Phosphorylation of insulin receptors solubilized from rat skeletal muscle.
A method has been developed to solubilize insulin receptors from skeletal muscles. Rat hindlimb muscles were rapidly frozen in liquid nitrogen, powdered, extracted with buffered Triton X-100, and partially purified by differential centrifugation followed by wheat germ agglutinin affinity chromatography. The solubilized receptors exhibit typical curvilinear Scatchard plots in insulin binding assays: rapid, Mn2+-dependent autophosphorylation of the beta-subunit on exposure to insulin as well as insulin-stimulated kinase activity toward histone H2B. Furthermore, when intact soleus muscles were incubated in phosphate-depleted medium containing Na2H[32P]PO4, addition of insulin stimulated the in situ phosphorylation of the beta-subunit of the insulin receptor. The ability to rapidly and efficiently isolate insulin receptors from skeletal muscle may permit investigation of factors that modulate insulin action in this tissue.
D000818:Animals; D001345:Autoradiography; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D037102:Lectins; D008297:Male; D009132:Muscles / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism*; D014909:Wheat Germ Agglutinins
D037102:Lectins; D014909:Wheat Germ Agglutinins; D011972:Receptor, Insulin