THE SYNTHESIS OF a-AMYLASE BY ROUGH AND IN VITRO RECON- STITUTED ROUGH ENDOPLASMIC RETICULUM DERIVED FROM RAT PAROTID GLAND BERNARD CWIKEL,RACHELAVNER, HENRYKH. CZOSNEK, ABRAHAM A. HOCHBERG, and NATHANDE GROOT Dept. o f Biological Chemistry, The Hebrew University o f Jerusalem, Jerusalem, lsrael (Received 12 March, 1976) Abbreviations. Fp: Free polyribosomes; Bp: Membrane-bound polyribosomes released by DOC; RM: Rough membrane; SM: Smooth membrane; RMst: Rough membrane stripped; RMrec: In vitro reconstituted rough membrane; DOC: Sodium deoxycholate. Summary. The isolation of rough and smooth endoplasmic reticulum from rat parotid salivary gland is described. The rough membrane was stripped of its bound ribosomes using the KCl-puro- mycin method. Rough endoplasmic reticulum was reconstituted from stripped-rough membrane and polyribosomes. The reconstituted rough membrane resembled the native rough membrane in the following aspects: RNA/protein ratio, buoyant density in a continuous sucrose gradient and amino acid incorporation capacity. The in vitro synthesis of a-amylase by both rough and in vitro reconstituted rough membrane was demonstrated using SDS polyacrylamide gel electrophoresis. The reconstituted rough membrane could be restripped by KCl-puromycin. The in vitro syn- thesized a-amylase remained associated with the rough or the in vitro reconstituted rough membrane, even after these membranes were stripped of their bound ribosomes. I. INTRODUCTION The exocrine gland represents an integrated and dynamic set of structural components active in the synthesis, processing and secretion of proteins destined for export. The site for this synthesis is the membrane-bound ribosome [ 1]. Many aspects of these steps have already been elucidated in studies using slice incubates of rat parotid gland [2]. The role of three types of neuroreceptors is implicated in regulating secretion of a.amylase, the major secretory enzymatic protein [3, 4]. The membrane dynamics of enzyme secretion, as revealed by electron microscopy, involve exocytosis - a rapid series of membrane fusions between the plasma membrane of the exocrine cells and that of the secretory granules held within. This reaction in vivo is a response to a set of dietary and environmental conditions [5]. An isolated cell-free system derived from parotid gland is a useful system for understanding the first stage of a series of consecutive subcellular processes, namely the synthesis of protein on the 455 Molecular Biology Reports 2 (1976) 455-463. All Rights Reserved. Copyright 9 1976 by D. Reidel Publishing Company. Dordrecht-Holland.