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J H Im, D J Pillion, E Meezan; Comparison of insulin receptors from bovine retinal blood vessels and nonvascular retinal tissue.. Invest. Ophthalmol. Vis. Sci. 1986;27(12):1681-1690.
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The isolation and characterization of insulin receptors from retinal microvessels and nonvascular retinal tissue was carried out. Proteins were solubilized with Triton X-102 from retinal microvessels and nonvascular retinal tissue. The solubilized proteins were fractionated by DEAE-Sephacel ion exchange chromatography and complexed with 125I-insulin. The 125I-insulin-protein complexes were covalently cross-linked with disuccinimidyl suberate and chromatographed on a Sepharose CL-6B column. Three 125I-insulin-protein complexes with molecular weights of 560,000, 220,000 and 95,000 were obtained from both retinal microvessels and nonvascular tissue samples. The relative amount of the three complexes in retinal microvessels was about six times greater than in nonvascular retinal tissue. When aliquots of the complexes were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, the 560,000-Da complex, which displayed the greatest specific binding of [125I]insulin, stayed at the origin; the 220,000-Da complex was dissociated into 180,000-190,000-Da and 80,000-86,000-Da components; and the 95,000-Da complex was dissociated into an 80,000-Da component. In contrast, when the cross-linked 125I-insulin-protein complexes were first reduced with dithiothreitol (DTT) and then subjected to SDS-PAGE, the 560,000-Da complex from retinal microvessels was dissociated into a 125,000-Da subunit, which is identical in size to the alpha-subunit of the insulin receptor reported in other tissues, while the 560,000-Da complex from nonvascular retinal tissue was dissociated into a 116,000-Da subunit. Upon SDS-PAGE under reducing conditions, both the 220,000-Da complex and the 95,000-Da complex from both types of retinal tissue were dissociated into 65,000-Da subunits. These results confirm that the insulin receptors of nonvascular retinal tissue exhibit structural differences from those in retinal microvessels and from insulin receptors in other tissues.
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