title: Isolation of insulin degradation products from endosomes derived from intact rat liver.
abstract: Rats were injected with [125I]iodoinsulin labeled at either the A14 or B26 tyrosine, and the animals were killed and livers subcellularly fractionated to yield light (early or neutral) endosomes and heavy (late or acidic) endosomes. 125I-Labeled material was extracted from endosomes and analyzed by Sephadex G-50 filtration and high performance liquid chromatography (HPLC). Radiolabeled material in both types of endosomes is comprised of high molecular weight, insulin-sized, and low molecular weight components, with B chain-labeled small molecular weight material in two peaks, one corresponding to iodotyrosine and one to small peptides (Mr less than 1500). As compared with A chain label, however, less of the B chain material appears in the degradation components (both high and low molecular weight fractions) suggesting that a fragment of B chain containing the B26 residue is lost from the endosomes. Analysis on HPLC shows that significant amounts of the insulin-sized and high molecular weight material have proteolytic cleavage(s) in the B chain with an intact A chain. The B chain-derived labeled peptides elute from HPLC identically with products generated by insulin protease. These results therefore show substantial insulin degradation occurring in light endosomes prior to endosomal acidification and to receptor dissociation, suggesting receptor-bound insulin is a substrate for insulin protease.
authors.affiliation: Veterans Administration Medical Center, Omaha, Nebraska 68105.
authors.forename: F G
authors.initials: FG
authors.lastname: Hamel
authors.forename: B I
authors.initials: BI
authors.lastname: Posner
authors.forename: J J
authors.initials: JJ
authors.lastname: Bergeron
authors.forename: B H
authors.initials: BH
authors.lastname: Frank
authors.forename: W C
authors.initials: WC
authors.lastname: Duckworth
mesh_terms: D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D051381:Rats; D014461:Ultracentrifugation / Q000379:methods
chemical_list: D007328:Insulin