Identification of a high affinity growth hormone receptor in rat adipocyte membranes.

摘要: Affinity cross-linking techniques have been used to identify a growth hormone (GH) receptor in rat adipocyte membranes. Adipocytes were incubated with 125I-human GH (125I-hGH) for 2 h at 37 degrees C and washed once remove unbound hGH. The bivalent reagent disuccinimidyl suberate (0.4 mM) was added the cells 15 min C. A plasma membrane-enriched fraction prepared subjected sodium dodecyl sulfate-polyacrylamide gel electrophoresis. autoradiographs of gels containing samples treated reductant revealed an intense band apparent Mr = 134,000 which not present when excess hGH or bovine GH. In contrast, intensity altered by presence similar insulin prolactin during binding step. Multiple lower molecular weight species displaying same sensitivity as also but much levels. absence reductant, affinity-labeled migrated broad 116,000-125,000 less 230,000. At low concentrations, both hGH-labeled complexes exhibited larger weights (Mr (X 10(3) ) 135 270), indicating intrachain disulfide bonds. higher 270,000 disappeared increased intensity. Use cleavable reagent, ethylene glycol bis(succinimidyl succinate), conjunction two-dimensional electrophoresis, that complex is composed iodinated monomeric 22,000) bound membrane protein. reduced protein itself calculated be 112,000, assuming 1:1 stoichiometry receptor.