarsine oxide (PAO), a trivalent arsenical, has been shown to inhibit insulin-stimulated glucose transport in 3T3-L1 adipocytes, implicating vicinal dithiols in signal transmission [Frost & Lane (1985) J. Biol. Chem. 260, 2646-2652]. To assist in the direct identification of a PAO-binding protein which might be involved in this process, we have synthesized [3H]acetylaminophenylarsine oxide [( 3H]APAO) from the amino derivative of phenylarsine oxide (NPAO). To assess the inhibitory effect of the product, a dual-labelling experiment was performed which showed that [3H]APAO inhibited insulin-stimulated 2-deoxy[1-14C]glucose transport in 3T3-L1 adipocytes with a Ki of 21 microM, identical with that of the parent compound, NPAO. Further characterization revealed that over a wide concentration range, uptake of the labelled arsine oxide was linear. Although the dithiol reagent 2,3-dimercaptopropanol (DMP) reversed PAO-induced inhibition of transport, it had no effect on the uptake of [3H]APAO. In a simple fractionation experiment approx. 50% of the radioactivity was associated with the cytosolic fraction and 50% with the total membrane fraction. Identification of radiolabelled proteins by non-reducing SDS/PAGE revealed fraction-specific binding, although many proteins were observed. Covalent modification was time-dependent and could be reversed by addition of DMP. These data further support a role for vicinal dithiols in insulin-stimulated glucose transport. Additionally, the probe described may offer a new means with which to identify the inhibitory protein or, more globally, to investigate mechanisms of action of vicinal dithiol-containing proteins.
Frost
S C
SC
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville 32610.
Schwalbe
M S
MS
D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D001152:Arsenicals / Q000378:metabolism / Q000493:pharmacokinetics*; D001692:Biological Transport; D002458:Cell Fractionation; D003847:Deoxyglucose / Q000493:pharmacokinetics; D007328:Insulin / Q000494:pharmacology; D007424:Intracellular Fluid / Q000378:metabolism; D007700:Kinetics; D013268:Stimulation, Chemical; D013997:Time Factors; D014316:Tritium
D001152:Arsenicals; D007328:Insulin; C029341:oxophenylarsine; D014316:Tritium; C065413:acetylaminophenylarsine oxide; D003847:Deoxyglucose