Methionine Biosynthesis in Lemna - Plant Physiology

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Plant Physiol. Vol. 69, 1982 Sulfate O-Acetylserine - Sulfide ------- 1078 THOMPSON ET AL. ---lt l~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I Aspartate~~~~~~~~~~~~~~~~~~~~~~~~I Aspartate |Lys+Thr| O-Phosphohomoserine.--------- Propargyl- I Threonine glycine Homocysteine - N'-Methyl H,-Folate Methionine I FIG. 1. Methionine biosynthesis in higher plants. The solid arrows represent the major pathway operative in Lemna and Chlorella. The dashed arrows represent an alternative pathway involving the direct sulfhydration of O-phosphohomoserine (reaction 2). Also indicated are the metabolic sites where lysine plus threonine, AVG, and propargylglycine block methionine formation (7). (N-morpholino)-propanesulfonic acid and L-methionine from Schwarz-Mann; L-cystathionine, O-succinyl-L-homoserine, and O-acetyl-L-serine from Calbiochem; adenosine from Nutritional Biochemicals; and Sephadex G-25 (medium) from Pharmacia Fine Chemicals. Baker Analyzed (J. T. Baker Chemical Co.) formic acid was used exclusively; Na2S was also from Baker. Dowex 50 was obtained from Bio-Rad Laboratories under the designation of AG 50W-X4, 200 to 400 mesh; the dye reagent concentrate used for protein assays (2) was also from Bio-Rad. 0- Phospho-L-homoserine was synthesized according to a method previously published (6); solutions of theO-acylhomoserine esters were prepared as described (6). AVG' was kindly provided to us by Dr. A. Stempel of Hoffman-LaRoche. L-[4CJCystine, L-[3S]cystine, and Na235S] were obtained from New England Nuclear. Preparationof L-[14CJcysteine and L-[35S]cysteine was described earlier (6). Na2[35S] (specific radioactivity, about 12 mCi/mmol) was dissolved in KCI and processed for storage described (8). L-[14CJCystathionine was prepared from L-[UT14C]serine L-homocysteine by the method of Mudd et aL (18). We are grateful to Mr. Henry Richards for samples of ['4CJadenosyl-L-homocysteine and S-adenosylhomocysteine hydrolase which were prepared by the method of de la Haba and Cantoni (11). Plants. Lemna paucicostata Hegelm. was grown as described (7, 9). In most experiments, the plants were harvested and stored frozen at -40°C until used for extraction and assay, as both cystathioniney-synthase andO-phosphohomoserine sulfliydrylase activities were found to be essentially unchanged between fresh and frozen samples. Because freezing the plants was found to diminish 0-acetylserine sulfhydrylase activity, Lemna was used fresh for determination of this enzyme. Methods. 'Abbreviations: AVG, L-aminoethoxyvinylglycine (i.e. L-2-amino-4-(2'aminoethoxy)-trans-3-butenoic acid). General. Paper electrophoresis of enzyme reaction products was performed as described previously (6). Protein was determined by a modification of the method of Bradford (2). Absorbance measurements were converted to protein (JAg) by comparison with a standard curve constructed from measurements performed on stock solutions of BSA. Preparation of Enzyme Extracts. Lemna samples were homogenized in glass tissue grinders in 20 mm Hepes, pH 7.8 (0.75-1.5 Id/frond for most samples, about 3 ,il/frond for plants in which O-acetylserine sulfhydrylase determinations were to be performed). It was not necessary to add agents which protect against inactivation by phenolic compounds. Neither PVP, PEG-4000, borate, Tween 80, diethyldithiocarbamate, caffeine, isoascorbate, BSA, imidazole, metabisulfate, or Amberlite XAD-4 gave significant improvements in activity yields (data not shown). The homogenates were centrifuged at 48,000g at 4°C for 30 to 60 min. The supernatant fluid was immediately desalted into 20 mm Hepes, pH 7.8, on Sephadex G-25 (medium) using a centrifuge technique described by Penefsky (19). The column size used was always 7 to 12 times greater than the sample volume. Protein measurements demonstrated that 75 to 90%o of the protein could be routinely recovered in this fashion and when ['4CJcysteine was present, greater than 98% of the radioactivity, representing unbound small molecules, was removed. The eluates were collected into 1.5-ml polypropylene vials (Eppendorf), and volumes were determined gravimetrically. Using this volume to calculate approximate extracted fronds per id of eluate, it was possible to make a preliminary estimate of protein content (about 3-4 jig of protein/extracted frond) and then to decide the amount of extract to be used in an assay. Assay of Cystathionine y-Synthase. The assay and incubation were performed essentially as described (6). L-[mS]Cystine was substituted for L-['4Cjcystine in some experiments. Between 4 and 10 x 105 dpm were used per assay. To facilitate assay of a large number of samples, the procedure for isolating the product was changed. Each stopped reaction mixture was centrifuged to remove TCA-insoluble material, and cystathionine was isolated by chromatography on a column of Dowex 50-H+ as described previously (18). A suitable aliquot of the NH40H eluate from the column was taken to dryness and dissolved in 200 p1 of performic acid. Each tube was stoppered and placed in a water bath at 30°C for 1 h. Oxidations were terminated by the addition ofld 10 of 48% HBr. After bubbling ceased and the color had turned from orange to yellow, each sample was diluted with 10 ml of H20 and reapplied to the regenerated Dowex 50-H+ column. Each column was washed with 12 ml of H20 to remove cysteic acid, and then the oxidized cystathionine was eluted with 3.9 ml of 2 N NH40H. Cystathionine was determined from the amount of radioactivity in this eluate. Assay ofO-Phosphohomoserine Suflhydrylase. The assay, which involves coupling homocysteine formation to S-adenosylhomocysteine formation by use of S-adenosylhomocysteine hydrolase, was performed as described (8) through the step in which each sample was applied to a Dowex 50-H+ column (2.9 x 0.9 cm). Each column was then washed with 10 ml of H20 and eluted with 4.5 ml of 2 N NH40H. Aliquots of the eluates were taken to dryness and oxidized in performic acid by the procedure described for cystathioniney-synthase but with the volumes scaled upward 2.5-fold. After oxidation, each sample was diluted with 10 ml of H20 and reapplied to regenerated Dowex 50-H+. Each column was then washed with 10 ml of H20 and eluted with 4.5 ml of 2 N NH40H. The amount of S-adenosylhomocysteine (at this point in an oxidized form) produced during the assay incubation was determined from the amount of radioactivity in the eluate. Assay ofO-Acetylserine Suflhydrylase. The assay, which uses Gaitonde's acid ninhydrin reagent (12) to determine cysteine formed, was modified from Chambers and Trudinger(5). The
eaction mixtures contained, in a volume of 1.0 ml, the following (,umol): Hepes, pH 7.8 (200), DTT (5), O-acetylserine (20), freshly prepared Na2S (5), and Lemna extract containing 3 to 40 ,ug of protein. No KCN was added. Incubations were performed in a fume hood in 2-ml-capacity conical bottom plastic analyzer cups by a procedure analogous to the one used for O-phosphohomoserine sulfhydrylase (8). All components except the sulfide were placed in the tubes, and the mixtures were overlaid with 500 pd of paraffin oil. Each reaction was started by adding sulfide solution through the layer of paraffin oil with a micropipet. The tubes were then incubated at 30°C in a Reacti-Therm Heating Module (Pierce Chemical Company) for 15 min. The reactions were terminated by removing a 500-td aliquot from each aqueous reaction mix and adding it to 500 ,ul of glacial acetic acid contained in 12-ml graduated tubes. To each sample was added 500 ,ul of Gaitonde's acid ninhydrin reagent 2 (12). The tubes were covered with glass marbles and placed in a boiling water bath for 10 min. After cooling with cold water and dilution to 10 ml with 95% ethanol, the absorbance of each sample was measured at 560 nm on a Beckman DU spectrophotometer. Measurements were compared to a standard curve from authentic cysteine subjected to the same analysis procedure. RESULTS Validity of Enzyme Assays. Cystathionine y-Synthase. The assay used in these studies differed from an earlier procedure (6) in the method of analysis for cystathionine. Table I shows that, although the radioactivity in the blanks was higher in the new method, significant activity (A) was observed only when both enzyme extract and O-phosphohomoserine were present and that the values obtained by each method were quantitatively similar. Activity was linearly proportional to the amount of extract added up to at least 50 utg protein (data not shown). For the first 15 min of incubation, activity was Table I. A Comparison of Proceduresfor Isolating and Measuring Cystathionine Formed during a Cystathionine y-Synthase Reaction Equal portions of a first ammonia eluate from a complete reaction mix and from a blank (pooled from reactions lacking either the extract or the O-phosphohomoserine) were treated either by the oxidation procedure and second Dowex 50 column described in "Methods," or by the method described previously involving oxidation on paper followed by electrophoresis (6). The radioactivity initially present in each sample before treatment by either method is given under first ammonia eluate. Values indicated under second ammonia eluate are those obtained by the current procedure. Values for the old procedure, given under electrophoresis, were calculated by two methods because authentic samples of ['4C]cystathionine which were added to blank eluate and treated by this procedure gave not only cystathionine sulfone, but also at least two minor unidentified products which move slower than cystathionine sulfone. In the first method, only radioactivity which comigrated with authentic cystathionine sulfone (approximately 20 cm toward the cathode) was counted and recovery was taken into account with a correction factor of 0.70. In the second method (parentheses), all of the radioactivity which moved toward the cathode was counted and a recovery factor of 0.86 was applied. A is the difference in radioactivity between the complete and blank values. Radioactivity Sample First Second ammonia ammonia Electrophoresis eluate eluate dpm Complete 32,118 24,607 20,282 (23,653) Blank 7,425 857 34 (2,735) A 24,693 23,750 20,247 (20,918) ENZYMES OF METHIONINE SYNTHESIS IN LEMNA Table II. A Comparison of Proceduresfor Isolating and Measuring Homocysteine (S-Adenosyihomocysteine) Formed during an 0- Phosphohomoserine Suflhydrylase Reaction Equal portions of a first ammonia eluate from a complete reaction mix and from appropriate blanks lacking either extract, 0-phosphohomoserine, adenosine, or adenosine and hydrolase were treated either by the oxidation and second Dowex 50 column procedure (see "Methods") or by the protocol described previously for oxidation and electrophoresis (8). The radioactivity initially present in each sample before treatment by either method is given under first ammonia eluate. Values designated second ammonia eluate are those obtained by the current procedure. Values from the old procedure, given under electrophoresis, were calculated by two methods because authentic samples of [3Hl- and ["4C]-S-adenosylhomocysteine which were added to a blank eluate and treated by this procedure gave not only a peak that comigrated with oxidized S-adenosylhomocysteine, but also one other peak which moves only slightly from the origin toward the cathode. In the first method, only radioactivity that comigrated with oxidized S-adenosylhomocysteine (6-11 cm toward the cathode) was counted and a correction factor of 0.32 was applied, representing the fraction of [3H1-S-adenosylhomocysteine which migrates in this zone after identical treatment. In the second method (parentheses), all of the radioactivity which migrates toward the cathode was counted and a recovery factor of 0.75 was used. A is the difference in radioactivity between the complete reaction mix and the blank lacking enzyme extract. Radioactivity Sample First Second Electroammoma ammoma eluate eluate phoresis dpm Complete 13,882 4,116 3,283 (3,010) -Enzyme extract 12,282 758 73 ( 844) -O-Phosphohomoserine 11,935 1,822' 228 (1,239) -S-Adenosyl-homocysteine hydrolase 10,964 856 108 ( 229) -Adenosine and S-adenosylhomocysteine hydrolase 7,644 687 NDb A 1,600 3,358 3,210 (2,166) ' In other experiments, this blank gave a value very close to those obtained with the other incomplete reaction mixtures. b ND, not determined. 5: i- I- U.) ULJ en w 0 C.) -J 0 100 80 60 40j 201 l 1079 I 0.25 0.50 0.75 1.00 2.00 METHIONINE, MICROMOLAR FIG. 2. Cystathionine y-synthase (0) and 0-phosphohomoserine sulfhydrylase (A) activities in Lemna grown in the presence of external methionine for 119 h. The activities are presented as percentages of the specific activity measured in a control culture grown and extracted at the same time. also linear with time; incubations for 30 min showed a slight (
Page 1: Plant Physiol. (1982) 69, 1077-1083
Page 5 and 6: Table IV. The Effect of Various Com
Page 7: that of O-phosphohomoserine sulfhyd

Methionine Biosynthesis in Lemna - Plant Physiology

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