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Amino Acid Transport in Suspension-cultured Plant Cells

II. CHARACTERIZATION OF L-LEUCINE UPTAKE ¹

Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12181

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 Q₁₀ 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 Q₁₀ of 1.3.

¹ This work was supported in part by grants from the National Institutes of Health (1R01 GM 25838-01), the National Science Foundation (PCM78-11793), and United States Department of Agriculture (5901-0410-8-0075-0 from the Competitive Research Grants Office) to C. N. M. M. S. B. gratefully acknowledges the financial support of the United States Army. This paper will be a part of the thesis submitted by M. S. B. in partial fulfillment of the requirements for a Ph.D. in Biology at Rensselaer Polytechnic Institute.

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