Effect of oxidative iodination of epidermal growth factor on its binding and secretion by hepatocytes

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Abstract
Experiments were undertaken to determine whether the method of iodination of epidermal growth factor (EGF) affects its binding to rat liver plasma membranes and its uptake, processing, and secretion into bile by intact rat hepatocytes. EGF was iodinated using one of three oxidative reagents: chloramine T (CT), lactoperoxidase (LP), or monochloride (MC). Quantitative receptor binding studies on plasma membranes isolated from male rat livers with either CT-, LP-or MC-125I-EGF indicated no significant difference in the apparent binding constants of the three preparations. To determine whether these three preparations were capable of forming a covalent-like complex with the EGF receptor, they were individually incubated with isolated plasma membranes and subjected to polyacrylamide gel electrophoresis under reducing conditions, followed by autoradiography. Each preparation formed a major radioactive protein band of ∼180 kD, identified as the EFG receptor by immunoprecipitation with monoclonal anti-EGF receptor antibodies. Furthermore, even unlabeled EGF incubated with plasma membranes formed this same 180 kD band, as revealed on Western blots using anti-EGF antibody. The biliary secretion of CT-, LP-, and MC-125I-EGF was compared by injecting each one into rat portal veins and measuring the total and immunoprecipitable radioactivity in bile. The amount of immunologically intact CT-125I-EGF in bile was significantly greater than the others, whereas MC-125I-EGF transport was significantly reduced. We conclude that the method of iodination does not affect the covalent-like binding properties of EGF. Furthermore, since unlabeled EGF displayed these same binding properties, oxidative iodination procedures per se do not account for the covalent-like association between EGF and its receptor. However, the method of iodination used did affect the intracellular transport and processing of EGF by hepatocytes. The structural modification responsible for this alteration in transport properties has yet to be determined.