Purification of the insulin receptor from human placenta by chromatography on immobilized wheat germ lectin and receptor antibody.

Human placental membranes were labeled with Na’”1 using lactoperoxidase, solubilized in 1% Triton X-100, and applied to a column of immobilized wheat germ lectin. After washing, 1 to 2% of the applied radioactivity could be eluted from the column with 0.3 M N-acetyh-glucosamine, of which 0.38 to 0.44% was immunoprecipitated by autoantibodies specific for the insulin receptor. Acrylamide-sodium dodecyl sulfate gel electrophoresis of the immunoprecipitates under reducing conditions and autoradiography revealed three bands with apparent molecular weights of 126,000, 90,000, and 42,000. The elution of unlabeled membrane glycoproteins from wheat germ lectin resulted in an -20-fold purification of the insulin receptor with near complete recovery, and exclusion of “insulinase” activity. The wheat germ eluate was passed through a column of immobilized normal IgG and then applied to a column of immobilized IgG containing antibodies to the insulin receptor. Insulin receptors desorbed from the antibody column with 2.5 M MgC12, pH 6.5, showed a further 20-fold increase in binding capacity and a 38-fold increase in immunoreactivity. However, these elution conditions were also shown to decrease irreversibly the binding capacity of crude receptors by up to 80%. Electrophoresis of the antibodypurified receptor revealed two major Coomassie-staining bands with apparent molecular weights of 126,000 and 42,000, and a minor band of 90,000 to 94,000 which may be a dimer of the M, = 42,000 species. Assuming a molecular weight for the insulin-binding subunit of -126,000, the antibody-purified receptor is -20% functionally pure, which is substantially more than that achieved by conventional insulin affinity chromatography. Molecular purification may, however, be close to homogeneity given that the deleterious effect of MgC12, pH 6.5, leads to an underestimate of receptor activity and that only two or three major proteins are recovered. The molecular weights of these purified proteins are identical with those determined for insulinbinding subunits using affinity-labeling methods.

• Publication year

  1980

• Venue

  Journal of Biological Chemistry

• Publication date

  1980-12-25

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1016/s0021-9258(19)70245-2PMID 7440588
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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