Leucine: tRNA Ligase from Cultured Cells of Nicotiana tabacum var. Xanthi

Abstract

Leudne:tRNA 1gmn was asayed in extracts from cultured tobacco (coVatiana tabacwn) XD Of the component steps in the de novo synthesis of proteins from amino acids in higher plants, the first step-that is the activation and attachment of amino acid to tRNA -has received the most attention (see review by Lea and Norris [17]). The enzymes catalyzing this step, the amino acid:tRNA ligases (amino acid-activating enzymes, aminoacyl tRNA synthetases, EC 6.1.1.), have been shown to change in activity during germination of pea (13), bean (1), and wheat (21), during differentiation of pea (7) and bean (14) roots, and during senescence of soybean cotyledons (5) and tobacco leaves (19), and several workers have suggested that changes in the activity of these enzymes, coupled with changes in isoaccepting species of tRNA, may regulate the temporal development of specific enzymes (2,4,23,27). However, although these developmental changes in ligase activity are well characterized and may play an important role in cellular differentiation nothing is known about how these changes are brought about. 3To whom requests for reprints should be addressed.Many of the recent advances in our understanding of the basic control mechanisms involved in plant nitrogen metabolism have been realized due to the utilization of cell suspension cultures (e.g. 9, 29, 30). Such cultures possess several obvious advantages over the intact plant especially the investigators' ability to manipulate and control the environment of the cells. In view of this, and the fact that specific temporal changes in the activity levels of most amino acid:tRNA ligases occur during growth of batch suspension cultures of tobacco XD cells (33), cell suspension cultures seem to be ideally suited to an investigation of the regulatory mechanisms underlying the development of these enzymes. Here, we present density labeling experiments which indicate that at least one of these enzymes, leucine:tRNA ligase, is synthesized de novo and that its cellular activity is controlled by a balance between the synthesis and loss of functional enzyme molecules. A preliminary account of some of this work has been presented (11).MATERIALS AND METHODS Chemicals. Ninety-nine mol % deuterium oxide and 99 mol % [15Nlsodium nitrate were obtained from Bio-Rad Laboratories Ltd., Bromley, Kent. L-[4,53Hlleucine (53 Ci/mol) was obtained from the Radiochemical Centre, Amersham, Bucks; 2,5-diphenyloxazole (PPO) and 1,4-di-2-(5-diphenyloxazolyl)benzene (POPOP) and toluene were obtained from Koch-