Figure 3.

Autoradiographs of enzymatic and chemical processing of the photoconjugated ¹²⁵I-all-R-K13–hPTH/PTHrP receptor complex. Route A: (a) The intact radiolabeled ¹²⁵I-all-R-K13–hPTH/PTHrP receptor photoconjugate was exhaustively digested by Lys-C (lane 1; IA ≈18 kDa) and analyzed by 16.5% (wt/vol) Tricine SDS/PAGE. The radiolabeled band was excised, eluted from the gel, and treated by Endo F (see Materials and Methods) (lane 2, IIA ≈12 kDa). Arrows indicates the positions of the radioligand crosslinked Lys-C-derived fragment (≈18 kDa) and its deglycosylated form (≈12 kDa). Molecular mass markers are indicated at the left. (b) Exhaustive CNBr digestion of Lys-C treated radiolabeled band. Deglycosylated Lys-C-derived band before treatment with CNBr (lane 1; IIA ≈12 kDa). The excised and eluted sample from lane 1 after exhaustive digestion by CNBr (see Materials and Methods) (lane 2; IIIA ≈6 kDa). Arrows indicates the positions of the deglycosylated radioligand crosslinked Lys-C-derived fragment (≈12 kDa) and its CNBr-generated fragment (≈6 kDa). Molecular mass markers are indicated at the left. Route B: (c) The intact radiolableled ¹²⁵I-all-R-K13–hPTH/PTHrP receptor photoconjugate before (lane 1) and after an exhaustive CNBr digestion (fragment IB ≈46 kDa, position indicated by arrow on the right) resolved on a 7.5% (wt/vol) SDS/PAGE in the absence (lane 2) or presence of 2-mercaptoethanol (lane 3). (d) The ¹²⁵I-all-R-K13–hPTH/PTHrP receptor photoconjugate CNBr-derived fragment before (lane 1; IB ≈46 kDa) and after exhaustive Endo F treatment (lane 2; IIB ≈19 kDa), resolved on a 16.5% (wt/vol) Tricine SDS/PAGE. The arrows indicate the position of the 46-kDa CNBr-derived fragment (IB) and its ≈19-kDa deglycosylated form (IIB). Molecular mass markers are indicated at the left.