Dithiothreitol activation of the insulin receptor/kinase does not involve subunit dissociation of the native. cap alpha. /sub 2/. beta. /sub 2/ insulin receptor subunit complex

Journal Article · Tue Nov 04 00:00:00 EST 1986 · Biochemistry; (United States)

OSTI ID:6477462

Sweet, L J; Wilden, P A; Pessin, J E

The subunit composition of the dithiothreitol- (DTT) activated insulin receptor/kinase was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and gel filtration chromatography under denaturing or nondenaturing conditions. Pretreatment of /sup 32/P-labeled insulin receptors with 50 mM DTT followed by gel filtration chromatography in 0.1% SDS demonstrated the dissociation of the ..cap alpha../sub 2/..beta../sub 2/ insulin receptor complex (M/sub r/ 400,000) into the monomeric 95,000 ..beta.. subunit. In contrast, pretreatment of the insulin receptors with 1-50 mM DTT followed by gel filtration chromatography in 0.1% Triton X-100 resulted in no apparent alteration in mobility compared to the untreated insulin receptors. Resolution of this complex by nonreducing SDS-polyacrylamide gel electrophoresis and autoradiography demonstrated the existence of the ..cap alpha../sub 2/..beta../sub 2/ heterotetrameric complex with essentially no ..cap alpha beta.. heterodimeric or free monomeric ..beta.. subunit species present. This suggests that the insulin receptor can reoxidize into the M/sub r/ 400,000 complex after the removal of DTT by gel filtration chromatography. To prevent reoxidation, the insulin receptors were pretreated with 50 mM DTT. Under the conditions the insulin receptors migrated as the M/sub r/ 400,000 ..cap alpha../sub 2/..beta../sub 2/ complex. These results demonstrate that treatment of the insulin receptors with high concentrations of DTT, followed by removal of DTT by gel filtration, results in reoxidation of the reduced ..cap alpha../sub 2/..beta../sub 2/ insulin receptor complex. Further, these results document that although the DTT stimulation of the insulin receptor/kinase does involve reduction of the insulin receptor subunits, it does not result in dissociation of the native ..cap alpha../sub 2/..beta../sub 2/ insulin receptor subunit complex.

• Research Organization: Univ. of Iowa, Iowa City
• OSTI ID: 6477462
• Journal Information: Biochemistry; (United States), Vol. 25:22
• Country of Publication: United States
• Language: English

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