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Purification of the adipocyte insulin receptor by immunoaffinity chromatography.

1980; Elsevier BV; Volume: 255; Issue: 4 Linguagem: Inglês

10.1016/s0021-9258(19)86093-3

1083-351X

Julia Heinrich, Paul F. Pilch, Michael Czech,

Lipid metabolism and biosynthesis

Adipocyte plasma membranes were equilibrated with 5 n~ '261-insulin, and the hormone was covalently crosslinked to a high molecular weight protein with properties characteristic of the insulin receptor as previously described (Pilch, P. F., and Czech, M. P. (1979) J.BioL Chem 254,[3375][3376][3377][3378][3379][3380][3381].When membranes crosslinked with "'1-insulin were solubilized in Triton X-100 and incubated with anti-insulin serum, about half of the label could be precipitated upon addition of anti-IgG serum or protein A isolated from Staphylococcus aureus.Plasma membranes were iodinated with lZ6I and cross-linked to '311-insulin to yield a 1261:'311 ratio of about 7000.The doubly labeled membranes, solubilized in 1% Triton X-100, were chromatographed on Sepharose 6B and receptor fractions, exhibiting a Stokes radius of about 70 A, were collected, and pooled.This material was divided into two groups and incubated with anti-insulin or control IgG at 23°C for 6 h before incubation with protein A-Sepharose 4B for 1 h and loading into columns.Following exhaustive washing of these affinity columns, 30% of the applied '311-insulin-'261-receptor cross-linked complex treated with anti-insulin IgG could be eluted with 3 M potassium thiocyanate, and this fraction exhibited a 12sI:'311 ratio of 2.4.Only 2% of the 13' 1 label could be eluted from the affinity column with thiocyanate when normal IgG was substituted for anti-insulin IgG.Dodecyl sulfate gel electrophoresis of the purified fraction followed by autoradiography revealed a major labeled band which migrated at 125,000 daltons in the presence of reductant.These data indicate that purification was approximately 3,000-fold and that the insulin receptor is the major protein species present in the purified preparation. ~Purification of the insulin receptor from detergent-solubilized extracts of rat liver and fat cell membranes by affinity chromatography using insulin-linked agarose has been described by several laboratories (1-4).These studies have enabled the description of certain physicochemical properties of the insulin receptor including a subunit molecular weight of 135,000 and a Stokes radius of 72 A (2).However, amounts of receptor sufficiently large to enable detailed chemical and physical description of its properties have not been obtained