Separation of the high affinity insulin-like growth factor I receptor from low affinity binding sites by affinity chromatography.
We have identified high and low affinity insulin-like growth factor I (IGF I)-binding sites with mean dissociation constants of 0.37 and 6.25 nM, respectively, in solubilized placental membranes. We have separated these sites and purified the high affinity IGF I receptor 1,300-fold, with an overall yield of 9.9%, using wheat germ agglutinin-Sepharose chromatography, insulin affinity chromatography, and IGF I affinity chromatography. The Scatchard plot of IGF I binding to the high affinity receptor is linear, suggesting the purification of a single homogeneous class of binding sites. Insulin is two orders of magnitude less effective than IGF I in competitively inhibiting IGF I binding to this receptor. The high affinity IGF I receptor is composed of alpha and beta subunits with apparent molecular weights of 135,500 and 96,200, respectively. IGF I at concentrations of greater than or equal to 50 ng/ml stimulates autophosphorylation of the beta subunit of the purified high affinity receptor 4.6-fold. Low affinity IGF I-binding sites run through the IGF I affinity column or are eluted from the insulin affinity column. The separation of IGF I receptors with different binding affinities by sequential affinity chromatography will make it possible to examine directly the determinants of receptor affinity.
D001665:Binding Sites; D002846:Chromatography, Affinity; D006801:Humans; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight; D010920:Placenta / Q000378:metabolism; D011972:Receptor, Insulin / Q000302:isolation & purification*; D017451:Receptors, Somatomedin; D012995:Solubility; D013002:Somatomedins / Q000378:metabolism
D017451:Receptors, Somatomedin; D013002:Somatomedins; D011972:Receptor, Insulin