Protein Synthesis in a Cell-Free System Derived from Rabbit Liver

Abstract

Cell-free extracts of bacteria, plants and animals have been utilized to investigate the mechanism of protein synthesis. The ability of polyuridylic acid (poly U) and other synthetic polyribonucleotides to direct specific polypeptide synthesis by these cell-free extracts has provided a tool not only for investigating the nature of the genetic code but also for elucidating the overall mechanism of protein synthesis. However, the regulation of protein synthesis at the transcriptional and translational levels has been studied in relatively few mammalian tissues (1)(2)(3)(4)(5)(6)(7)(8). In this report we describe a subcellular protein-synthesizing system that utilizes components derived from rabbit liver tissue which is capable of incorporating amino acids into protein at levels greater than that described for other mammalian cell-free systems.Materials and Methods. Materials. Foiyuridylic acid (poly U), with 250/260 and 280/260 optical density ratios of 0.78 and 0.3 5, respectively, was purchased from Calbiochem (Los Angeles, CA). Uniformly labeled L-[ 14C] -phenylalanine (4 14 mCi/ mmole) was purchased from New England Nuclear Corp. (Waltham, MA). Crystalline phosphoenolpyruvate kinase, potassium phosphoenolpyruvate, and sodium ATP and GTP were obtained from Sigma Chemical Corp. (St. Louis, MO). Nonidet P-40 was the gift of the Shell Chemical Co. (New York) . Methods. Preparation of ribosomes. San Juan rabbits were exsanguinated, their livers were removed and rinsed 3 times in cold buffer A (0.025 M KCl, 0.005 M MgC12, 0.006 M 2-mercaptoethanol, and 0.05 M Tris, pH 7.6).The tissue was resuspended in buffer A containing 0.2 5 M sucrose (homogenizing buffer), homogenized with a motor driven loose-fitting Teflon pestle and rehomogenized by 6 strokes each in a loose-and a tight-fitting Dounce homogenizer. The homogenizations and all subsequent preparative steps were carried out a t 4' . The homogenate was centrifuged at 15,000g for 10 min and the supernate was centrifuged at 105,OOOg for 2 hr in a type 50.1 rotor on a Spinco L2-65B ultracentrifuge. The resulting supernate (S-105) was used to prepare pH 5 enzyme and transfer factors (see below). The microsomal pellet was resuspended in buffer A containing 0.5% Nonidet P-40, carefully layered over an equal volume of 0.5 M sucrose in buffer A and centrifuged at lOS,OOOg for 2.5 hr. The ribosomal pellet was resuspended in buffer A, dispensed into aliquots, rapidly frozen and stored a t -70". Ribosomal RNA (rRNA) was assayed by a modification of the procedure of Fleck and Munro (9).Preparation of pH 5 enzyme. The S-105 supernatant was diluted with 2 vol of cold, sterile glass-distilled water containing 0.006 M 2-mercaptoethanol, made pH 5.1 with 1 M acetic acid and incubated at 4' for 15 min. The preparation was centrifuged at 10, OOOg for 20 min and the clear supernatant was used for the preparation of transfer facnis remwh was supported by u.s. PubEc tors ( 1 1). The pellet was resuspended in buffer B (0.07 M KCI, 0.006 M 2-mercapto-MgC12, 0.35 k ! SWrOSe and 0.05 & i ? ...