41] Transcellular transport of proteins studied in vivo

Abstract

This chapter focuses on the transcellular transport of proteins studied in vivo . This approach follows the distribution of radioactively labeled proteins injected into the blood circulation of unanesthetized rats. Samples of blood, urine, bile, and pancreatic juice are collected over time, and individual radioactively labeled proteins contained in these samples are analyzed after their separation by two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). The chapter describes the methods used in this analysis and gives several examples of data derived from study of the transcellular transport of pancreatic exocrine proteins. Proteins for injection into the blood circulation can be labeled, either exogenously or endogenously, to high specific activities. In exogenous radioactive label, highest specific radioactivities can be achieved by iodination of proteins with Na 125 I. Iodination of tyrosine residues occurs with either the enzymic method or the chloramine-T method. After the iodination procedure it is necessary to separate, usually by Sephadex G-25 chromatography, the iodinated protein from the unreacted iodide and to establish that the iodinated protein has retained its biological activity. The enzymic and conjugation methods preserve biological activity to the greatest extent.