Subunit structure of insulin receptor of rat adipocytes as demonstrated by photoaffinity labeling.
Isolated rat adipocytes were incubated in the dark with either one or two radioiodinated photoreactive insulin derivatives, N epsilon B29-(azidobenzoyl) insulin (B29-MABI) and N alpha B1-(azidobenzoyl) insulin (B1-MABI), and were then exposed to light. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and radioautography of the crude plasma membrane fraction after reduction showed that B29-MABI labeled specifically three proteins of Mr 130 000, 90 000, and 40 000 whereas B1-MABI labeled specifically two proteins of Mr 130 000 and 40 000. B1-MABI also variably labeled some bands of intermediate Mr between 130 000 and 90 000. In contrast, the labeling of the 40-kilodalton protein was not observed in our previous studies in which photolabeling was carried our on isolated plasma membrane preparations [Yip, C. C., Yeung, C. W. T. & Moule, M. L. (1980) Biochemistry 19, 70-76; Yeung, C. W. T., Moule, M. L., & Yip, C. C. (1980) Biochemistry 19, 2196-2203]. Without reduction, an Mr 300 000 band and a larger band which barely entered a 5-15% gradient gel were specifically labeled by both photoreactive insulins. Reduction of these two high molecular weight bands gave rise to the 130-, 90-, and 40-kilodalton bands. The labeling of these proteins was affected neither by the time or temperature of incubation nor by the addition of methylamine, chloroquine, bacitracin, phenylmethanesulfonyl fluoride, p-(chloromercuri) benzenesulfonic acid, Trasylol, N-ethylmaleimide, or benzamidine. The labeling of these proteins by the photoreactive insulin derivatives was inhibited by first incubating the adipocytes with a human autoimmune serum to insulin receptor. We therefore conclude that these proteins are subunits of the insulin receptor in intact adipocytes.
D000273:Adipose Tissue / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D007328:Insulin / Q000031:analogs & derivatives; D008970:Molecular Weight; D010777:Photochemistry; D011487:Protein Conformation; D011506:Proteins / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000302:isolation & purification*
D000345:Affinity Labels; D001386:Azides; D007328:Insulin; D011506:Proteins; C025300:insulin, 4-azidobenzoyl(B29)-; D011972:Receptor, Insulin