Polyribosomes from Peas AN IMPROVED METHOD FOR THEIR ISOLATION IN THE ABSENCE OF RIBONUCLEASE INHIBITORS ' Received for publication

Profiles of polyribosomes were obtained from etiolated stem segments of Pisum sativum L. var. Alaska isolated in various buffers. Tissue homogenized in a medium containing 0.2 M tris-HCI, pH 8.5, 0.2 M sucrose, 30 mM MgCL, and 60 mM KCI yielded polyribosomes exhibiting far less degradation than tissue homogenized in conventional media containing tris-HCl at lower ionic strength and pH. A further decrease in degradation was found when polyribosomes were sedimented through a sucrose pad buffered at pH 8.5 prior to centrifugation. Increased separation was obtained using heavy (125-500 mg/ml), linear sucrose gradients. Using these techniques, messenger RNA species bearing up to 12 ribosomes (dodecamers) were resolved, with messenger RNA chains bearing 9 ribosomes (nonamers) being the most abundant (having the highest absorption peak). The data presented suggest that buffer of high ionic strength and high pH was more effecetive in preventing degradation of polyribosomes than was diethyl pyrocarbonate and, furthermore, that ratios involving large polyribosomes (hexamers and larger) were more accurate indices of degradation than were ratios involving total polyribosomes. Weeks and Marcus (9) and Anderson and Key (1) reported that many published polyribosomal proffles were indicative of considerable degradation due to endogenous ribonuclease acting during isolation. Conclusions concerning the functional status of the tissue based on information from degraded polyribosomes may, therefore, be misleading. Because the proportion of polyribosomes increased when tissue was ground in the presence of the ribonuclease inhibitor, diethyl pyrocarbonate, Weeks and Marcus (9) and Anderson and Key (1) suggested that such an inhibitor should be employed routinely for work involving polyribosome distribution in plants. This inhibitor might be used profitably with peas, as they have been shown to contain high levels of endogenous RNase (5), some of which is firmly associated with the microsomes (2, 6). Situations exist, however, which preclude the use of DEP.' These include assay of polyribosome-associated enzymes such as RNase itself (2), assay of other enzymes which may be DEP1This research was supported in part by a Biomedical Sciences Support Grant RT-07055 through the University of Nebraska Research Council, and a National Science Foundation Fellowship to B.A.L. 'Abbreviation: DEP: diethyl pyrocarbonate. sensitive, e.g., cellulase (unpublished data), and measurement of protein synthetic capacity in vitro (1, 9). In such cases, therefore, methods must be made available which permit the isolation of undegraded polyribosomes in the absence of DEP. We report the development of such a method. MATERIALS AND METHODS Ten to 20 apical 10-mm segments (200 to 450 mg tissue) from the third internode of dark-grown Alaska pea seedlings (Pisum sativum L.) were frozen on Dry Ice and ground in a mortar in at least 1O volumes of grinding buffer (buffer A). The composition of buffer A is given in the text for each experiment. The resulting brei was clarified by centrifugation for 20 min at 29,000g, and the supernatant was gently layered over a 4-ml pad of 1.5 M sucrose in buffer B (40 mM tris-HCI, pH 8.5; 10 mm MgCl,; 20 mm KCI) and centrifuged for 90 min at 95,000g (average) in the 40 rotor of a Spinco Model L ultracentrifuge. The pellet was rinsed gently and resuspended in 0.5 ml buffer B by means of a Vortex-Genie mixer. Aliquots (usually 0.5 ml) of resuspended polyribosomes were layered on linear (125-500 mg/ml) sucrose gradients in buffer C (20 mM tris-HCl, pH 8.5; 10 mm MgCl,; 20 mm KCI) and spun for 75 min at 122,000g (avg) in an SW-36 rotor. The gradients were prepared by layering 2 ml of sucrose at 500 mg/ml in cellulose nitrate tubes followed by 4 ml at 375 mg/ml, 4 ml at 250 mg/ ml, and 2 ml at 125 mg/ml and equilibrated for 24 to 48 hr at 2 C (3). Lower concentration gradients were used (e.g., 75-300 mg/ml and 100-400 mg/ml) and spun for shorter periods of time, but poor resolution was obtained. All operations were conducted at 0 to 4 C except where stated otherwise. After density gradient centrifugation, the contents of the tubes were analyzed in an ISCO Model 640 fractionator and the A. nm monitored continuously. The areas in different regions of the polyribosomal profiles were determined from the average of three measurements with a planimeter. The different regions were: total absorbing material (T), i.e., polyribosomes plus monosomes plus sub-units; polyribosomes (P), i.e., material sedimenting faster than monosomes; and large polyribosomes (LP), i.e., material sedimenting faster than pentamers. The ratios P/T, LP/T and LP/P were calculated. Equilibrated blank gradients were always monitored because it was found that the baseline varied from time to time. The baseline is reported for each figure and the area below excluded from calculations. Sucrose solutions were clarified by filtering through activated charcoal to remove UV-absorbing materials and endogenous RNase (3). Diethyl pyrocarbonate (diethyl oxydiformate) was obtained from Fisher Scientific Co., and bovine pancreatic ribonuclease (type II-A) was obtained from Sigma Chemical Co.