The binding of 125I-insulin to specific receptors in IM-9 human lymphocytes. Detection of radioactivity covalently linked to receptors.
GA Saviolakis, LC Harrison, J Roth
J Biol Chem | Published : 1981
We used the cultured human IM-9 lymphocyte to characterize nondissociable radioactivity present after binding of 125I-insulin. Cells were incubated ate 37°C with 5 x 10-11M 125I-insulin, extensively washed, and solubilized with 1% Triton X-100 in 50 mM Hepes, 150 mM NaCl buffer, pH 7.8. After gel chromatography on Sephadex G-50 up to 45% of the solubilized, nondissociable radioactivity was present as a high molecular weight fraction. The formation of this fraction was time- and temperature-dependent, and could not be inhibited by bacitracin, phenylmethylsulfonyl fluoride, sodium azide, or methylamine. Unlabeled insulin (4 x 10-5M) completely abolished the high molecular weight fraction when ..View full abstract