Cysteine Metabolism in Cultured Tobacco Cells. Received for publication April 2, 1979 and in revised form September 2, 1979. H. MICHAEL HARRINGTON' ...
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Cysteine Metabolism in Cultured Tobacco Cells Received for publication April 2, 1979 and in revised form September 2, 1979
H. MICHAEL HARRINGTON' AND IVAN K. SMITH Department of Botany, Ohio University, Athens, Ohio 45701 The maximum internal cysteine pool was similar when the cells were fed either sulfate or cysteine as a sulfur source. These results suggest that sulfate transport is regulated by the internal sulfate pool while the cysteine pool is regulated by degradative enzymes
Transported L-135Sicysteine was rapidly metabolized by cultured tobacco cells when supplied to the cells at 0.02 millimolar or 0.5 millimolar. The internal cysteine pool was expandable to approximately 2400 nmoles per gram fresh weight. The 35S label derived from cysteine was found in several metabolites. The amount of label in glutathione and sulfate was directly proportional to the internal L-536SIcysteine, while the levels of labeled methionine and protein were apparently independent of internal labeled cysteine. Cysteine was more rapidly metabolized when the external cysteine concentration was low (0.02 millimolar) with up to 90% of the 36S label present as compounds other than cysteine. The initial step in cysteine degradation yielded pyruvate, suffide, and presumably NH4'. Stoichiometry studies using extracts prepared from acetone powders of tobacco cells indicated that pyruvate and sulfide were produced in a 1:1 ratio. The catabolic reaction was linear with respect to time and amount of protein and had a pH optimum of 8 in crude extracts. Preliminary kinetic data indicated the K. to be approximately 0.2 millimolar. The extractable degradative activity was enhanced 15- to 20-fold by preincubating the cells for 24 hours in 0.5 millimolar cysteine. The extractable specific enzyme activity roughly reflected the growth curve of the cells in culture. Maximal cysteine degradation was observed in extracts prepared from late log phase cultures that were preincubated in cysteine, while little activity was found in similar extracts from stationary phase cultures. These results are consistent with an inducible catabolic enzyme similar to the cysteine desulfhydrase from bacteria.
The regulation of sulfate reduction and the biosynthesis of the
sulfur-containing amino acids has been reviewed recently by Wilson and Reuveny (29). Cysteine or a close metabolite is thought to be an important regulatory compound in bacterial and
fungal systems (2, 15, 16, 20, 27). At least one enzyme involved in sulfate reduction (ATP-sulfurylase) in tobacco cells is thought to be subject to end product repression by cysteine or a close metabolite and derepression during growth on more slowly assimilated sulfur sources (22, 23). In Lemna, adenosine 5'-phosphosulfate sulfotransferase activity is inhibited in vitro by cysteine, and sulfide pretreatment results in decreased extractable activity of this enzyme (3). These observations caused these workers to conclude that the internal cysteine and/or sulfide pool regulate the sulfur assimilation pathway in Lemna (3). Cysteine inhibits sulfate transport into cultured tobacco cells (14) and potato tubers (19). Smith (25) has demonstrated that cysteine may contribute sulfur to the sulfate pool in tobacco cells. When tobacco cells are incubated in L-[35S]cysteine, the internal cysteine pool is elevated to a "maximum level" and the excess cysteine is degraded with the sulfur being metabolized to sulfate.
' Present address: Comparative Animal Research Laboratory, 1299 Bethel Valley Road, Oak Ridge, Tennessee 37830.
(25). Cysteine is actively transported into cultured cells (13). Either L- or D-cysteine can serve as the sole sulfur source for tobacco cell cultures (14) and can support growth rates comparable to cultures grown on sulfate as the sole sulfur source (23). The sulfur derived from cysteine can be oxidized to the level of sulfate in cultured tobacco cells (25). Cysteine is also metabolized in potato tuber discs, but no determination of products has been made (19). In Salmonella, cysteine is degraded by an inducible cysteine desulfhydrase, the products being pyruvate, NH4', and sulfide (4, 5, 16, 17). Little information is available on cysteine degradation by higher plant material; however, Tishel and Mazelis (26) have described the degradation of cystine to pyruvate, S-sulfocysteine, and NH4' by preparations from cabbage. The above-mentioned metabolism of cysteine and the possible importance of cysteine in the regulation of sulfur assimilation led us to investigate the distribution of3S label derived from cysteine under conditions where transport was more favorable. The results of an earlier 3S label distribution study (25) and the present experiments provide evidence for cysteine catabolism and a rationale for studying cysteine degradation. MATERIALS AND METHODS L-[35SICysteine Feeding Experiments. Tobacco cells (Nicotiana tabacum) var. Xanthi (XD line) were dark-grown on liquid B-5 medium (10) for 8 days before experiments. Amino acids, derivatives, and the diamine reagent were obtained from Sigma Chemical Co., L-[35S]cysteine and L-[U-'4C]cysteine from Amersham/ Searle Corp. Handifluor scintillation fluid and analytical grade chemicals were obtained from conmmercial suppliers. Approximately 0.5 g fresh weight of cells from 8-day-old cultures (mid-log phase) were collected by vacuum filtration and washed with 50 ml one-tenth strength (0.1) B-5 under sterile conditions. The cells were then transferred to 125-ml flasks containing 40 ml 0.1 B-5, 10 mm sodium citrate (pH 5.5), and 0.5 or 0.02 mM L-[35S]cysteine (1.25 ,tCi/,umol). The cells were incubated for the specified time on a rotary shaker maintained at 23 C in the dark. Transport experiments were terminated by filtering the cells and washing them with 50 ml of unlabeled cysteine (concentration 10-fold higher than incubation). The cells were then split into two duplicate fractions; each was weighed and placed in 5 ml ice-cold 70%o ethanol extraction medium which contained 2 L.mol/25 ml each of cysteine, cystathionine, reduced glutathione, homocysteine thiolactone, and methionine. The mixture was sonicated for 2 min in an ice bath with a Bronwill Biosonic IV, then centrifuged for 20 min, 20,000 g. The pellet was resuspended twice in 5 ml fresh extraction medium and centrifuged as before. The resultant supernatants were pooled and made up to a total of 25 ml. The alcohol-insoluble pellet was placed in a scintillation vial with 0.5 ml tissue solubilizer and allowed to stand 24 h before addition of scintillation fluid.
HARRINGTON AND SMITH
The cell extract was applied to a Dowex 50-H+ column (0.9 x 3 cm) and washed twice with 5 ml 70o ethanol. ['S]Sulfate was determined in the column effluents by barium precipitation (25). Since sulfate is soluble to at least 2 mm in 70% ethanol, it is unlikely that appreciable amounts of sulfate were precipitated in the alcohol insoluble pellet. The sulfate fraction as determined by the above methods probably does not contain significant amounts of BaS and BaSO3. Both of these compounds are readily soluble in the Dowex 50-H+ column effluent and the acidic nature of the effluent caused the odors of H2S (from BaS) and S02 (from BaSO3) to be detected. Thus, it is unlikely that either sulfide or sulfite contributes significantly to the sulfate fraction. Amino acids were eluted from the column with 10 ml 3 M NH40H in 700o ethanol and treated essentially by the method of Giovanelli et aL (12). The amino acid fractions were dried under an air stream at 30 C and then oxidized in performic acid for 1 h. The sample was again dried as above, redissolved in 5 ml H20, and applied to a similar Dowex 50-H+ column. The column was washed twice with 2 ml H20, and the acidic oxidation products (cysteic, homocysteic, and glutathione sulfonic acids) were obtained in the effluent. The column was eluted with 10 ml 3 NH40H, to obtain the neutral oxidation products (methionine sulfone and cystathionine sulfone). Both column fractions were again dried as above and redissolved in 0.5 ml H20. The components of the fractions were separated by ascending TLC on Bakerflex sheets (Silica Gel lB) in methanol-pyridine-1.25 M HCI (37:4: 8; v/v/v). Sulfur-containing oxidation products were identified by co-chromatography with authentic compounds. The ninhydrinreactive spots were scraped into scintillation vials containing 1 ml H20 and 10 ml Handifluor. Radioactivities were determined by liquid scintillation counting with external standardization. Autoradiography of chromatograms failed to detect any radioactive
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