Isolation of a proteolytically derived domain of the insulin receptor containing the major site of cross-linking/binding.

摘要: Radiolabeled insulin was affinity cross-linked to purified receptor with six separate bifunctional N-hydroxysuccinimide esters of different lengths. Results were qualitatively identical for each cross-linker in that predominantly through its B chain the receptor's alpha subunit. The maximum efficiencies cross-linking 10-15% most effective reagents, and this value dependent upon concentration length cross-linker. In an effort locate site, monoiodoinsulin affinity-purified disuccinimidyl suberate. Limited proteolysis hormone/receptor adduct Staphylococcus aureus V8 protease, chymotrypsin, or thermolysin SDS-containing buffer rapidly generated a 55-kDa, insulin-labeled fragment as shown by SDS-polyacrylamide gel electrophoresis. We reported earlier 55-kDa chymotryptic contained multiple internal disulfide bonds evidenced shifting mobility on SDS after dithiothreitol treatment [Boni-Schnetzler et al. (1987) J. Biol. Chem. 262, 8395-8401]. Here we show is also formed absence prior cross-linking. This prepared amounts sufficient sequence analysis passage successively over permeation reverse-phase HPLC columns. fragment's amino terminus corresponds reacts antibody raised against synthetic peptide corresponding residues 242-253 subunit.(ABSTRACT TRUNCATED AT 250 WORDS)