Amino Acid Transport in Suspension-cultured Plant Cells: II. CHARACTERIZATION OF l-LEUCINE UPTAKE

Abstract

l-Leucine (l-Leu) transport into suspension cultured Nicotiana tabacum L. cv. Wisconsin 38 cells 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 l-Leu 43, 90, and 10 times when the initial l-Leu concentration was 20 micromolar, 100 micromolar, and 1.0 millimolar, respectively; (e) phases had K(m) values of 17.6 micromolar, 60.1 micromolar, and 1.38 millimolar, respectively; and (f) in the temperature range of 17 to 27 C phases had Q(10) 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 Q(10) of 1.3.