Identification of liver cell membrane galactoglycoproteins involved in the process of insulin binding.
The glycoproteinic nature of the insulin receptor was indicated using two different approaches: 1. [125I] insulin binding to soluble receptors from mouse liver was inhibited by digestion with beta-galactosidase or pretreatment with Ricinus communis I or concanavalin A. An other enzyme (neuraminidase) and lectins (wheat germ agglutinin, Dolichos biflorus) did not affect the binding reaction. These data confirmed that insulin directly interacts with the galactoglycoproteins of liver membranes. 2. The galactose oxidase-sodium boro[3H] hydride technique, previously used for labeling accessible membrane galactoglycoproteins, was again utilized to discern the components that interact with insulin. When liver membranes were equilibrated with 10-7 M insulin prior to labeling, the SDS gel radioactive profiles were specifically modified with two galactoglycoprotein of apparent molecular sizes 195 000 and 145 000, compatible with their participation in the insulin binding interaction. Membrane pretreatment with beta-galactosidase or Sophora japonica lectin reduced the labeling in most peaks, thus supporting the argument for labeling sensitivity. Preincubation of membranes with 10-7 M proinsulin slightly hindered labeling, while pretreatment with 10-7 M glucagon was ineffective, suggesting a specificity of the insulin effect. These data indicate that glycoprotein nature of the insulin receptor for two reasons: alteration of insulin binding after modification of the galactoglycoproteins, and alteration of galactoglycoprotein labeling after insulin binding. Two galactoglycoproteins, with apparent molecular weights 145 000 and 195 000, respectively, were identified and they are suggested to have insulin binding properties.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D005690:Galactose / Q000031:analogs & derivatives / Q000302:isolation & purification / Q000378:metabolism; D005934:Glucagon / Q000494:pharmacology; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D037102:Lectins / Q000494:pharmacology; D008099:Liver / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D011384:Proinsulin / Q000494:pharmacology; D011972:Receptor, Insulin / Q000187:drug effects / Q000378:metabolism*
D006023:Glycoproteins; D007328:Insulin; D037102:Lectins; D008565:Membrane Proteins; C017336:galactoproteins; D005934:Glucagon; D011384:Proinsulin; D011972:Receptor, Insulin; D005690:Galactose