Polyribosome Isolation in the Presence of Diethyl Pyrocarbonatel

Isolation of polyribosomes from wheat embryos and corn root tips in the presence of diethyl pyrocarbonate showed this reagent to have a protective effect on polyribosome structure. In addition, the use of diethyl pyrocarbonate allowed initial homogenization to be performed under less stringent conditions than those normally employed for polyribosome isolation. The use of the reagent is however limited, in that it is deleterious to in vitro ribosomal amino acid incorporation. Numerous studies have been reported in which the size distribution of polyribosomes has been correlated with a particular phvsiological state (1,2,4, 9, 11, 12, 18, 22, 23, 25). An implicit requirement for such studies has been a method for obtaining accurate representation of polyribosome size distribution. The widespread occurrence of ribonuclease (RNAase), particularly in plant tissues, has hampered efforts to develop such a method. The inclusion of the ribonuclease inhibitors, bentonite (24) or polyvinyl sulfate (2), in the homogenizing medium is not always effective (2,6,16) and, in addition, may result in considerable loss of polyribosomes (6,20). Recent reports (5,17) describing a more promising nuclease inhibitor, diethyl pyrocarbonate (DEP), prompted an examination of its use in polyribosome isolation. This report describes the results of those studies. Materials and Methods Plant Materials and Germination Conditionts. Wheat embryos (Triticum vulgare) were isolated by a modification of the method described by Johnston and Stern (7). Samples of 250 mg were imbibed in water for 5 hr at 260. Corn seeds (Zea mays) were germinated for 40 hr at 26° in a solution of 0.1 mm CaCl2. 1 This investigation was supported by Grant No. GB-7880 from the National Science Foundation, by Public Health Service Post-Doctoral Fellowship 1-F2-CA39084 from the National Cancer Institute, and by grants awarded to this Institute: U.$.P.H.S. Grants CA-06927 and FR-05539 from the National Cancer Institute and by an appropriation from the Commonwealth of Pennsylvania. Polyribosomge Isolation. Imbibed wheat embrvos were blotted dry, frozen on dry ice, and ground in a mortar and pestle packed in dry ice. Corn root tip sections (3 mm) were cut, immediately frozen on dry ice, and ground similarly. The ground wheat embryos (250 mg) or corn root tips (40 tips) were homogenized in a Duall homogenizer in 7 ml of a solution containing 0.25 m sucrose, 20 mM KCl, 5 mM MgAc2, 50 mM tris-CI (pH 7.7), 1 mM Cleland's reagent. If no diethyl pyrocarbonate (DEP) was to be added during homogenization, the final tris-Cl concentration was brought to 0.1 M with 1.0 M tris-Cl (pH 7.7). If DEP was included, the tris-CI con-centration was brought to 0.1 M by adding 1.0 M untitrated tris. Diethyl pyrocarbonate has a short half-life in water (5 hr at 0°), and was therefore added (0.1 ml per 10 ml grinding solution) immediately before homogenization. The remainder of the isolation procedure was similar to the procedure described by Lin, Key, and Bracker (10). The homogenates were cleared by centrifugation for 10 min at 17,000g. They were then layered over 3 ml of 1.66 M sucrose containing 20 mm KCI, 5 mm MgAc2, 50 mM tris-Cl (pH 7.7), 1.0 mM Cleland's reagent and centrifuged for 90 min at 225,000g. Microsomal pellets were resuspended in 0.5 ml of 20 mM KCl, 5 mM MgAc2, 50 mM tris-Cl (pH 7.7), 1.0 mm Cleland's reagent. Approximately 1 mg of ribosomes was layered on a 10 to 34 % sucrose density gradient containing 20 mM KCI, 5 mm MgAc2, 50 mM tris-Cl (pH 7.7), and 1.0 mM Cleland's reagent and centrifuged 2 hr at 90,000g in a SW25 rotor. The gradients were fractionated using a modified ISCO density-gradient fractionator with continual monitoring of absorbance at 254 mM. In Vitro Amino Acid Incorporation. Polyribosomes were obtained from microsomal pellets prepared as described above. Supernatant-(S-23) was obtained from dry wheat embryos (13) and added to 1291 www.plantphysiol.org on August 29, 2017 Published by Downloaded from Copyright © 1969 American Society of Plant Biologists. All rights reserved.