Characterization of the somatogenic receptor in rat liver. Hydrodynamic properties and affinity cross-linking

Rat liver somatogenic receptors have been characterized by gel permeation chromatography, sucrose density gradients in H₂O and D₂O, and affinity cross-linking using ¹²⁵I-bovine growth hormone (bGH) as a specific somatogenic receptor ligand. Cross-linking of ¹²⁵I-bovine growth hormone to a Triton X-100-treated low density fraction isolated from livers of late pregnant rats followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis under reducing conditions showed three major binders with M(r) 95,000, 86,000, and 43,000 and a minor binder of M(r) 55,000, after correction for bound ligand assuming a 1:1 binding ratio of ligand-receptor. The M(r) 86,000, 55,000, and 43,000 species were recovered in the detergent-soluble supernatant after high-speed centrifugation, whereas the M(r) 95,000 species remained Triton X-100 insoluble. Detergent-soluble ¹²⁵I-bGH-receptor complexes were further analyzed by sedimentation into sucrose density gradients. The sedimentation coefficient was s_20,w = 5.2 S and the partial specific volume v̄ = 0.72 ml/g. Gel permeation chromatography on a Sepharose S-400 column indicated a Stokes radius of 61 Å for the ¹²⁵I-bGH-receptor-Triton X-100 complex. Based on these figures, the molecular weight of the complex was calculated as 131,100. The molecular weight of the ligand-free receptor-Triton X-100 complex was calculated as M(r) 109,100. Affinity cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 61 Å peak from Sephacryl S-400 chromatography (cf. above) showed two binding entities, one major and one minor with M(r) values 86,000 and 43,000, respectively, in the absence of reductant. When electrophoresis was run in the presence of reductant the M(r) 43,000 species was the major binding entity. Furthermore, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (first dimension, nonreducing and second dimension, reducing) showed that a disulfide-linked binder at M(r) 43,000 is contained within the M(r) 86,000 species. As with pregnant rats, female and male rats both showed ¹²⁵I-bovine growth hormone binders of M(r) 95,000, 84,000, 55,000, 43,000, and additionally an M(r) 35,000 binder.