Effect of Ethionine on Invertase Development and Methylation of Ribonucleic Acid

Abstract

Ethionine, an analogue of methionine, has been used as an antimetabolite in animals and plants. It inhibits the induction of flowering in Xanthium (5) and Lemna (20) and auxin-stimulated cell elongation in the Avena coleoptile (14). Synthesis of enzymes such as a-amylase in barley (21), invertase in yeast (6), and phenylalanine ammonia lyase in potato disks (23) are also inhibited by ethionine. To our knowledge, all processes which are inhibited by ethionine are reversed by the addition of methionine to the plant.The development of invertase in the sugar beet disk was studied according to the methods used by Cherry (3). The aeration of disks was carried out in sterile 50-mm plastic Petri dishes containing 1 ml of 0.01 M phosphate buffer, pH 6.5, 10-4 M penicillin, and 10-4 M streptomycin. The incubation medium was changed every 8 to 10 hr. For incorporation of labeled precursors into RNA, 10 g of sugar beet disks were incubated for 6 hr in sterile aeration buffer containing the amount of labeled precursors indicated in the results. Adenosine and guanosine, 10-4 M, were added to the incubation medium to suppress methyl group incorporation from L-(methyl-3H)-methionine into the purine ring. Total nucleic acids were extracted from the tissue by the phenoldupanol method of Cherry et al. (4) and fractionated on a Sephadex G-100 column (105 cm X 1.0 cm) by the method of Galibert et al. (10).A leucyl-tRNA synthetase preparation was extracted from sugar beet by grinding 100 g of tissue in liquid nitrogen with subsequent suspension of the powdered material in 100 ml of a solution containing 0.1 M tris-HCl, pH 7.8; 0.04 M KCI; 0.04 M MgCl2; 0.01 M 2-mercaptoethanol; 0.2 M ascorbate; and 1.0 M sucrose. Following cheesecloth filtration and centrifugation at 15,000g for 15 min, the supernatant was brought to 70% saturation with respect to ammonium sulfate. The protein precipitate was dissolved in, and dialyzed overnight against, a solution containing 0.05 M tris-HCl, pH 7.8; 0.02 M KCI; 0.02 M MgCl2; and 0.005 M 2-mercaptoethanol. Subsequently, the protein concentration of the preparation was adjusted to 1 to 2 mg/ml for the aminoacylation assay.Soluble RNA was isolated by the method of Anderson and Cherry (1), except that the RNA was solubilized in 2 M potassium acetate, pH 6.5, instead of in 1 M sodium chloride. Leucine acceptor activity was assayed by the method of Anderson and Cherry (1).Invertase activity in the beet disks increased linearly over a 72-hr incubation period (Fig. 1) (3) with actinomycin D and suggest that ethionine affects some early stages of invertase development.The observed inhibition of invertase by ethionine may be due to the substitution of ethionine for methionine during invertase synthesis, resulting in altered biological activity or reduced rate of synthesis, or both. Yoshida and Yamosaki (22) reported that one-third of the methionine in a-amylase from Bacillus subtilis was substituted by ethionine without alteration of the physiochemical and biological properties of the isolated enzyme. During...