Purification and properties of a membrane-bound insulin binding protein, a putative receptor, from Neurospora crassa.
The protein that is responsible for specific, high-affinity binding of insulin to the surface of Neurospora crassa cells has been purified to homogeneity. The insulin binding activity of solubilized plasma membranes resembled that of intact cells with regard to affinity of binding, specificity for mammalian insulins, and amount of insulin bound per cell. Insulin binding activity was purified from Triton X-100 solubilized membranes in two steps: FPLC on a MonoQ HR5/5 column; and affinity chromatography on insulin-agarose. The pure material migrated as a single band of ca. 66 kDa on SDS gels, pI = 7.4 by isoelectric focusing. The protein bound 5.34 pmol of insulin/micrograms, or 35% of that expected for univalent binding. Cross-linking of 125I-insulin to pure protein or to solubilized membranes revealed a single labeled band of 67-70 kDa on SDS gels. In nonreducing native gels, two labeled bands of ca. 55 and 110 kDa were produced after cross-linking, and two bands of similar molecular weight bound iodinated insulin after transfer to nitrocellulose filters. These may correspond to active monomer and dimer forms. The pure protein possessed no protein kinase activity against itself, or against exogenous substrates (histone H2, casein, or the synthetic peptide Glu80-Tyr20), and possessed no detectable phosphorylated amino acids. It is suggested, however, that this 66-kDa protein is the "receptor" that mediates insulin-induced downstream metabolic effects.
Kole
H K
HK
Department of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854-5635.
Muthukumar
G
G
Lenard
J
J
D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D007328:Insulin / Q000378:metabolism*; D007525:Isoelectric Focusing; D009492:Neurospora crassa / Q000032:analysis*; D017830:Octoxynol; D011092:Polyethylene Glycols; D011972:Receptor, Insulin / Q000302:isolation & purification*; D015398:Signal Transduction
D003432:Cross-Linking Reagents; D007328:Insulin; D011092:Polyethylene Glycols; D017830:Octoxynol; D011972:Receptor, Insulin