T. Betsche, K. Bosbach, and B. Gerhardt Botanisches Institut der Universit~it, Schlol3garten 3, D-4400 Mfinster, Federal Republic of G e r m a n y
Abstract. By ammonium sulfate fractionation and gel filtration an enzyme preparation which catalyzed NAD § L-lactate oxidation (10-4 kat kg- 1 protein), as well as NADH-dependent pyruvate reduction ( 1 0 . 3 katkg -1 protein), was obtained from leaves of Capsella bursa-pastoris. This lactate dehydrogenase activity was not due to an unspecific activity of either glycolate oxidase, glycolate dehydrogenase, hydroxypyruvate reductase, alcohol dehydrogenase, or a malate oxidizing enzyme. These enzymes could be separated from the protein displaying lactate dehydrogenase activity by gel filtration and electrophoresis and distinguished from it by their known properties. The enzyme under consideration does not oxidize D-lactate, and reduces pyruvate to L-lactate (the configuration of which was determined using highly specific animal L-lactate dehydrogenase). Based on these results the studied Capsellaleaf enzyme is classified as L-lactate dehydrogenase (EC 188.8.131.52). It has a Kmvalue of 0.25mmo11-1 (pH7.0, 0.3 mmol 1- 1 NADH) for pyruvate and of 13 mmol 1-1 (pH 7.8, 3 mmol 1-a NAD +) for L-lactate. Lactate dehydrogenase activity was also detected in the leaves of several other plants. Key words:
Capsella L-Lactate dehydrogenase - Leaf
Information regarding the occurrence, properties, and metabolic function of plant lactate dehydrogenases is scarce as compared to that on animal and bacterial lactate dehydrogenases (Everse and Kaplan, 1973). In higher plants, lactate dehydrogenase has only been Abbreviation: F M N = flavin adenine mononucleotide
reported for achlorophyllous tissues. The enzyme has been isolated from potato tubers (Davies and Davies, 1972; Rothe, 1974), roots (Davies et al., 1974; Oba et al., 1977), soybean cotyledons (King, 1970; Barthov~ et al., 1977), and pea seedlings up to an age of 48 h (Barthov~ et al., 1976). The function of the enzyme is seen in relation to the anaerobic metabolism in these tissues. Considering photosynthetically active cells, lactate dehydrogenase has been found in green algae and in a few lower land plants, but could not be detected in the green tissues of higher plants (Gruber et al., 1974). On the other hand, there are a number of reports on the isolation of lactate from the leaves of higher plants (Schneider, 1960). Enzyme reaction(s) forming lactate in leaves are unknown, while the oxidation of lactate may occur by the peroxisomal L-~-hydroxy acid oxidase, gtycolate oxidase (Gruber et al., 1974). In connection with studies on population genetics of Capsella bursa-pastoris (Bosbach, 1979), lactate dehydrogenase activity has now been observed in crude leaf extracts. This paper presents results indicating that this activity is due to an authentic L-lactate dehydrogenase. In addition, some properties of the enzyme, and its occurrence in the leaves of other higher plants, are reported.
Materials and Methods
Plant Material Capsella bursa-pastoris (L.) Med. was collected in the field or grown in a greenhouse. For enzyme preparations, basal leaves of the plants were used. To obtain axenic plants, seeds were surface sterilized and germinated under sterile conditions in Erlenmeyer flasks on 0.8% agar supplemented with K n o p ' s solution at half strength. The axenic cultures were maintained at 20 ~ C and under a regime
T. Betsche et al. : L-Lactate Dehydrogenase from Capsella
568 of 14 h light and 10 h darkness. Before harvesting axenic plants for enzyme preparations, a few plants were withdrawn from each culture and grown separately on 0.8% agar supplemented with 2% sucrose for several days. If microorganisms developed in such a single-plant control, the whole culture, from which the corresponding control plant was selected, was discarded.
Lactuca sativa L., Raphanus sativus L., Spinacia oleracea L., and Zea mays L. were grown in a greenhouse. Leaves of Cardamine hirsuta L., Rubus idaeus L., Taraxaeum officinale Web., and Thlaspi arvense L. were collected in the field.
Preparation of Crude Extracts 30 g of leaves with the petioles and main veins removed were ground in a cooled mortar with quartz and 90 ml of a medium consisting of 50 mmol 1- 1 Tris-acetate, pH 7.4, and 5 mmol 1-1 2mercaptoethanol. The homogenate was filtered through cheesecloth and the filtrate centrifuged at 20,000 g for 20 min. The resulting supernatant was defined as crude extract. To protect lactate dehydrogenase activity from inhibition by phenolic compounds, an altered grinding medium (" anti-phenolic" medium) was used in some cases. This medium was composed essentially as described by Kelley and Adams (1977). In addition, the medium contained insoluble polyvinylpyrrolidone (Polyclar AT; Serva, Heidelberg) soaked in H20 and added to the medium at an amount of 1 g per 2 g of leaf tissue.
Partial Purification of Lactate Dehydrogenase Protein precipitated from the crude extract between 30 and 50% ammonium sulfate saturation was collected by centrifugation (20,000 g, 20 min) and redissolved in 2.5 ml of 50 mmol 1- 1 potassium phosphate, pH 7.0. This solution, which contained 50 to 80% of the lactate dehydrogenase activity of the crude extract, was clarified by centrifugation and ultrafiltration (ultrafilter SM 11308 ; Sartorius, G6ttingen) and then added to a column (80 cm- 5.3 cm 2) of Sephadex G-200 previously equilibrated with 20 mmol I i potassium phosphate, pH 7.0. The protein was eluted with the same buffer. Fractions of 4 ml were collected. The fractions with the highest lactate dehydrogenase activity were pooled and the enzyme solution, normally 40 ml, was concentrated over an ultrafiltration membrane (Diaflo PM10; Amicon, Lexington, USA) to one tenth of its volume. The yield of lactate dehydrogenase activity accounted for about 30% of the activity of the crude extract. All operations were done at 2 to 4 ~ C.
Electrophoresis Analytical electrophoresis was performed on 7.5% polyacrylamide gel slabs. The gels were prepared and the electrophoresis (400 V, 5 h, 0~ C) was carried out essentially according to Loeschcke and Stegemann (1966). The applied samples contained 15 50 pkat lactate dehydrogenase activity where the activity was to be detected with pyruvate as substrate; the activity was raised to 80 500 pkat in cases where the detection medium contained lactate as substrate. The glycolate oxidase activity of the samples varied between 30-300 pkat where the activity was to be detected with glycolate. The methods for detecting enzyme activities were as follows: Lactate dehydrogenase: (A) : With lactate as substrate, the method described by Shaw and Prasad (1970) was used. (B): With pyruvate as substrate, gels wer incubated for 10rain in a solution of 200 mmol l-a potassium phosphate, pH 7.0, 90 mmol 1 ~ pyrurate, and 1.3 mmol I 1 N A D H . Then the gels were immediately viewed under u.v.-light. On sites where N A D H was consumed
the NADH-dependent fluorescence disappeared and, therefore, dark bands on a fluorescent background indicated the location of dehydrogenase activity. Glycolate oxidase: Either the staining procedure described by Grodzinski and Colman (1972) was employed, or method (A) for lactate dehydrogenase detection was used except that the lactate was replaced by 10 mmol 1 I glycolate. Hydroxypyruvate reductase: Method (B) for lactate dehydrogenase detection was employed except that the pyruvate was replaced by 90 mmol i ~ glyoxylate. Malate oxidizing enzymes : The staining medium was as described by Brinkman and Meer (1975),
Identification of the Reaction Product of NADH-Dependent Pyruvate Reduction by Capsella Leaf Enzyme Preparations The reaction mixture for pyruvate reduction contained, in a final volume of 1 ml, 100 pmolpotassium phosphate, pH 7.0, 0.5 gmol [1-14C]pyruvate (533 GBq mol- 1; Amersham Buchler, Braunschweig), 0.3 gmol N A D H , and an aliquot of the partially purified Capsella enzyme preparation, or an aliquot of equal activity of highly purified and dialyzed L-lactate dehydrogenase from pig heart (Boehringer, Mannheim). Pyruvate reduction was allowed to proceed until the added N A D H was consumed. The consumption of the N A D H was followed spectrophotometrically. Then 1 ml of hot ethanol was added to the reaction mixture. Following centrifugation (20,000 g, 10 min), the clear supernatant was placed onto a Dowex50W-X8 (H+-form) cation exchange column (2 cm- 3.1 cm2), and washed in and then eluted with a small volume of HzO. Components of the concentrated elute were separated by thin-layer chromatography on plates coated with cellulose M N 300 (Macherey and Nagel, Dfiren) and using a solvent system 1-butanol : propionic acid : HzO (50:40 : 25). [1-14C]pyruvate, unlabeled pyruvate, L-lactate, and malate as single components or as mixtures were cochromatographed with aliquots of the elute. In the control experiments, a mixture of the acids, or the individual acids, were added to the pyruvate reduction mixture before passing it through the cation exchange column. Organic acids were located on the plates by spraying with 0.1% bromcresol green in weak alkaline ethanol. Radioactive compounds were detected by scanning the chromatograms with a Berthold LB 2723 thin-layer scanner. The radioactive compound formed from [1-14C]pyruvate by the Capsella enzyme preparation or the animal lactate dehydrogenase was eluted, concentrated and rechromatographed in the same solvent system as used before. The rechromatographed labeled reaction product was used as the substrate for the highly purified animal L-lactate dehydrogenase in an assay mixture containing 50 pmol Tris-HC1, ph 9.0, 3 btmol N A D +, 15 gmol hydrazine, and 0.2 mg L-lactate dehydrogenase in a total volume of 1 ml. The reaction was followed spectrophotometrically and stopped by the addition o f 1 ml of hot ethanol when the reduction of the added N A D + began to cease. Then, the reaction mixture was subjected again to thin-layer chromatography as described.
Organelle Isolation 10 g of leaves were chopped and then carefully ground in a mortar in a solution (2 mI per g leaves) containing 1 mol 1-1 sucrose, 170 m m o l l - 1 Tricine, pH 7.5, 1 0 m m o l l - 1 KC1, 1 mmol 1-1 MgC12, 1 mmol 1-1 EDTA, 10 mmol 1-1 2-mercaptoethanol, and 0.9% bovine serum albumine. The homogenate was strained through cheesecloth and centrifuged at 500 g for 10 rain. 7 ml of the resultant supernatant were layered onto linear sucrose density gradients (32 ml, 30 to 60% sucrose). Following centrifugation (Beckman SW 27 rotor, 25,000 rev. rain- 1 ~ 113,000 g . . . .
T. Betsche et al. : L-Lactate Dehydrogenase from
3 h), 1 ml fractions were collected. Each fraction was assayed for
lactate dehydrogenase activity,fumarase, catalase, and chlorophyll. All operations were at 2-4 ~ C.
Assays Enzyme activities were measured spectrophotometrically at 20 ~ C. Lactate dehydrogenase activity was assayed following either N A D § reduction (forward reaction) or N A D H oxidation (reverse reaction) at 340 nm. The reaction mixture for assaying the forward reaction consisted of 50 m m o l 1-1 Tris-HC1, p H 9.0, 60 mmol 1 1 L-lactate, 3 m m o l 1- t N A D + , and 65 m m o l 1 ~ hydrazine. The Km value for lactate was determined using a buffer system of 100 m m o l 1-1 Tris-
HC1, pH 7.8, and without hydrazine in the reaction mixture. The reverse reaction was measured essentially according to Bergmeyer et al. (1974). Hydroxypyruvate reductase (NADH-dependent glyoxylate reductase) was assayed under the same conditions as lactate dehydrogenase in the reverse reaction, except 43 mmol i- 1 glyoxylate replaced the pyruvale. NADPH replaced NADkI when the activity of the NADPH-dependent glyoxylate reductase was determined. Glycolate oxidase was assayed as described by Gerhardt (1974). Alcohol dehydrogenase activity was measured with ethanol (Bergmeyer et al., 1974) or acetaldehyde (Wignarajah and Greenway, i976) as substrate. Catalase was assayed according to Gerhardt and Beevers (1970), and fumarase as described by Racker (1950). Chlorophyll was determined according to Arnon (1949), and protein by the Lowry method.
Front Start Fig. 1A and B. Radiochromatograms from reaction mixtures in which [1-14C]pyruvate was reduced by the partially purified enzyme .
preparation from Capsellaleaves (A) or by animal L-lactate dehydrogenase (B). The identification of the compounds I-IV is described in the text Results and Discussion
Crude extracts from leaves of Capsella bursa-pastoris catalyze a low but measurable N A D H - d e p e n d e n t reduction of pyruvate. Partial purification of this enzyme activity increased its specific activity ten-fold to 10-3 kat kg-1 protein. When assayed with L-lactate as substrate, the partially purified enzyme was found to have an activity of 10 -4 kat kg -1 protein. The enzyme preparation showed no activity with Dlactate. The rate of pyruvate reduction proceeded 12 times faster with N A D H (0.3 m m o l 1- 1) than with N A D P H (0.3 m m o l 1 1). Leaves from axenic cultures of Capsella bursa-pastoris had the same enzymatic capacity to reduce pyruvate with N A D H as leaves from plants grown under field or greenhouse conditions. Thus, the enzyme activity under consideration did not result from bacterial or fungal infection of the leaves.
Identification of the Reaction Product Incubation of partially purified enzyme with [1-14C]pyruvate and N A D H , and separation of the deproteinized reaction mixture by thin-layer chromatography, resulted in a r a d i o c h r o m a t o g r a m as shown in Fig. 1 A. An identical r a d i o c h r o m a t o g r a m was ob-
tained in a parallel experiment in which the Capsella enzyme was replaced by highly purified animal L-lactate dehydrogenase and in which the reaction was allowed to consume an equal amount of N A D H (Fig. 1 B). C o c h r o m a t o g r a p h y experiments (see Materials and Methods) showed that radioactive compound I (for numbering of the radioactive compounds compare Fig. 1) of the separated reaction mixture and lactate, and pyruvate and radioactive c o m p o u n d III, had identical Rf values (0.73 and 0.47, respectively). C o m p o u n d IV (Rf 0.33) was an impurity in the commercial [1-14C]pyruvate preparation and not a product of the enzyme reactions. Radioactive compound II (Rf 0.53) was not identified by the cochrom a t o g r a p h y experiments. No radioactive malate was formed during the reaction catalyzed by the Capsella leaf enzyme preparation. C o m p o u n d I (lactate), formed by the Capsella leaf enzyme preparation, served equally well as substrate for the highly stereospecific animal L-lactate dehydrogenase as did the product (compound I) formed previously from [1-14C]pyruvate by the animal enzyme itself. The initial rates of N A D + reduction followed spectrophotometrically were comparable with both substrates, the concentration of which were adjusted based on their radioactivity. The radiochromatogram
T. Betsche et al. : L-Lactate Dehydrogenase from
u O O
Fig. 2A. Radiochromatogram from a reaction mixture in which rechromatographed compound I served as substrate for animal Llactate dehydrogenase. Compound V is the product of this reaction. Compound I was previously formed from [1-14C]pyruvate by the partially purified enzyme preparation from Capsella leaves (Fig. 1). B As (A) except that the compound I was previously formed from [1-14C]pyruvate by animal L-lactate dehydrogenase
was identical for both reaction mixtures (Fig. 2); the Re value (0.47) of the reaction products (compounds V) corresponded to that of pyruvate. The results of the outlined experiments demonstrate that pyruvate is reduced to lactate by the Capsella leaf enzyme and indicate that this lactate has L-configuration.
Proof of Lactate Dehydrogenase The lactate dehydrogenase activity demonstrated for Capsella leaves may be attributed to the presence of lactate dehydrogenase. Since, up to now, the occurrence of this enzyme has not been reported for the photosynthetically active tissues of higher plants, the interpretation can be questioned. However, in the following, strong evidence is provided that the studied lactate dehydrogenase (pyruvate reductase) activity is associated with a specific protein which belongs to the group of lactate dehydrogenases and that the activity is not due to a known leaf enzyme with specifity for substrates "structurally related to lactate and pyruvate, respectively. The partially purified enzyme preparation from Capsella leaves contained glycolate oxidase, a leaf enzyme known to oxidize L-lactate (Zelitch and Ochoa, 1953 ; Frederick et al., 1973). But as an oxidase, glyco-
late oxidase transfers electrons to oxygen via its more or less tightly bound FMN. Under reaction conditions measuring the enzyme activity as a dehydrogenase, only artificial two electron acceptors are reduced, but not NAD § . The enzyme preparation studied here, on the other hand, transfers electrons to NAD + and then only from L-lactate, but not from glycolate: In the presence of 3 mmol 1-1 NAD § and at pH 9.0, the pH optimum of L-lactate oxidation, glycolate (60 mmol 1- l) was not oxidized by the enzyme preparation whether hydrazine was included in the reaction mixture or not. This result would not be expected if a glycolate oxidase was present in the leaves of Capsella which could use NAD-- as electron acceptor. In addition, glycolate oxidase does not catalyze a reverse reaction (Zelitch and Ochoa, 1953), i.e., in this case a pyruvate reduction. Also, 2-pyridyl-hydroxymethanesulfonate, an inhibitor of glycolate oxidase (Zelitch, 1959; Corbett and Wright, 1971), had no effect on the lactate dehydrogenase activity (whether the forward or reverse direction was measured) at a concentration of 2.5 gmol 1- 1 which inhibited completely the glycolate oxidase activity of the preparations. That the studied lactate dehydrogenase activity cannot be attributed to glycolate oxidase is also evident from the following results: On a Sephadex G200 column, the elution profiles of glycolate oxidase and lactate dehydrogenase activity are not identical (Fig. 3), and both activities were separated by electrophoresis. Electrophoresis was performed with fractions of the Sephadex G-200 elute containing high glycolate oxidase activity besides the lactate dehydrogenase activity (e.g. fractions 47 50 of the experiment shown in Fig. 3). When the polyacrylamide gels were stained in a detection medium containing glycolate as the enzyme substrate and dichlorophenolindophenol as the electron acceptor (Grodzinski and Colman, 1972), two bands of enzyme activity with extremely low electrophoretic mobility were obtained. In addition to these, a third band of somewhat higher electrophoretic mobility was observed when nitroblue tetrazolium was used as the electron acceptor (Fig. 4). This third band had an atypical redish color and was also observed when glycolate was absent from the staining mixture, therefore, it is considered to be unrelated to the studied problem. The band may be due to the presence of fraction-1 protein in the enzyme preparation (O'Sullvian and Wedding, 1972). In positions identical to the two specific bands of glycolate oxidizing activity, enzyme activity was observed when L-lactate was used as substrate in the staining mixture. However, replacing the glycolate of the detection medium by L-lactate also resulted in the appearance of two additional activity bands with
T. Betsche et al. : L-Lactate Dehydrogenase from
.2 -0,6 1,0= ".2
Fig. 3. Elution pattern of glycolate oxidase ( o - o), lactate dehydrogenase activity (8 - Q), NADH-dependent glyoxylate reductase (A-zx), and NADPH-dependent glyoxylate reductase (A--A) during filtration, on Sephadex G-200, of the 30 50% ammonium sulfate fraction from a Capsella leaf extract. Activity 1 corresponds to 16.7 nkat
much higher electrophoretic mobility (Fig. 4); the one with the tower Re value was judged to be the main band of lactate oxidizing activity. The band with the g r e a t e r R f value was only demonstrable if very high lactate dehydrogenase activity was applied to the gels. Also, only when a large amount of enzyme was applied were the bands of glycolate oxidizing activity detectable with L-lactate as the enzyme substrate. Since glycolate as well as L-lactate are oxidized by glycolate oxidase (Zetitch and Ochoa, 1953; Frederick et al., 1973), the activity bands detectable with either of these substrates indicate the location of this enzyme on the gels. The other two activity bands, demonstrable only with L-lactate, are the expression of enzyme protein(s) different from glycolate oxidase. The separation, by electrophoresis, of the lactate dehydrogenase activity from the glycolate and lactate oxidizing enzyme excludes not only glycolate oxidase as the enzyme responsible for the studied lactate dehydrogenase activity but also glycolate dehydrogenase. This enzyme, functionally analogous to glycolate oxidase, has been found mainly in algae but also in two higher plants (Tolbert, 1976). The protein exhibiting lactate dehydrogenase activity also differs from glycolate dehydrogenases with respect to the fact that the latter oxidize D-lactate which does not serve as substrate for the former, rather than L-lactate (Frederick et al., [973), and that glycolate dehydrogenases are inhibited by 2 mmol l-1 KCN (Frederick et al., 1973) and by sulphonates (Tolbert, 1974) while the studied enzyme is insensitive to these compounds.
Fig, 4a and b, Separation of glyco/ate oxidase and lactate dehydrogenase by electrophoresis on polyacryIamide gel. Different aliquots of the fraction, contaimng the highest glycolate oxidase activity, of a Sephadex G-20O e|ute (compare Fig. 3) were applied to the gel. Staining of the gel with nitroblue tetrazolium and glycolate (a) or L-lactate (b)
The enzyme preparation from the leaves of Capsella enriched in lactate dehydrogenase (pyruvate reductase) activity was not free from NADH-dependent glyoxylate (hydroxypyruvate) reductase activity. Since hydroxypyruvate reductase does not use pyruvate (or lactate) as a substrate (Zelitch, 1953 ; Holzer and Holldorf, 1957; Laudahn, 1963; Kohn and Warren, 1970) and, further, is competitively inhibited by pyruvate (Holzer and Holldorf, 1957; Kohn and Warren, 1970), the pyruvate reductase activity of the leaves should not be due to this enzyme. In fact, the activities for glyoxylate and pyruvate reduction differed in their elution profiles from a Sephadex G-200 column (Fig. 3). Electrophoresis of a fl'action of the Sephadex G-200 elute, which contained equal activities of glyoxylate reductase and pyruvate reductase (e.g., fraction 60 of the experiment shown in Fig. 3), resulted in different band patterns for glyoxylate reductase and pyruvate reductase activity (Fig. 5). The sole band of pyruvate reductase activity always had a very slightly higher mobility than the faster moving band
T. Betsche et al.: L-Lactate Dehydrogenase from Capsella
result indicates that the studied enzyme activity was not due to an unspecific reaction catalyzed by an NAD-dependent malate oxidizing enzyme such as the NAD-dependent malate dehydrogenase and the NAD-
Fig.5a and b. Separation of glyoxylate reductase and pyruvate reductase by electrophoresis on polyacrylamide gel. Aliquots of a fraction, containing equal activity for both subtrates, of the Sephadex G-200 elute (compare Fig. 3) were applied to the gel. Staining of the gel with pyruvate (a) or glyoxylate (b) as substrate
of glyoxylate reductase activity. Thus, hydroxypyruvate reductase could be distinguished from the protein with pyruvate reductase activity. The inhibition of hydroxypyruvate reductase by pyruvate was also demonstrated on the gels. In this case, the gels were stained in a medium which contained pyruvate as well as glyoxylate as substrate (90 mmol 1-t each). Only one band of activity appeared on the gels. It had a position identical to that of the enzyme activity obtained with pyruvate as the sole substrate. Concerning the chloroplastic NADPH-dependent glyoxylate reductase, no substantial activity was detectable in the partially purified enzyme preparation from Capsella leaves. But more important, pyruvate is no substrate for the NADPH-dependent glyoxylate reductase (Zelitch and Gotto, 1962), and this enzyme is highly specific for NADPH (Zelitch and Gotto, 1962; Tolbert et al., 1970) which was a poor reductant in the studied enzyme reaction. Following electrophoresis of the dialyzed 30-50% ammonium sulfate fraction from CapselIa leaves, gels were stained by the nitroblue tetrazolium method in the presence of malate or lactate as substrate and NAD § as oxidant. The patterns of malate and lactate oxidizing activities were different. This
Following electrophoresis of fractions of the Sephadex G-200 elute and staining the gels for pyruvate reductase activity, one band of enzyme activity appeared on the gels. In contrast, two bands specific for lactate dehydrogenase activity were observed. To prove whether the lactate oxidizing activity and the pyruvate reductase activity of the Capsella leaf enzyme preparation are due to the same protein, gels of identical runs were stained either for lactate oxidizing or for pyruvate reductase activity (Fig. 6). The band of pyruvate reductase activity appeared on the gel in a position identical to the location of the strong colored band of lactate oxidizing activity. A counterpart to the weak band of lactate oxidizing activity was not observed after staining the gel for pyruvate reductase activity. However, there are plausible reasons, resulting from the different staining methods, why a second band of pyruvate reductase activity may not be observed. To detect activity for lactate oxidation, the gels were incubated in the staining medium for several hours and thereafter the color intensity of the second activity band was still low. Since a non-diffusible reaction product is formed during staining for lactate oxidation, a certain overloading of the gels which still gives distinguishable bands was also possible. On the other hand, staining for pyruvate reductase activity resulted in a blurred band due to the rapid diffusion of NADH from the surroundings to the reaction site and the resulting decrease in fluorescence around the reaction site proper. Therefore, bands closely located cannot be distinghuished and only low protein concentrations can be loaded onto the gel to minimize rapid band broadening. When alcohol dehydrogenase was assayed either with ethanol or with acetaldehyde as substrate, no activity was detected in the Capsella leaf enzyme preparation. This result excludes alcohol dehydrogenase as the source of the leaf lactate dehydrogenase activity.
Properties of the Lactate Dehydrogenase The studies on some properties of the leaf lactate dehydrogenase were performed using the partially purified protein. With standard assay conditions, the pH optimum for pyruvate reduction was 7.0 and that for lactate oxidation 9.0 (Fig. 7). Preliminary kinetic studies did not show deviations from Michaelis-Menten kinetics. At pH 7.0 and in the presence of
T. Betsche et aI. : L-Lactate Dehydrogenase from Capselta
pH Fig. 6a and b. Electrophoresis of partially purified Capsetta leaf lactate dehydrogenase and of crude extract from Lactuca leaves. Staining of the gels with L-lactate (a) or pyruvate (b). Starting from the left, Capsella enzyme and Lactuca crude extract were applied alternately to the lanes of the gel. Concerning the Lactuca crude extract, the band with the lowest mobility is due to an unspecific staining Table 1. Lactate dehydrogenase (LDH) activity of crude leaf extracts. The extracts were prepared using the "anti-phenolic" homogenisation medium. The activity was assayed with pyruvate as substrate and is expressed as nkat g 1 fresh weight Species
0.3 m m o l 1- 1 N A D H , the K m value for pyruvate was 0 . 2 5 m m o l l - t . It increased to 0.78 m m o l t - 1 at p H 7.8; at this p H the Km value for L-lactate was 13 m m o l 1- 1 in the presence of 3 m m o l 1- a N A D +. The K m value for L-lactate was determined at p H 7.8 in order to determine it at a p H more physiologically relevant than that of the p H optimum of lactate oxidation. Under standard assay conditions, the enzyme did not show substrate inhibition by pyruvate or L-lactate (at concentrations up to 6 m m o l 1- 1 and 100 m m o l 1-1, respectively). As reported for animal (Everse and Kaplan, 1973) and plant (Davies and
Fig. 7. Influence of pH on the activity of Capsella leaf lactate dehydrogenase. At pH 9.2 100 mmol 1-1 pyrophosphate, o - o : Reaction measured as lactate oxidation; 100% activity correspond to 0.5 nkat/reaction mixture. 9 9 Reaction measured as pyruvate reduction; 100% activity correspond to 6.7 nkat/reaction mixture Davies, 1972) L-lactate dehydrogenases A T P inhibited pyruvate reduction; 0.25 m m o l 1- 1 A T P reduced the reaction rate by 60%. The enzyme was not found associated with either chloroplasts, mitochondira, or microbodies from the leaves of CapseIla. At present, the enzyme is considered to be a component of the cytosol.
Lactate Dehydrogenase Activity in Leaves of Other Plants When crude extracts from leaves of the plants listed in Table 1 were tested for their capability to catalyze a N A D H - d e p e n d e n t pyruvate reduction, only extracts from Lactuca sativa and Capsella bursa-pastoris showed such an activity. Following gel electrophoresis of the crude lettuce leaf extract, lactate oxidation could also be demonstrated. Figure 6 shows a comparison of the pattern of lactate dehydrogenase activity from leaves of Lactuca and Capsella. By replacing the standard homogenisation medium with the " a n t i - p h e n o l i c " medium (see Materials and Methods), pyruvate reductase (lactate dehydrogenase) activity could also be demonstrated in crude extracts from the leaves of several further plants (Tab. 1). Concerning Capsella and Lactuca, homogenisation of the leaves in the '~anti-phenolic" medium resulted in a lactate dehydrogenase activity about
30% lower than that obtained with the standard homogenisation medium. Conclusion
Based on the outlined results and for the reasons discussed, we conclude that the lactate dehydrogenase activity of leaves from Capsella bursa-pastoris and the other investigated plants is due to a protein which has to be classified as lactate dehydrogenase. More specifically, this enzyme belongs to the group of L-lactate dehydrogenases (EC 184.108.40.206) since it does not oxidize D-lactate and reduces pyruvate to L-lactate. The kinetic properties, as far as determined at present, of the CapselIa leaf L-lactate dehydrogenase agree well with those reported for L-lactate dehydrogenases from achlorophyllous tissues of higher plants (Davies and Davies, 1972; Rothe, 1974; Barthovfi et al., 1976) and those reported for the extensively studied mammalian L-lactate dehydrogenases (Everse and Kaplan, 1973). References Arnon, D.I. : Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (1949) Barthovfi, J., Borvfik, J., Leblovfi, S.: Isolation and properties of lactate dehydrogenase from germinating pea plants. Phytochemistry 15, 75 77 (1976) Barthovfi, J., Wilhelmovfi, N., Leblov~t, S. : The regulation of lactate dehydrogenase activity in soy-bean seedlings. Biol. Plant. 19, 190 195 (1977) Bergmeyer, H.U., Gawehn, K., Gral31, M.: Enzyme als biochemische Reagentien. In: Methoden der enzymatischen Analyse, vol. 1, S. 454-558, Bergmeyer, H.U., ed. Weinheim: Verlag Chemic 1974 Bosbach, K. : Enzympolymorphismus in nattirlichen Populationen des Hirtentfischelkrautes. Ein Beitrag zur Biosystematik der Gattung Capsella (Brassicaceae). Thesis, Univ. Mfinster 1979 Brinkman, F.G., Meer, L.J. yon der: Dehydrogenases in the potato tuber (Solanum tuberosum). Identity, coenzyme-specifity and isoenzyme composition of malic enzyme, malate dehydrogenase and lactate dehydrogenase. Z. Pflanzenphysiol. 75, 322-331 (1975) Corbett, J.R., Wright, B.J.: Inhibition of glycollate oxidase as a rational way of designing a herbicide. Phytochemistry 10, 2015 2024 (1971) Davies, D.D., Davies, S. : Purification and properties of L(+)-lactate dehydrogenase from potato tubers. Biochem. J. 129, 831 839 (1972) Davies, D.D., Grego, S., Kenworthy, P. : The control of the production of lactate and ethanol by higher plants. Planta 118, 297 310 (1974) Everse, J., Kaplan, N.O.: Lactate dehydrogenases: structure and function. Adv. Enzymol. 37, 61-133 (1973) Frederick, S.E., Gruber, P.J., Tolbert, N.E. : The occurrence of glycolate dehydrogenase and glycolate oxidase in green plants. Plant Physiol. 52, 318 323 (1973) Gerhardt, B.: Studies on the formation of glycolate oxidase in developing cotyledons of Helianthus annuus L. and Sinapis alba L. Z. Pflanzenphysiol. 74, 14-21 (1974) Gerhardt, B., Beevers, H. : Developmental studies on glyoxysomes in Ricinus endosperm. J. Cell Biol. 44, 94 102 (1970)
T. Betsche et al. : L-Lactate Dehydrogenase from Capsella Grodzinski, B., Colman, B. : Disc electrophoresis of glycollate oxidizing enzymes. Phytochemistry 11, 1281-1285 (1972) Gruber, P.J., Frederick, S.E., Tolbert, N.E.: Enzymes related to lactate metabolism in green algae and lower land plants. Plant Physiol. 53, 167 170 (1974) Holzer, H., Holldorf, A.: Isolierung yon D-Glycerat-dehydrogenase, einige Eigenschaften des Enzyms und seine Verwendung zur enzymatisch-optischen Bestimmung yon Hydroxypyruvat neben Pyruvat. Biochem. Z. 329, 292 312 (1957) Kelley, W.A., Adams, R.P.: Preparation of extracts from juniper leaves for electrophoresis. Phytochemistry 16, 513-516 (1977) King, J. : The isolation, properties, and physiological role of lactic dehydrogenase from soy-bean cotyledons. Can. J. Bot. 48, 533-540 (1970) Kohn, L.D. Warren, W.A.: The kinetic properties of spinach leaf glyoxylic acid reductase. J. Biol. Chem. 245, 3831-3839 (1970) Laudahn, G.: Vergleichende Untersuchungen zum Umsatz yon Pyruvat, Hydroxypyruvat und Glyoxylat durch die NAD-Oxydoreduktasen Lactat-Dehydrogenase, Glyoxyls/iure-Reduktase und D-Glycerat-Dehydrogenase. Biochem. Z. 337, 449-461 (1963) Loeschcke, V., Stegemann, H.: Polyacrylamid-Elektrophorese zur Beurteilung yon Proteinen der Kartoffel (Solanum tuberosum L.). Z. Naturforsch. 21b, 879 888 (1966) Oba, K., Murakami, S., Uritani, J. : Partial purification and characterization of L-lactate dehydrogenase isozymes from sweet potato roots. J. Biochem. 81, 1193-1201 (1977) O'Sullvian, S.A., Wedding, R.T.: Malate dehydrogenase isoenzymes from cotton leaves. Molecular weights. Plant Physiol. 49, 117 123 (1972) Racker, E.: Spectrophotometric measurement of the enzymatic formation of fumaric and cis-aconitic acid. Biochim. Biophys. Acta 4, 211 214 (1950) Rothe, G.M. : Catalytic properties of three lactate dehydrogenases from potato tuber (Solanum tuberosum). Arch. Biochem. Biophys. 162, 17-21 (1974) Schneider, A. : Milchs/iure in h6heren Pflanzen. In: Encyclopedia of Plant Physiology, vol. XII, pt. 1, pp. 1009-1022, Ruhland, W., ed. Berlin, Heidelberg, New York: Springer 1960 Shaw, C.R., Prasad, R. : Starch gel electrophoresis - a compilation of recipes. Biochem. Genet. 4, 297 320 (1970) Tolbert, N.E.: Photorespiration. In: Algal Physiology and Biochemistry, pp. 474-504, Stewart, W.D.P., ed. Oxford, London, Edinburgh, Melbourne: Blackwell Scientific Publications 1974 Tolbert, N.E.: Glycollate oxidase and glycollate dehydrogenase in marine algae and plants. Aust. J. Plant Physiol. 3, 129-132 (1976) Tolbert, N.E., Yamazaki, R.K., Oeser, A. : Localization and properties of hydroxypyruvate and glyoxylate reductases in spinach leaf particles. J. Biol. Chem. 245, 5129-5136 (1970) Wignarajah, K., Greenway, H. : Effect of anaerobiosis on activities of alcohol dehydrogenase and pyruvate decarboxylase in roots of Zea mays. New Phytol. 77, 575-584 (1976) Zelitch, I. : Oxidation and reduction of glycolic and glyoxylic acids in plants. II. Glyoxylic acid reductase. J. Biol. Chem. 201, 719-726 (1953) Zelitch, I.: The relationship of glycolic acid to respiration and photosynthesis in tobacco leaves. J. Biol. Chem. 234, 3077 3081 (1959) Zelitch, I., Gotto, A.M. : Properties of a new glyoxylate reductase from leaves. Biochem. J. 84, 541 546 (1962) Zelitch, I., Ochoa, S.: Oxidation and reduction of glycolic and glyoxylic acids in plants. I. Glycolic acid oxidase. J. Biol. Chem. 201,707 718 (1953)
The chlorogenic acids, chlorogenic acid glycosides and flavonoids of the leaves of Lonicera henryi L. (Caprifoliaceae) were investigated qualitatively by liquid chromatography tandem mass spectrometry. Thirty-one chlorogenic acids and their glycoside
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Juvenile hormones have attracted attention as safe and selective targets for the design and development of environmentally friendly and biorational insecticides. In the juvenile hormone III biosynthetic pathway, the enzyme farnesol dehydrogenase cata
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In order to isolate high yields of pyrenoids from the brown alga Pilayella littoralis it is necessary to pretreat them with 0.1% HgCl2 in sea water for 3 h. Without this pretreatment there is a substantial loss of pyrenoid ground substance and yields
This research was carried out to determine biochemical properties of β-glucosidase (β-D-glucoside glucohydrolase, EC 18.104.22.168) isolated from Turkish tea leaves. Two protein peaks containing β-glucosidase activity were recovered and characterized, whi