Isolation of functional alpha beta heterodimers from the purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex. A structural basis for insulin binding heterogeneity.
To investigate the role of subunit communication in the insulin binding and tyrosine-specific protein kinase activities of the purified human placental insulin receptor, we have developed the methodology to isolate a functional alpha beta heterodimeric insulin receptor complex from the native alpha 2 beta 2 heterotetrameric disulfide-linked state. The dissociation of the alpha 2 beta 2 heterotetrameric insulin receptor into an alpha beta heterodimer was found to be approximately 50% efficient by treatment with alkaline pH (8.75) and dithiothreitol (2 mM). Removal of the dithiothreitol and pH neutralization (pH 7.60) by rapid Sephadex G-50 gel filtration resulted in the preservation of tracer insulin binding activity. The nondissociated alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes could then be effectively separated by Bio-Gel A-1.5m gel filtration. Scatchard analyses of insulin binding to the alpha 2 beta 2 heterotetrameric control or dithiothreitol-treated but nondissociated alpha 2 beta 2 heterotetrameric insulin receptor complexes demonstrated a curvilinear binding isotherm with a maximum of 1 mol of insulin bound/mol of alpha 2 beta 2 heterotetrameric complex. However, binding analyses performed on the isolated alpha beta heterodimeric complex yielded a nearly linear binding curve also, with 1 mol of insulin bound/mol of alpha beta heterodimeric complex at saturation. These data demonstrate that the insulin half-site binding reactivity observed in the alpha 2 beta 2 heterotetrameric insulin receptor complex results from either an asymmetric assembly of identical alpha beta heterodimers or from absolute negative cooperativity.
Sweet
L J
LJ
Morrison
B D
BD
Pessin
J E
JE
D002850:Chromatography, Gel; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism*; D046911:Macromolecular Substances; D010920:Placenta / Q000032:analysis*; D011247:Pregnancy; D011972:Receptor, Insulin / Q000302:isolation & purification* / Q000378:metabolism
D004220:Disulfides; D007328:Insulin; D046911:Macromolecular Substances; D011972:Receptor, Insulin; D004229:Dithiothreitol