Cysteine Transport into Cultured Tobacco Cells

Abstract

Cysteine transport by tobacco cells (Nicotiana tabacum L. var. Xanthi) cultured on liquid B-5 medium was examined. Transport was linear with time or amount of tissue and had a pH optimum of 4.5. Cysteine transport over a wide concentration range was biphasic. The isotherm, for descriptive convenience, was divided into 2 segments both of which obeyed Michaelis-Menten kinetics. The Km for high affinity transport was in the range 1.7 .times. 10-5 M (.+-. 0.17) while the Km for low affinity transport was in the range 3.5 .times. 10-4 M (.+-. 0.13). Maximum velocities were 3-6 nmol/g fresh wt/min and 13-16 nmol/g fresh wt/min, respectively. Azide and 2,4-dinitrophenol caused more than 90% inhibition of net transport by either system. N,N''-Dicyclohexylcarbodiimide was not inhibitory while the inhibition by carbonylcyanide m-chlorophenylhydrazone was dependent on the cysteine concentration. Only those compounds that were inhibitory to transport caused significant efflux of labeled material from preloaded cells. Tobacco cells that were preincubated in iodoacetamide or N-ethylmaleimide did not transport cysteine while similar treatments with dithiothreitol were only inhibitory or had no effect on transport. Transport by either system was, to some extent, inhibited by all other tested amino acids and analogs. Alanine, methionine and S-methyl cysteine were most effective in inhibiting cysteine transport. Both alanine and methionine were competitive inhibitors of cysteine transport by either system with inhibition constants that were similar to the Km for the particular system.