The Effects of Diethyl Pyrocarbonate on the Stability and Activity of Plant Polyribosomes

Abstract

Although polyribosomes have been isolated from several plant systems, the data are suggestive of considerable degradation of large polyribosomes. Weeks and Marcus (18) showed that much of the degradation could be prevented by isolation with diethyl pyrocarbonate, a compound previously used by Fedorcsak et al. (2,3,16,17) as a nuclease inhibitor during the isolation of nucleic acids. The results of Weeks and Marcus (18) showed that there was a small number of monoribosomes and a much larger population of subunits in DEP'-treated preparations and that DEP inhibited the ability of the ribosomes to support in vitro amino acid incorporation. Although interpreted differently by Weeks and Marcus (18), the very high proportion of subunits relative to 80S ribosomes was suggestive of a DEP-induced dissociation of ribosomes into subunits. Some of the first experiments in our laboratory showed that DEP did have dissociative effects on the polyribosomes and that the effects were concentration dependent. In order to make meaningful comparisons between ribosome preparations from different physiological states, the amount of DEP-induced dissociation or ribonuclease-induced degradation of the ribosomes must be known. Accordingly, a detailed analysis of the effects of DEP on polyribosome isolation and functional stability has been made, and the results are reported here. MATERIALS AND METHODSMung bean seeds (Phaseolus aureus Roxb.) were germinated for 16 hr in rolls of moist paper (4). Soybean seeds (Glycine max, var. Hawkeye) were germinated between layers of moist vermiculite for 72 hr. Three hundred mung bean embryonic axes were used, without cotyledons, when they reached 0.75 to 1.0 cm in length. Two hundred 0.5-cm sections were cut from the apex of the soybean hypocotyl or 100 1.0-cm sections were cut from the basal hypocotyl and incubated in 80 /M 2, 4-D for 8 hr (6). hydrolysis (2,18). DEP was always added immediately before homogenizing the tissue.The homogenate was centrifuged at 20,000g for 15 min and filtered through one layer of Miracloth (Calbiochem). Polyribosomes were prepared by centrifuging the samples through 3.0 ml of 1.5 M sucrose containing tris-buffer A at 229,000g for 85 min (Spinco type 65 rotor). The polyribosome pellets were suspended in 1.0 ml of the tris-buffer A, using a glass pestle ground to fit the centrifuge tube. Preparations used for sucrose gradient centrifugation were suspended in tris-buffer A containing 1.0% Triton X-100 (v/v). The Triton caused dissociation of some nonribosomal material, which floated on the sucrose gradients (see Fig. 1). A 0.4-ml sample of ribosomes containing approximately 16 A,., was layered on a 35-ml, 10 to 30% w/v linear sucrose gradient and centrifuged at 81,500g for 105 min (Spinco SW 27 rotor, 1 x 3.5 in tube). Gradients were fractionated as previously described (7,10).When the effects of DEP or ribosome dissociation were studied, the DEP could be added during homogenization of the tissue or during suspension of the 229,000g pellet with the same results. The lat...

Polyribosomes from Peas

“…III or by showing that its effects on polysome breakdown are identical to those caused by exogenous RNase). Conditions which have been used recently to prevent (or lessen) RNase activity (especially during isolation of polysomes) include the addition of DEP (2,31), high levels of trisHCl (10), and high concentrations of K+ (7) to the grinding buffer. Figures 4, A and C, show the results of grinding the tissue in the absence and presence of 0.1% DEP when no Ca' was added.…”

“…DEP, which has been used as a ribonuclease inhibitor in other tissues (2,31), prevents the calcium-stimulated polysome degradation in peas (Fig. 4) (30).…”

Polyribosomes from Peas

“…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.…”

“…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).…”

“…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.…”