Plauta
Planta 139, 119-125 (1978)
9 by Springer-Verlag 1978
Aspartate Kinase and the Synthesis of Aspartate-derived Amino Acids in Wheat Simon W.J. Bright, Peter R. Shewry, and Benjamin J. Miflin BiochemistryDepartment, Rothamsted ExperimentalStation, Harpenden, Herts. AL5 2JQ, U.K.
Abstract. Aspartate kinase (EC 2.7.2.4.) has been purified from 7 day etiolated wheat (Triticum aestirum L. var. Maris Freeman) seedlings and from em-
bryos imbibed for 8 h. The enzyme was 50% inhibited by 0.25 mM lysine. In this study wheat aspartate kinase was not inhibited by threonine alone or cooperatively with lysine; these results contrast with those published previously. In vivo regulation of the synthesis of aspartate-derived amino acids was examined by feeding [l~C]aeetate and [3SS]sulphate to 2 3 day germinating wheat embryos in culture in the presence of exogenous amino acids. Lysine (1 mM) inhibited lysine synthesis by 86%. Threonine (1 mM) inhibited threonine synthesis by 79%. Lysine (1 mM) plus threonine (1 raM) inhibited threonine synthesis by 97%. Methionine synthesis was relatively unaffected by these amino acids, suggesting that there are important regulatory sites other than aspartate kinase and homoserine dehydrogenase. [35S]sulphate incorporation into methionine was inhibited 50% by lysine (2 raM) plus threonine (2 raM) correlating with the reported 50% inhibition of growth by these amino acids in this system. The synergistic inhibition of growth, methionine synthesis and threonine synthesis by lysine plus threonine is discussed in terms of lysine inhibition of aspartate kinase and threonine inhibition of homoserine dehydrogenase. Key words: Aspartate kinase - Feedback regulation - Lysine - Methionine - Threonine - Triticurn.
Introduction
Exogenously supplied lysine and threonine have been shown, in a number of plant systems, to inhibit growth in a synergistic manner. This inhibition is relieved by methionine and its metabolic precursors Abbreviations: AEC=S-(2-aminoethyl)cysteine
homoserine and homocysteine (Dunham and Bryan, 1969; Henke et al., 1974; Bright et al., 1978). This has been interpreted in terms of feedback inhibition of enzymes responsible for the synthesis of aspartatederived amino acids. It has been suggested that the synergistic effect of lysine plus threonine could be due to their cooperative inhibition of aspartate kinase (Dunham and Bryan, 1971; Wong and Dennis, 1973a) shutting off methionine biosynthesis and hence inhibiting growth. The incorporation pattern of radioactivity from [14C]aspartate into soluble and protein amino acids in Marchantia polymorpha has been interpreted in the same way (Dunham and Bryan, 1971). However, the validity of this interpretation has, recently, been questioned in barley and maize (Bright et al., 1978; Miflin, 1977) because although the growth of seedlings of these cereals is inhibited by lysine plus threonine (Green and Phillips, 1974; Bright et al., 1978) the isolated aspartate kinase from both species is not cooperatively inhibited by lysine plus threonine (Cheshire and Miflin, 1975; Shewry and Miflin, 1977; Aarnes, 1977). However, Wong and Dennis (1973b) have reported that aspartate kinase from wheat is cooperatively inhibited by lysine and threonine although the primary effect is by lysine alone. Since there is little difference in the effects of these two amino acids on the growth of wheat, barley and maize seedlings (Green and Phillips, 1974; Bright et al., 1978) we have examined the properties of aspartate kinase from wheat and the pattern of in vivo incorporation of radioactive precursors in order to define more closely the regulation of synthesis of the aspartate-derived amino acids in this important crop plant.
Materials and Methods
Aspartate Kinase
(EC 2.7.2.4.) was extractedfrom 7 day dark grown shoots of wheat (Tricitcum aestivum L. variety Maris Freeman, 1975 harvest) and
0032-0935/78/0139/0119/$01.40
120 assayed by the m e t h o d of Shewry a n d Miflin (1977) except that 0.4 M NaCI was used to etute from DEAE-cellulose. The assay mixture in 1 ml contained 0-3.2 m g protein, 25 m M aspartic acid (K salt), 10 m M MgSO4, 10 m M ATP, 500 m M N H 2 O H . HC1 (pH adjusted to 7.5 with KOH). Wheat embryos for enzyme extraction were prepared by a modification of the method of Johnston a n d Stern (1957) omitting solid CO2. After being imbibed for 8 h on moistened filter paper, the embryos were frozen in liquid nitrogen, ground a n d extracted in 0.1 M K-phosphate buffer p H 7.5 containing 20% glycerol, 10 m M mercaptoethanol, 1 m M EDTA. The purification buffer contained 0.05 M K-phosphate buffer and 5 m M mercaptoethanol with other ingredients as in the extraction buffer.
S.W.J. Bright et al. : Synthesis of Aspartate-derived Amino Acids 100
80
60 o
Wheat embryos were hand dissected from a subsample of the seed used for enzyme extraction by the method of Bright et al. (1978). Embryos were incubated, after sterilisation, on Murashige and Skoog (1962) m e d i u m without hormones, 6 g/1 agar, 5 g/1 sucrose for [l~C]acetate labelling, 30 g/1 for [35S]sulphate labelling. After 3 days on m e d i u m with no amino acids, under growth conditions as described previously (Bright et al., 1978), the plants (shoots 1 5 m m , roots 2-8 mm) were transferred to m e d i u m with amino acid supplements and incubated for 16 h. Radioactive, carrier-free sodium [14C]acetate 4 gl of 100 gCi/ml, 56 m C i / m m o l ; (Radiochemical Centre, A m e r s h a m U.K.) was injected under the scutellum of each plant. K O H , 0.2 ml 20% w/v was placed in a glass centre well and the dishes sealed and incubated for up to 8 h. After 8 h most of the exess solution around the scutellum had been taken up. Plants were rinsed in distilled water, blotted dry and immersed in liquid nitrogen before freeze-drying. K O H was diluted ten-fold before counting in 10 ml of the scintillant of Fricke (1975). After weighing, the dried plants from one petri dish were ground with a small volume of diethyl ether which was allowed to evaporate before the powder was transferred to a tube, extracted three times with diethyl ether a n d hydrolysed under nitrogen for 21 h at l l 0 ~ in 10 ml 6N HC1 and 7 . 2 r a M mercaptoethanol. The hydrolysate was filtered through a glass fibre disc, evaporated to dryness at 40~ taken up in 25 ml HC1 p H 2.5 a n d passed through 3 ml Bio-Rad A G 50-W-X4 resin, H + form. The resin was washed with 10 ml p H 2.5 HC1 then 10 ml water before amino acids were eluted with 7 M a m m o n i a and evaporated to dryness. Methionine was oxidised by performic acid. The sample was dissolved in 4 ml formic acid, cooled to 0~ and 6 ml performic acid reagent added (1 vol 30% v/v or 0.6 vo] 50% H ~ O ~ + 9 or 9.4 vol formic acid, kept at r o o m temperature for 30 min then cooled to 0 ~ C). After 2 h the performic acid was removed by evaporation at 25 ~ C. Samples for chromatography were in a final volume of 0.2 ml. Two dimensional thin layer chromatography on cellulose ( M N 300, Macherey and Nagel) was run with butanol:acetone: diethylamine: triethylamine: water (10 : 10: 1 : 1 : 5) in the first dimension and iso-propanol: formic acid :water (20 : 1 : 5) in the second. A m i n o acids were located under u.v. light after heating the plates for 15 min at 90~ and were scraped from the plates and the radioactivity determined (Davies, 1977). The total counts in each sample was determined by drying an aliquot onto glass fibre paper and counting in the same manner. For [35S]sulphate feeding embryos were grown for 2 days (shoots 3 10 m m , roots 5-20 ram) before transfer, 18.5 h preincubation on a m i n o acids a n d addition of sodium [3SS]sulphate 3 ~xl, (1.02mCi/ml, 1.63 mg/ml). After grinding and extraction in ether, soluble amino acids were extracted by boiling 3 times for 5 rain with 5 ml 75% v/v ethanol. Soluble amino acids, but not the protein hydrolysates, were purified by ion exchange chromatography. Performate oxidation was for 2 h with 10 ml reagent. One dimensional thin layer chromatography
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Planta 139, 119-125 (1978)
9 by Springer-Verlag 1978
Aspartate Kinase and the Synthesis of Aspartate-derived Amino Acids in Wheat Simon W.J. Bright, Peter R. Shewry, and Benjamin J. Miflin BiochemistryDepartment, Rothamsted ExperimentalStation, Harpenden, Herts. AL5 2JQ, U.K.
Abstract. Aspartate kinase (EC 2.7.2.4.) has been purified from 7 day etiolated wheat (Triticum aestirum L. var. Maris Freeman) seedlings and from em-
bryos imbibed for 8 h. The enzyme was 50% inhibited by 0.25 mM lysine. In this study wheat aspartate kinase was not inhibited by threonine alone or cooperatively with lysine; these results contrast with those published previously. In vivo regulation of the synthesis of aspartate-derived amino acids was examined by feeding [l~C]aeetate and [3SS]sulphate to 2 3 day germinating wheat embryos in culture in the presence of exogenous amino acids. Lysine (1 mM) inhibited lysine synthesis by 86%. Threonine (1 mM) inhibited threonine synthesis by 79%. Lysine (1 mM) plus threonine (1 raM) inhibited threonine synthesis by 97%. Methionine synthesis was relatively unaffected by these amino acids, suggesting that there are important regulatory sites other than aspartate kinase and homoserine dehydrogenase. [35S]sulphate incorporation into methionine was inhibited 50% by lysine (2 raM) plus threonine (2 raM) correlating with the reported 50% inhibition of growth by these amino acids in this system. The synergistic inhibition of growth, methionine synthesis and threonine synthesis by lysine plus threonine is discussed in terms of lysine inhibition of aspartate kinase and threonine inhibition of homoserine dehydrogenase. Key words: Aspartate kinase - Feedback regulation - Lysine - Methionine - Threonine - Triticurn.
Introduction
Exogenously supplied lysine and threonine have been shown, in a number of plant systems, to inhibit growth in a synergistic manner. This inhibition is relieved by methionine and its metabolic precursors Abbreviations: AEC=S-(2-aminoethyl)cysteine
homoserine and homocysteine (Dunham and Bryan, 1969; Henke et al., 1974; Bright et al., 1978). This has been interpreted in terms of feedback inhibition of enzymes responsible for the synthesis of aspartatederived amino acids. It has been suggested that the synergistic effect of lysine plus threonine could be due to their cooperative inhibition of aspartate kinase (Dunham and Bryan, 1971; Wong and Dennis, 1973a) shutting off methionine biosynthesis and hence inhibiting growth. The incorporation pattern of radioactivity from [14C]aspartate into soluble and protein amino acids in Marchantia polymorpha has been interpreted in the same way (Dunham and Bryan, 1971). However, the validity of this interpretation has, recently, been questioned in barley and maize (Bright et al., 1978; Miflin, 1977) because although the growth of seedlings of these cereals is inhibited by lysine plus threonine (Green and Phillips, 1974; Bright et al., 1978) the isolated aspartate kinase from both species is not cooperatively inhibited by lysine plus threonine (Cheshire and Miflin, 1975; Shewry and Miflin, 1977; Aarnes, 1977). However, Wong and Dennis (1973b) have reported that aspartate kinase from wheat is cooperatively inhibited by lysine and threonine although the primary effect is by lysine alone. Since there is little difference in the effects of these two amino acids on the growth of wheat, barley and maize seedlings (Green and Phillips, 1974; Bright et al., 1978) we have examined the properties of aspartate kinase from wheat and the pattern of in vivo incorporation of radioactive precursors in order to define more closely the regulation of synthesis of the aspartate-derived amino acids in this important crop plant.
Materials and Methods
Aspartate Kinase
(EC 2.7.2.4.) was extractedfrom 7 day dark grown shoots of wheat (Tricitcum aestivum L. variety Maris Freeman, 1975 harvest) and
0032-0935/78/0139/0119/$01.40
120 assayed by the m e t h o d of Shewry a n d Miflin (1977) except that 0.4 M NaCI was used to etute from DEAE-cellulose. The assay mixture in 1 ml contained 0-3.2 m g protein, 25 m M aspartic acid (K salt), 10 m M MgSO4, 10 m M ATP, 500 m M N H 2 O H . HC1 (pH adjusted to 7.5 with KOH). Wheat embryos for enzyme extraction were prepared by a modification of the method of Johnston a n d Stern (1957) omitting solid CO2. After being imbibed for 8 h on moistened filter paper, the embryos were frozen in liquid nitrogen, ground a n d extracted in 0.1 M K-phosphate buffer p H 7.5 containing 20% glycerol, 10 m M mercaptoethanol, 1 m M EDTA. The purification buffer contained 0.05 M K-phosphate buffer and 5 m M mercaptoethanol with other ingredients as in the extraction buffer.
S.W.J. Bright et al. : Synthesis of Aspartate-derived Amino Acids 100
80
60 o
Wheat embryos were hand dissected from a subsample of the seed used for enzyme extraction by the method of Bright et al. (1978). Embryos were incubated, after sterilisation, on Murashige and Skoog (1962) m e d i u m without hormones, 6 g/1 agar, 5 g/1 sucrose for [l~C]acetate labelling, 30 g/1 for [35S]sulphate labelling. After 3 days on m e d i u m with no amino acids, under growth conditions as described previously (Bright et al., 1978), the plants (shoots 1 5 m m , roots 2-8 mm) were transferred to m e d i u m with amino acid supplements and incubated for 16 h. Radioactive, carrier-free sodium [14C]acetate 4 gl of 100 gCi/ml, 56 m C i / m m o l ; (Radiochemical Centre, A m e r s h a m U.K.) was injected under the scutellum of each plant. K O H , 0.2 ml 20% w/v was placed in a glass centre well and the dishes sealed and incubated for up to 8 h. After 8 h most of the exess solution around the scutellum had been taken up. Plants were rinsed in distilled water, blotted dry and immersed in liquid nitrogen before freeze-drying. K O H was diluted ten-fold before counting in 10 ml of the scintillant of Fricke (1975). After weighing, the dried plants from one petri dish were ground with a small volume of diethyl ether which was allowed to evaporate before the powder was transferred to a tube, extracted three times with diethyl ether a n d hydrolysed under nitrogen for 21 h at l l 0 ~ in 10 ml 6N HC1 and 7 . 2 r a M mercaptoethanol. The hydrolysate was filtered through a glass fibre disc, evaporated to dryness at 40~ taken up in 25 ml HC1 p H 2.5 a n d passed through 3 ml Bio-Rad A G 50-W-X4 resin, H + form. The resin was washed with 10 ml p H 2.5 HC1 then 10 ml water before amino acids were eluted with 7 M a m m o n i a and evaporated to dryness. Methionine was oxidised by performic acid. The sample was dissolved in 4 ml formic acid, cooled to 0~ and 6 ml performic acid reagent added (1 vol 30% v/v or 0.6 vo] 50% H ~ O ~ + 9 or 9.4 vol formic acid, kept at r o o m temperature for 30 min then cooled to 0 ~ C). After 2 h the performic acid was removed by evaporation at 25 ~ C. Samples for chromatography were in a final volume of 0.2 ml. Two dimensional thin layer chromatography on cellulose ( M N 300, Macherey and Nagel) was run with butanol:acetone: diethylamine: triethylamine: water (10 : 10: 1 : 1 : 5) in the first dimension and iso-propanol: formic acid :water (20 : 1 : 5) in the second. A m i n o acids were located under u.v. light after heating the plates for 15 min at 90~ and were scraped from the plates and the radioactivity determined (Davies, 1977). The total counts in each sample was determined by drying an aliquot onto glass fibre paper and counting in the same manner. For [35S]sulphate feeding embryos were grown for 2 days (shoots 3 10 m m , roots 5-20 ram) before transfer, 18.5 h preincubation on a m i n o acids a n d addition of sodium [3SS]sulphate 3 ~xl, (1.02mCi/ml, 1.63 mg/ml). After grinding and extraction in ether, soluble amino acids were extracted by boiling 3 times for 5 rain with 5 ml 75% v/v ethanol. Soluble amino acids, but not the protein hydrolysates, were purified by ion exchange chromatography. Performate oxidation was for 2 h with 10 ml reagent. One dimensional thin layer chromatography
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