Alterations of intermolecular disulfides in the insulin receptor/kinase by insulin and dithiothreitol
Dithiothreitol (DTT) was observed to increase the insulin receptor (IR) ..beta.. subunit autophosphorylation and exogenous substrate phosphorylation in the presence and absence of insulin. The DTT and insulin stimulation of the IR/kinase was found to increase the initial rate of autophosphorylation without any change in the extent of phosphorylation. Similarly, the V/sub max/ of exogenous substrate phosphorylation was observed to increase without any alteration in the apparent Km of substrate binding. In the presence of relatively low concentrations of DTT, insulin was found to potentiate the IR subunit dissociation of the native ..cap alpha../sub 2/..beta../sub 2/ complex into ..cap alpha beta.. halves, when observed by silver staining of SDS-polyacrylamide gels and autoradiograms of IR first autophosphorylated. (/sup 3/H)NEM labeling in the absence of DTT pretreatment demonstrated that only the ..beta.. subunit had accessibly sulfhydryl group(s), and that insulin has no effect on the total amount of (/sup 3/H)NEM incorporation. In contrast, IR that were pretreated with DTT demonstrated (/sup 3/H)NEM labeling of both the ..cap alpha.. and ..beta.. subunits. Further, incubation of IR with both DTT and insulin was also found to potentiate the IR subunit dissociation without any change in the total amount of (/sup 3/H)NEM labeling. These results suggest that the insulin activation of the IR/kinase involves an increased sensitivity of the IR to reducing agents without alteration in the total number of accessible sulfhydryl groups.
- Research Organization:
- Univ. of Iowa, Iowa City
- OSTI ID:
- 6956366
- Report Number(s):
- CONF-8606151-; TRN: 87-004071
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
DITHIOLS
BIOLOGICAL EFFECTS
INSULIN
RECEPTORS
PHOSPHOTRANSFERASES
ENZYME ACTIVITY
PHOSPHORYLATION
AUTORADIOGRAPHY
DISULFIDES
ELECTROPHORESIS
NEM
TRITIUM COMPOUNDS
ANTIMITOTIC DRUGS
CHEMICAL REACTIONS
DRUGS
ENZYMES
HORMONES
IMIDES
LABELLED COMPOUNDS
MEMBRANE PROTEINS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PEPTIDE HORMONES
PHOSPHORUS-GROUP TRANSFERASES
PROTEINS
RADIOSENSITIZERS
REAGENTS
THIOLS
TRANSFERASES
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