The effect of some antibiotics on protein synthesis activity of in vitro reconstituted rough membrane from rat liver

Abstract

In order to learn about the ribosome-membrane interaction, rough membranes have been stripped from their ribo- somes by different methods and experiments have been carried out to reconstitute the rough membrane from these stripped membranes and polyribosomes [2,3]. Ribosomes do reattach to the stripped rough membrane but the major question is whether the reconstituted rough membrane is identical to the original rough membrane before it was stripped. There are several reports about some differences between the amino acid incorporation activity of free polyribosomes compared to that of the rough membrane ribosomes, differences such as optimal Mg” concentration [4] and sensitivity towards pro- tein synthesis inhibitors [S] . In this communication we compare the effect of some inhibitors on protein synthesis promoted by rough to that of in vitro reconstituted rough membrane. 2. Materials and methods Preparation of rat liver fractions: Free polyribo- homes (FP), rough (RM) and smooth (SM) endoplas- mic membranes were prepared as described by Czosnek and Hochberg [6]. For reconstitution purpose, rough membrane was stripped using the KCl-puromycin method (RMst) and free polyribosomes were bound to this stripped rough membrane according Borgese et al. (RMrec) [3]. The different membrane fractions were pelleted and suspended in 5 ml 2 M sucrose TKM (Tris-HCl pH=7.6,50 mM; KCl. 25 mM;