2Sl-lnsulin. 125I-Insulin bound mainly to two bands of microvillous membrane protein (data not shown). The less intense band (A) had a molecular mass higher than 250 kDa, and the label was displaced by IGF-I. The most intense band migrated at 135 kDa (E) and was displaced only by insulin; neither IGF-I nor IGF-II displaced the labeled ligand. Labeled insulin incubated with basal plasma membranes was bound essentially to two bands (Fig. 6). The slower band (A) had a molecular mass higher than the band (molecular mass > 250 kDa) to which the 125I-insulin bound. It was displaced by neither IGF-I nor IGF-II, but was completely displaced by insulin. However, this electrophoretic profile also occurred when a high membrane protein concentration was used in the reaction mixture. The second labeled band had a molecular mass of 135 kDa (E) and was not displaced by IGF-I or IGF-II, only by unlabeled insulin.
Binding Site Specificity
Each labeled band in the 135-130 kDa region was assigned to its main ligand, and the labeled bands in the highest molecular mass region were assigned to possible associations of the various subunits (Fig. 6) by using higher amounts of membrane proteins for binding (120-150 fxg). The basal plasma membrane was analyzed for the three types of binding sites it contained. 125I-IGF-I and 125I-IGF-II both gave two bands in the 135- to 130-kDa region (E and F bands); their intensities were attenuated by unlabeled IGF-I and IGF-II (lanes 2,3,6,7). Insulin did not displace the fastest band (F), but did displace the E band (lane 4) or competed for the two bands (lane 8). 125I-Insulin gave only one band (E), which was displaced by unlabeled insulin (lane 12). ventolin inhalers
I25I-IGF-I gave two bands (> 250 kDa) (B) and (< 250 kDa) (D) (lane 5); both were displaced by IGF-I (lane 6). 125I-Insulin gave a very slow band (A) with a molecular mass higher than that of band В (lane 9); it was displaced only by unlabeled insulin (lane 12).