BIOLOGIA PLANTARUM (PRAHA) 27 (2--3) : 154--158, 1985 Tryptophan Aminotransferase and Tryptophan Dehydrogenase Activities in some Cell Compartments of Spinach Leaves: the Effect of Calcium Ions on Tryptophan Dehydrogenase KV~TA VACKOV~(, ARC~ANA MEHTA and M. KUT~-CEK Institute of Experimental Botany, Czechoslovak Academy of Sciences, Ke dvoru 15, 166 30 Praha 6, Czechoslovakia Abslracl. The content of spinach-leaf cells was compartmented by diffbrential centrifi~gation. Three fractious were obtained, i.e. chloroplasts, pellet of rernainiug organelles sedirnenting at 97 000 g and cytosol. Enzyme activities of L-tryptophan aminotransferase (TAT) as well as L-tryptophan dehydrogenase (TDH) were demonstrated in all cell fractions. The highest activities of both enzymes were found in the pellet of organelles followed by the enzyme ~ctivities in the chloroplasts. The cytosol had the lowest enzyme activities. Chloroplasts are characterized by a relatively higher TDH activity, organelles sedimenting at 97 000 g were marked by a relatiwdy higher TAT activity. In all frac'tions both pyridine nucleotide coenzymes catalyzed the TDH activity. Ca '-)~ in a concentration of 0.8 m~oI 1-1 increased markedly the T1)H activity in both directions of its activity. There is a general agreement that the main way of auxin synthesis in higher plants is the indolylpyruvate (IPyA) pathway. The introductory reaction can be directly catalysed by two enzymatic systems: L-tryptophan aminotransferase (TAT) (e. g. WI~TMA~" and CO1~E~r 1968, TRUELSEN 1972) and by a newly described L-tryptophan dehydrogenase (TDH) (KuT2[~EK and D~IMov_<, in print). Little is known about the eompartmentation of IAA synthesis in the plant cell and even less about the subcellular localization of the enzymes included in it. In a pioneer study, WIG~T~ and COl~E~- (1968) referred to the mito- chondrial and cytosolic transamination of L-tryptophan (L-trp) in mung bean. Analysing another plant material, NOGVC~ and HArAsm (1980) reported that TAT is localized in spinach peroxisomes and to some extent in the soluble fraction. The positive effect of bivalent ions on glutamate dehydrogenase activity was reported by several authors (i.e. E~rK~ and ]-IAI~TI~IANN ] 976, FAWOLE and BOULTE~ 1977, YA.~k~YAet al. 1984). MATERIAL AND METHODS Plant Material Spinach (,_qpinacia oleracea L., cv. Matador) was grown under field condit- ions. Leaves were harvested from mature rosettes. 154 TRYPTOPHAN AMINOTRANSFERASE AND TRYPTOPHAN DEHYDROGENASE 155 Protoplasma Fractionation The cell content was fractionated into the following fractions: 1) chloroplasts isolated by the procedure of L~s and D~NIS (1981), 2) remaining organelles, 3) eytosol. Isolation of chloroplasts, first cellular fraction: Deribbed leaf sections were cut into chilled isolation medium, pH 6.4 and homogenized in a blendor 3 times for 10 s. The medium for isolation of chloro- plasts consisted of [mmol 1-1]: MES buffer 45; sorbitol 330; NaC1 10; MgC12 1 ; MnC12 ]; Na2EDTA 2; KH2P04 0.5; ascorbate 2; and dithiothreitol 2. The crude homogenate was squeezed through eight layers of nylon net and free draining filtrate was centrifuged (200 g, 2 rain, 4 ~ After discarding the pellet (cellular debris and nuclei), the supernatant was centrifuged (1700 if, 5 min, 4 ~ The ehloroi)last-rich pellet was washed by resuspending in the sorbitol medium and separated by centrifugation (same conditions). Isolation of remaining organelles, second cellular fraction: The fraction Was separated as pellet by centrifugation (97 000 if, 60 rain, --30 ~ of the supernatant after removing the chloroplasts. Cytosol, third cellular fraction: As cytosol we call the supernatant passed through a Sephadex G-15 column for removing sorbitol and other ingredients of the medium used for isolation. The portion of proteins was lyophilized and used for assays. Extraction and Determination of Enzymes and Protein Content Acetone powders were prepared by the method of CEE~ and :BOLL (1968) from pellets of chloroplast and organelles after rupturing them in a hypotonic medium (medium used for isolation without sorbitol). Acetone powder was extracted with 50 mmol 1 ~ tris-HC1 buffer pH 8.5. After 30 min extraction the samples were centrifuged (20 000 g, 15 rain, 4 ~ The supernatant was used as a crude enzyme for testing the activity. Tryptophan aminotransferase: TAT activity was estimated by the method of TRUELSEN (1972). Substrates were L-trp and 2-oxoglutarate. The product of the reaction IPyA, stabilized as a borate complex, was determined spectrophotometricMly at 328 nm. Tryptophan dehydrogenase : TDH activity was determined in both directions of the enzymic reaction -- amination of IPyA and deamination of L-trp ~ on the basis of the oxidation or reduction of the coenzymes NAD(P) or NAD(P)H (KvT~I~ and DIMOV~,0 in print). Protein content of enzyme preparations was estimated by the method using Coomassie brilliant blue G 250 (BR~_DFORn 1976). Effect of Calcium Ions In the enzyme assays CaC12 in a scale of 0.2--1.6 mmol 1-1 final concen- tration was added. Abbreviations used: 2-(N-morpholino) ethanesulfonie acid = ME,q: Na2-ethylenediamino- tetraacetate dihydrate -- NazEI)TA. 2 HeO. 156 K. VACKOV-~ ET AL. TABLE 1 Distribution of tryptophan dehydrogenase and tryptophan aminotransferase specific activities in cell fractions of spinach* TDH Statistical Deamination Amination TAT Fraction value NAD NADP NADH NAI) PH Spec. % of Spec. % of Spec. ~ o of Spec. ~ of Spec. ~ of act. total act. total act. total act. total act,. total Chloroplasts Mean 7.9 32.1 4.1 18.0 19.1 30.3 31.2 24.8 11.5 13.0 SE 0.98 1.10 4.38 4.02 2.81 Organelles Mean 14.0 56.9 15.5 68.0 35.9 57.1 64.8 5].5 54.7 6].7 SE 4.13 4.79 2.89 5.38 5.75 Cytosol Mean 2.7 11.0 3.2 14.0 7.9 12.6 '-)9.8 23.7 22.5 25.3 SE 0.64 0.66 (t.65 1.80 2.80 * Mean is based on 3--6 observatmns; specific activity of TDH is in [nmol (product) rain -1 mg-t (protein)] and TAT in [nmol (IPyA) h -1 mg -I {protein)]; SE = standard error. RESULTS AND DISCUSSION Compartmentation of L-trp Deaminating Enzymes The distribution of TAT and TDH activities compared in units of specific activity in the three tested cellular fractions is shown in Table 1. In the chloroplast fraction both primary enzymes of the IAA synthesis, TAT and TDH, are localized. The role of TDH seems to be much more import- ant, TAT activity in chloroplasts is particularly weak. In the organelle fraction, TAT activity seems to be decisive for auxin bio- synthesis. The activities of both enzymes are in this fraction the highest. In the cytosol are present soluble forms of both enzymes. TAT and TDH activities are relatively weak. Concluding from the distribution of the enzymes deaminating L-trp, the site of the main IPyA synthesis is the fraction of cell organelles, where the activity of TAT is significant. However, in chloroplasts a separate, not negligible IPyA synthesis is proceeding with a prominent role of TDH. The activity of the cytosolic enzymes is inferior. Our results are in agreement with the reported site of TAT activity in spinach peroxisomes found by NoGvcm and Hivism (1980). The results are also in agreement with investigations of another type, i.e. comparing the yield of 14C-IAA formed from radioactive precursor in different cell compart- ments. In the studies of HEILMAN~ et al. (1982) using spinach, and SANDBERG et al. (1982) using barley, chloroplasts and crude cytoplasmic fraction were separated from protoplasts. Both fractions of protoplasts were able to transform L-trp into IAA at approximatively similar rate. FI~EGEAV and WmltTMA~ (1983) compared the biosynthesis of IAA in chloroplasts and in mitochondrial fractions of sunflower. The capacity of IAA biosynthesis was found in both compartments, the amount of IAA and phenylacetic acid synthesized per mg protein being greater in mitochondria than in chloropl asts T[:~YPTOPHAN AMINOTRANSFERASE AND TRYPTOPHAN 1)EHYDROGENASE 157 40 30 -~ 2o 4 Fig. 1. The effect of Ca 2e on tryptophan dehydrogenase activity in deaminating di- rection. Reaction mixture contained in final concentration 6 mmol 1-1 L-tryptophan, 0.16 mmol 1-1 NAD(P), scale of CaC12 and 3 S 7 10 14 18 enzyme prepared from cytosolic fraction. TINE OF SUBCULTURE [d] In the newly described enzyme TDH we could not prove a specific depen- dence on a unique pyridinnucleotide coenzyme as it was reported in the case of microsomal form of indolylacetaldehyde rcductase (BowE~ et al. 1976). Both coenzymes NAD and NADP are active in oxidation of L-trp; in the reduetive process the effect of NADPH compared to NADH was distinctly higher. The Effect of Calcium Ions on the TDH Activity The direct effect of calcium ions on the TDH activity was investigated An increasing activation of spinach TDH in both directions of its activity by Ca 2+ was observed in the range of Ca 2+ concentrations from 0.4 mmol 1 1 of CaC12 up to 1.4 mmol 1~1, with an optimum at 0.8 mmol 1-1 (Fig. 1). The relatively high pH of the enzyme assays could elicit in some cases a partial flocculation at higher concentrations of Ca ~+. The most probable explanation of the stimulating effect of Ca 2+ on TDH is the role of Ca 2+ in the binding of the substrate or eoenzyme to the active site of the enzyme. Another suggestion are conformational changes in the binding site of the enzyme or less specific conformational changes of the apoenzyme molecule. The aim of our work was to contribute to the recognition of the site of auxin biosynthesis in the cell.