A procedure for the quantitative recovery of homogeneous populations of undegraded free and bound polysomes from rat liver

Abstract

A procedure is described for the preparation of free and bound polysomes from whole homogenates of rat liver tissue. Liver is homogenized in a conventional medium containing glutathione; then after a 12-min centrifugation at 131000g, the free polysomes in the supernatant are saved, while the membrane-bound polysomes in the pellet are suspended in a mixture of ribonuclease inhibitors (cell sap, 250 mM KCl, and glutathione), homogenized in the presence of detergent (Triton X-100), centirfuged for 5 min at 1470g, decanted, and treated with deoxycholate; the polysomes in the two supernatants are harvested by centrifugation through sucrose gradients containing 250 mM KCl and cell sap. Free and bound polysomes prepared in this manner are undegraded, equally active in cell-free protein synthesis, and virtually free of ribonuclease, membranous material, glycogen, deoxycholate, completed protein, and cross-contamination. The recovery of polysomes is approximately 95% and the distribution between the free and membrane-bound state is 25 and 75%, respectively. The molecular weight profiles after sodium dodecyl sulfate-acrylamide gel electrophoresis of the polypeptides completed and released by free and bound polysomes in vitro are different, indicating that there are quantitative differences in the synthesis of various size polypeptides between the two polysome classes. The differential centrifugation procedure is rapid and reproducible, requires much less ultracentrifugation than the isopycnic technique, and provides a nearly quantitative means of separating free and bound polysomes.

Differences in size, structure and function of free and membrane-bound polyribosomes of rat liver. Evidence for a single class of membrane-bound polyribosomes

“…The pellet was suspended in 50% cell sap/HK(250)M medium, treated with 1 % Triton X-100, and centrifuged for 5 min at 1470gmax. to remove nuclei as described previously (Ramsey & Steele, 1976a (Table 1), the extract and pellet fractions obtained from the initial centrifugation of the homogenate were treated with Triton X-100 and deoxycholate and centrifuged for 10 min at 27000gmax. to remove deoxycholate-insoluble material.…”

“…(5OTi or 65 rotor, Beckman). The RNA in the 2M-sucrose layer and pellet was determined as described previously (Ramsey & Steele, 1976a (Ramsey & Steele, 1976a). The assignments of sedimentation coefficients and polyribosome size classes were made by comparison with profiles obtained on isokinetic gradients (McCarty et al, 1974).…”

“…[6-14C]Orotate-labelled membrane-free polyribosomes were prepared in the presence of HK(250)M medium and used as described previously (Ramsey & Steele, 1976a), except that they were sonicated twice for 5s at 0°C (Branson model W-185-C, power supply at a setting of 1 with a micro-tip) after isolation to disperse aggregates arising from sedimentation.…”

“…Polyribosomes were isolated from the various fractions without previous treatment and assayed essentially as described previously (Ramsey & Steele, 1976a). Polyribosomal suspensions (6.8 A260 units/ml) were incubated for 30min with either [4,5-3H]leucine (10nmol/ml, 2Ci/mmol) or [U-14C]leucine (10nmol/ml, 312Ci/mol) in a complete cell-free system.…”

“…All of the chemicals used have been described elsewhere (Ramsey & Steele, 1976a). All solutions were made with glass-distilled I-piperazine-ethanesulphonic acid], pH7.4 at 4'C, 25-500mM-KCl and 5mM-MgCl2; (x) denotes the KCI concentration indicated in mm.…”