7] Methods for the study of protein translocation across the RER membrane using the reticulocyte lysate translation system and canine pancreatic microsomal membranes

Abstract

This chapter presents the methods developed to elucidate this mechanism for the transport of pancreatic exocrine proteins across the rough endoplasmic reticulum (RER) membrane. The methods are optimized for the simultaneous study of 17 discrete pancreatic secretory proteins, which include individual members of several families of enzymes and proenzymes. The results show that secretory proteins synthesized by dog pancreas mRNA in the presence of nuclease treated microsomal membranes were similar to those synthesized in vivo. The findings indicate that microsomal membranes isolated from dog pancreas and added to the reticulocyte lysate system are unstable in the presence of 50 μg/ml each of trypsin and chymotrypsin. Using polyacrylamide gel electrophoresis in SDS, the chapter demonstrates that the addition of micrococcal nuclease-treated rough microsomes from dog pancreas results in complete proteolytic processing of pancreatic presecretory proteins to polypeptide chains with apparent molecular weights identical with authentic secretory enzymes and zymogens.