Enhancement of insulin binding to rat white adipocytes at 15 degrees C by 5,5'-dithiobis-(2-nitrobenzoic acid). Independence of the reagent's sulfhydryl group reactivity.
Insulin binding to isolated rat white adipocytes at 15 degrees C, a temperature at which cellular degradation of insulin is negligible, has been found to be described by the Two-step Binding Model: R + I in equilibrium RI in equilibrium R'I (Lipkin, E. W., Teller, D. C., and de Haën, C. (1986) J. Biol. Chem. 261, 1702-1711). RI is the initially formed complex between the receptor, R, and insulin, I, and R'I is the complex in an altered state or cellular location. Here the possibility was examined that R'I results from disulfide exchange between the receptor and insulin, an exchange proposed by Clark and Harrison (Clarke, S., and Harrison, L. C. (1986) J. Biol. Chem. 257, 12239-12244) to occur at 37 degrees C. A number of sulfhydryl reagents representing various chemical reactivities did not affect insulin binding. The exception was 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), which enhanced the number of insulin-binding sites up to 2-fold with no effect on the equilibrium constant. The data suggested that this enhancement was due to activation of cryptic binding sites pre-existing on the cell surface, possibly by increasing the valency of the receptor from 1 to 2. Insulin binding was also enhanced by structural congeners of DTNB devoid of sulfhydryl reactivity, the simplest one being benzoic acid. It was concluded that the effects were not related to modification of sulfhydryl groups, that modification of sulfhydryl groups on the receptor either did not take place or was without effect on binding, and finally, that disulfide exchange between insulin and the receptor was an unlikely explanation for the formation of R'I. Also, since it is possible to show insulin action at 15 degrees C, contrary to the proposal by Clark and Harrison (Clark, S., and Harrison, L. C. (1983) J. Biol. Chem. 258, 11434-11437), disulfide exchange does not appear to be necessary for signal transmission by the occupied receptor.
D000273:Adipose Tissue / Q000378:metabolism*; D000818:Animals; D003080:Cold Temperature; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D009579:Nitrobenzoates / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D007328:Insulin; D009579:Nitrobenzoates; D004228:Dithionitrobenzoic Acid