The dissociation of the purified human placental α2β2 heterotetrameric insulin receptor complex into an αβ heterodimeric state was found to occur in a pH- and dithiothreitol (DTT)-dependent manner. Formation of the αβ heterodimeric complex, under conditions which preserved tracer insulin binding and protein kinase activities (pH 8.75 for 25 min followed by 2.0 mM DTT for 5 min) occurred with an approximate 50% efficiency. The resulting nondissociated α2β2 heterotetrameric complexes could then be separated effectively by Bio-Gel A-1.5 m gel filtration chromatography at neutral pH. The isolated DTT-treated but nondissociated α2β2 heterotetrameric complex was resistant to any further dissociation by a second round of DTT and alkaline pH treatment, whereas the isolated αβ heterodimeric complex was stable to spontaneous reassociation for at least 72 h at pH 7.60. Kinetic analyses of the insulin receptor protein kinase activity demonstrated that the insulin stimulation of glutamic acid: tyrosine (4:1) synthetic polymer phosphorylation for both the α2β2 heterotetrameric and αβ heterodimeric complexes occurred via an increase in V(max) without any significant change in K(m). Examination of β subunit autophosphorylation of the αβ heterodimeric complex, in the presence but not in the absence of insulin, demonstrated the appearance of the covalent 32P-labeled α2β2 heterotetrameric complex. Further, the initial rate of insulin-stimulated β subunit autophosphorylation in the isolated αβ hterodimeric complex occurred in a dilution-dependent (intermolecular) manner. These data demonstrate that the isolated αβ heterodimeric insulin receptor complex is fully capable of expressing insulin-dependent activation of the β subunit protein kinase domain with the covalent reassociation of the 2aβ heterodimeric complex into an α2β2 heterotetrameric disulfide-linked state.
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|State||Published - Dec 1 1987|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology