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

B. Husman, L. A. Haldosen, G. Andersson, J. A. Gustafsson

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23 Scopus citations

Abstract

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 s20,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.

Original languageEnglish (US)
Pages (from-to)3963-3970
Number of pages8
JournalJournal of Biological Chemistry
Volume263
Issue number8
StatePublished - 1988

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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