L-Leucine (L-Leu) transport into suspension cultured Nicotiana tabacum L. cv. Wisconsin 38 ceils has been investigated. Cells were batch-cultured and routinely assayed 3.5 to 4 days after subculturing. Uptake rates were measured over the concentration range of 10 micromolar to 150 millimolar. Kinetic analysis of the uptake rates indicated that uptake was multiphasic with three saturable phases and one unsaturable phase. The three saturable phases which occur in the concentration ranges of 10 to 40 micromolar, 50 to 100 micromolar, and 0.2 to 5.0 millimolar exhibited the following characteristics; (a) phases were energy-dependent as shown by 84 to 94% inhibition of uptake rates by metabolic inhibitors; (b) phases exhibited broad pH optima between 3.0 and 5.5; (c) phases showed stereospecificity for L-Leu; (d) over a 12-hour incubation period, phases concentrated LLeu 43, 90, and 10 times when the initial L-Leu concentration was 20 micromolar, 100 micromolar, and 1.0 nillimolar, respectively; (e) phases had Km values of 17.6 micromolar, 60.1 micromolar, and 1.38 millimolar, respectively; and (J) in the temperature range of 17 to 27 C phases had Qlo values of 2.1, 1.4, and 1.4, respectively. L-Leu uptake in the three saturable phases was inhibited by a 20-fold higher concentration of 18 other amino acids; phenylalanine, alanine, and methionine were the most effective inhibitors, whereas aspartic acid, asparagine, histidine, and arginine were the least effective. The nonsaturable phase which was responsible for increases in the uptake rate above 5.0 mm appeared to be primarily diffusional since it was minimally influenced by metabolic inhibitors and had a Qlo of 13.The transport of amino acids into animal and microbial cells has been extensively studied (7) but until recently has received minimal attention in plants (18). To date, the number of systems involved in amino acid uptake for a single plant or plant tissue is not known; nor has any single system been fully characterized (6,17). In the past several years however, there has been an increased interest in amino acid uptake by plant cells, as evidenced by the increased number of laboratories investigating amino acid uptake.These studies have primarily used kinetic analysis of uptake to study amino acid transport. Many amino acids appear to be taken up by a common system (6) and there are data suggesting that ' This work was supported in part by grants from the National Institutes Suspension-cultured plant cells have been used for analyzing transport phenomena, and Maretzki and Thom (17) have recently discussed the advantages of cultured cells. We selected suspensioncultured tobacco cells for study because they are easily cultured on a totally defined medium (16) which can be readily modified. In addition, plants can be regenerated from single cells (25), haploid plants can be produced (19), and mutants can be isolated from cultured cells and regenerated into fertile plants (5). Our first objective, to develop an assay for measuring amino acid uptake i...
