Acylhydrolases from parsley (Petroselinum hortense). Relative distribution and properties of four esterases hydrolyzing malonic acid hemiesters of flavonoid glucosides

ARCHIVESOF BIOCHEMISTRYAND BIOPHYSICS Vol. 224, No. 1, July 1, pp. 261-271, 1933

Acylhydrolases from Parsley (Petroselinum hortense). Relative Distribution and Properties of Four Esterases Hydrolyzing Malonic Acid Hemiesters of Flavonoid Glucosides’ ULRICH Znstitut fiir Biologic

MATERN

ZZ, Lehrstuhl fiir Biochemie ckr Pjlanzen, Universitiit 1, 07800 Freiburg, West Germany

F&burg,

Schiinzlestr.

Received November 19, 1982, and in revised form February 1’7,1983

In parsley, malonylated flavonoid glycosides are formed in response to ultraviolet irradiation and accumulate in the vacuoles. Involvement of malonyltransferases, which catalyze the transfer of malonic acid from malonyl-coenzyme A to either flavone/ flavonol’7-0-glucosides or flavonol3-0-glucosides, has been described previously. These enzymes are present in very young leaf buds, and their activities decrease rapidly when leaves begin to unfold, while at the same time esterase activity is developing. The latter enzyme activity continues to increase with tissue age. Four esterases, distinguished by PI’S of 3.8,3.9,4.0, and 4.05, were purified to apparent homogeneity from parsley leaves and shown to hydrolyze malonic acid hemiesters of flavonoid glucosides. These esterases are unspecific and are best described as one acetyl- and three arylesterases on the basis of inhibition studies by 4-chloromercuribenzoic acid and diisopropyl fluorophosphate. Esterases and malonic acid hemiesters appear to be separated from each other within the parsley leaf cell, and only on disruption of the cells do the respective substrates become available to the enzymes. Involvement of esterases in formation of wound periderm in parsley plants is suggested.

Enzymes hydrolyzing carboxylic esters appear to be widely distributed in nature and have been isolated frequently from bacteria (l), fungi (2), higher plants (3), and mammalian tissue (4). Besides the lipid acylhydrolases (5, 6), carboxylesterases, arylesterases, and acetylesterases have been differentiated (7, 8) on the basis of their preferred substrates and by their differential inhibition by mercury compounds or phosphate esters. With the exceptions of pectinesterase and an esterase involved in polyacetylene metabolism (9) all esterases so far isolated from plant sources are relatively nonspecific, but usually show some preference with respect to 1This work was supported by Deutsche Forschungsgemeinschaft and by Wissenschaftliche Gesellschaft Freiburg. 261

the acyl moiety of the substrates. Due to the fact that natural substrates have not been defined, no physiological function for plant esterases is apparent. Dicarboxylic acid diesters, on the other hand, are selectively degraded by esterases from plant, fish, or mammalian sources. Phthalic acid esters are hydrolyzed in fish and mammalian liver to the hemiesters prior to conjugation to glucuranic acid (10-12). Similarly, only the 12ester bond of phorbol diacetate was cleaved by murine esterases (13). In wheat and maize, selective hydrolysis of one of the two acetylester bonds of malaoxone was reported (14, 15). Using artificial substrates, Levy and Ocken (16) demonstrated that charged esters in general were very poor substrates for aryl-, acetyl-, or carboxy1 esterases. To date, no esterase from 0003-9861/83 $8.00 Copyright All rights

Q 1983 by Academic Press, Inc. of reproduction in any form reserved.

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a plant source has been shown to hydrolyze hemiesters of dicarboxylic acids. Numerous malonic acid hemiesters of pglucosides have been isolated from plants (17-26). Such malonylglucosides are resistant to glucosidases like almond emulsin (27). From uv-irradiated parsley cell cultures about 85% of the total flavonoid glycosides were isolated as their respective malonic acid conjugates (20), and two flavonoid specific malonyltransferases were purified from these cells (28). Since the malonylglucosides are labile, it has been suggested that all the flavonoids were present in the cells as malonic acid hemiesters (20). Recently, we have discovered that malonylated flavonoids are exclusively located within the vacuoles of protoplasts derived from irradiated parsley cell cultures (29). Davenport and DuPont (30) reported that malonylated flavonoid glycosides could also be isolated from parsley plants, if the plant material was extracted with boiling water. Unboiled plant extract, on the other hand, liberated malonic acid from the partially purified flavonoid fraction. These results suggest that some esterase activity active toward malonic hemiesters is present in parsley plants, but absent from parsley cell cultures. In the plant, esterases must be localized in a separate cell compartment from the malonylglucosides or be inactive in the presence of these glucosides. Moreover, such esterase activity must be strictly separated from the malonyltransferase activities which synthesize the respective flavonoid malonic acid conjugates (28), to avoid futile cycling. In this report we describe the isolation of four esterases hydrolyzing malonic acid hemiesters of either flavone 7-O-glucosides or flavonol3-0-glucosides. These esterases do not completely fit either one of the esterase categories suggested by Holmes and Masters (31), but are best described as an acetylesterase and three arylesterases. Their subunit composition and their distribution, as well as the distribution of flavonoid glycoside-specific malonyltransferases in parsley plants, is demonstrated.

MATERN MATERIALS

AND

METHODS

Chemicals and materials. Analytical grade chemicals were purchased from Sigma, Munich (cy-naphthy1 acetate, a-naphthyl propionate, cY-naphthyl myristate), Merck, Darmstadt (a-naphthol, malonic acid), Serva, Heidelberg (diisopropyl fluorophosphate (DFP): 97%, 4-ehloromercuribenzoic acid, sodium salt (PCMB), UDP-glucose), and Roth, Karlsruhe (methyl cY-D-mannoside). Radioactive substrates were from New England Nuclear, Dreieich ([1,3-‘%]malonylcoenzyme A, 51.6 Ci/mol), and from our collection ([2-%]apigenin, 2 Ci/mol). Apigenin 7-0-glucoside was purified from a commercially available mixture (Roth, Karlsruhe) as described (31). Isorhamnetin 3-0-glueoside was kindly provided by H. Wagner, Munich. Materials and equipment for enzyme purification were from Pharmacia, Freiburg (Con A-Sepharose, phenyl-Sepharose, Sephadex LH-20, ampholytes Pharmalyte, pH 2.5-5), LKB Instruments GmbH, GrSifelfing (Ampholine PAGplates, pH 3.59.5, Ultrodex gel for flat bed electrofocusing, LKB 2117 Multiphor system for electrophoresis equipped with a LKB 2103 power supply), Macherey Nagel & Co, Diiren (Acrylex P 100), Roth, Karlsruhe (DEAEcellulose, Union Carbide dialysis tubes), and Amicon GmbH, Witten (Diaflo ultrafilter membranes UM 10). TLC-Ready plastic sheets F-1700 Micropolyamide were purchased from Schleicher & Sehiill GmbH, Dassel. Bv&rs. The following buffers were used: (A) 100 mM Tris-HCl, pH 8, containing 2 mg/ml bovine serum albumin and 1 mM dithiothreitol; (B) 100 mM TrisHCI, pH 7.5; (C) 100 mM Tris-HCl pH 8; (D) 100 mM Tris-HCI, pH 7.5, containing 20% glycerol (v/v); (E) 200 mM McIlvain buffer, pH 5; (F) 50 mM Tris-HCl, pH 7.5; (G) 50 m&f potassium acetate, pH 6, containing 1 mM CaCI,, 1 mM MgC&, and 1 mM MnCl,; (H) 50 mM potassium acetate, pH 6; (I) 100 mM potassium phosphate, pH 7. Buffers B, F, and H contained 7 mM 2-mercaptoethanol, buffers C and D contained 14 mM 2-mercaptoethanol. Preparation of substrates. [l”‘$“-%]Apigenin 7-0(6-0-malonylglucoside) or malonylated isorhamnetin 3-0-glucoside was prepared enzymatically (28), employing 500 gg of the flavonoid substrate, 1 PCi [1,3‘“C]malonyl-CoA and 500 ~1 of the malonyltransferase preparation. The mixture (4 ml total in buffer A) was incubated for 2 h at 30°C. Malonylated glucosides

’ Abbreviations used: DFP, diisopropyl fluorophosphate; PCMB, 4-chloromercuribenzoic acid, sodium salt; ~1, isoelectric point; SDS, sodium dodecyl sulfate; MAT-7, malonyl-CoA:flavone/flavonol 7-O-glucoside malonyltransferase; MAT-3, malonylCoA:flavonol3-0-glueoside malonyltransferase.

ACYLHYDROLASES were purified from the incubation as described (29) and redissolved in 2-methoxyethanol prior to use. [2-VJApigenin ‘I-0-glucoside was prepared from [2-r4C]apigenin (2.03 &i in 300 pl2-methoxyethanol) and UDP-glucose (30 mg) with a partially purified UDP-glucose:flavone/flavonol 7-O-glucosyltransferase (1.5 ml) from irradiated parsley cell cultures (32). Incubation in buffer B (20 ml total) was carried out for 2 h at 3O’C. Acetic acid (20 ml, 5%, v/v) was added, and the mixture was extracted twice with 50ml aliquots of n-butanol. [2-“C]Apigenin 7-O-glucoside was purified from the butanol phase by chromatography on Sephadex LH-20 in methanol, yielding 0.37 pCi of product. [2-“C]Apigenin 7-0-(6-O-malonylglucoside) was prepared from [2-14C]apigenin 7-0-glucoside (0.26 nCi in 70 rl2-methoxyethanol) and malonyl-CoA (100 pg in 100 ~1 of buffer A) with a partially purified malonyltransferase (200 ~1). The mixture (4 ml total in buffer A) was incubated for 30 min at 3O”C, when an additional 100 pg malonyl-CoA and 200 ~1 malonyltransferase were added. Incubation was continued for 30 min, the reaction terminated by addition of 100 ~1 acetic acid, and the flavonoids were extracted with three portions of 5 ml n-butanol. [2-i4C]Apigenin 70-(6-0-malonylglucoside) was purified from the butanol phase by chromatography on Sephadex LH-20 in methanol, yielding 0.1 &i of product. Cell cultures. For preparative isolation of malonyltransferases and glucosyltransferases, parsley cell cultures were grown and irradiated, as described previously (29). Parsley plants. Mature parsley plants of the variety “moosgriin” were purchased from growers nearby and used not later than 2 h after harvest. Parsley seedlings of the same variety were grown in growth chambers at 65% relative humidity and irradiated with 6000 lux ((16 h a day) at 28°C daytime and 23°C nighttime temperatures. Preparation. of enzymes. Crude malonyltransferases were isolated from irradiated parsley cell cultures, as described previously (28). For determination of malonyltransferase activity in parsley plants, seedlings (between 9 and 25 days old) were separated into root, stern, cotyledon, and leaf bud or leaf, respectively. Leaf buds were harvested separately as early as possible (about 1 mm size). The tissue was homogenized as described for the cell cultures (28). Partially purified UDP-glucose:flavone/flavonol 70-glucosyltransferase was supplied by W. Heller in our laboratory, and was obtained in the course of chalcone synthase purification from irradiated parsley cell cultures (33). Protein (extracted from 1 kg of cells) which was not bound to hydroxyapatite was precipitated with ammonium sulfate (80% saturation), dissolved in buffer D (10 ml), and dialyzed against buffer B prior to use.

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For determination of esterase activity, parsley seedlings or mature parsley plants were subdivided as described above, and the tissue was homogenized in buffer E. For preparative isolation of esterases, mature parsley plants (5.8 kg of leaves and stems) were homogenized in buffer E (12 liters) and treated subsequently with a 1 M solution of MnClz to give a final concentration of 50 mM MnClz. Purijicatim of este-rases. Esterases were purified from crude enzyme extracts by chromatography on columns of DEAE-cellulose @O-ml bed volume), ConA-Sepharose (35 ml), Acrylex P-100 (200 ml), and phenyl-Sepharose (1 ml). Extracts were applied to DEAE-cellulose in buffer F, and eluted by a linear gradient from 50 to 500 ml Tris-HCI. Fractions containing high esterase activity were concentrated, dialyzed against buffer G, and applied to a ConA-Sepharose column. Esterase activity eluted with methyl a-D-mannoside (5% in buffer G) was subjected to isoelectric focusing either on Ampholine PAGplates, pH 3.5-9.5 or, on a preparative scale, in Ultrodex gel using ampholytes Pharmalyte, pH 2.5-5, at 10 W constant for 17 h. Appropriate zones of the gel were eluted with water, and specific esterase activity and pH of the solution were determined from aliquots. Esterases were further purified separately on a calibrated Acrylex P 100 column in buffer H. Fractions containing high esterase activity were concentrated, ammonium sulphate was added to give a 25% saturation, and the enzymes were applied to a phenylSepharose column. Esterases were eluted by a linear gradient of decreasing ammonium sulfate (25 to 0% saturation) and increasing concentration of ethylene glycol (0 to 50%, v/v) in buffer H. Enzyme assays. Malonyltransferase activities were determined as described (28). No distinction was made with respect to the relative proportion of malonylCoA and malonic acid in the incubation mixture, and therefore results are not corrected for esterase activity already present in the extracts of 19-day-old parsley seedlings. Standard incubation mixtures for determination of the desired esterase activity contained 5 ~1 of the malonylated flavonoid glucoside (equivalent to 20,000 dpm, labeled in the malonic acid portion) in 2-methoxyethanol, 50 ~1 buffer B and 10 pl enzyme of appropriate dilution in buffer B. The mixture was incubated for 10 min at 3O”C, the reaction terminated by addition of 50 ~1 acetic acid, and the mixture was applied to paper strips for chromatography. Esterase activity of purified enzymes was also determined fluorimetrically (34), using a-naphthyl esters as substrates, and measuring the a-naphthol released in a Perkin-Elmer MPF-2A fluorescence spectrophotometer at an excitation wavelength of 330 nm and an emission wavelength of 460 nm. a-Naphthyl acetate, a-naphthyl propionate, and a-naphthol were dis-

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solved in ethanol (0.25%, w/v) and diluted with buffer I under rigorous stirring to give 300-PM solutions (35). Alternatively, those substances, as well as a-naphthy1 myristate, were dissolved according to Norgaard and Montgomery (36) to give 300-pM solutions, but using Triton X-100 as the detergent. The inhibitors, PCMB or DFP, were added to a solution of a-naphthy1 acetate in Triton X-loo-phosphate buffer. Solutions of PCMB and a-naphthyl myristate were sonicated. The latter substrate did not dissolve completely, but rather formed a semistable suspension. Standard fluorimetric assay was carried out according to Thomas and Bingham (34), using 3- or 5-min incubation times at 25°C. For quantitative esterase determination with either the assay employing radioactive substrates or the fluorimetric assay, enzyme concentrations were chosen such that not more than 20% of the substrate was utilized. Chromatography. Separation of enzymatic products was achieved on Whatman 3MM paper in 10% acetic acid (malonylated products: R, about 0.18, maIonic acid and malonyl-CoA: R, approximately 0.96), and on a Sephadex LH-20 column (60-ml bed volume) in methanol. Malonic acid was identified by chromatography on silica gel in ethyl acetate/2-butanone/formic acid/water, 5:3:0.&l, using authentic material as reference, and by paper chromatography and electrophoresis as described (39). Apigenin 7-O-glucoside was identified as a product of the esterase reaction by cochromatography with authentic material on silica gel in the above mentioned solvent system (R, 0.32), on paper in 10% acetic acid (39) and on micropolyamide sheets in benzene/dioxane/formic acid, 4:5:1 (Rf 0.33). LIetermination of nubactivity. Radioactivity was measured by liquid scintillation counting in toluene containing 5 g 2,5-diphenyloxazole/liter. On paper strips, radioactivity was localized in a LB-280 paper scanner, Berthold Wildbad, and quantitated by liquid scintillation counting of appropriate paper sections. SDS-polyacrylamide gel electraphoresis. Slab gel electrophoresis was carried out according to LHmmli (37), using a 14% polyacrylamide running gel and a 5% polyacrylamide stacking gel. Bovine serum albumin, ovalbumin, and chymotrypsinogen A were taken as reference proteins. Protein determination Protein determination was carried out according to Sehaffner and Weissman (33). RESULTS

Identification and General Properties of Esterase Activity Search for esterases in parsley plants was intentionally limited to enzymes active toward malonic hemiesters. When ei-

MATERN

ther [1”‘,3 “‘-14C]apigenin 7-O-(6-O-malonylglucoside) (Fig. 1) or the correspondingly labeled malonylated isorhamnetin 3-0-glucoside was incubated with extracts from mature parsley plants in Tris-HCl buffer, pH 7.5, the malonic acid conjugates were hydrolyzed. Substrate hydrolysis was time-dependent and dependent on addition of plant extracts. Boiled plant extracts failed to catalyze this reaction. The enzyme activity was identified as an esterase activity by the following experiments. Using substrates labeled in the maionic acid portion, malonic acid was determined as one of the products by cochromatography with authentic malonic acid on silica gel plates, paper, and by electrophoresis (29). Rf value of malonic acid varied with the amount of material applied, e.g., from Rf 0.39 to 0.55 on silica gel. Furthermore, hydrolysis in alkali, as described previously (39), resulted in a radioactive product indistinguishable from the enzymatically prepared product. When instead a substrate labeled in the aglycon portion, [2-14C]apigenin 7-O-(6-O-malonylglucoside), was used in the incubation, the other reaction product was identified as apigenin 7-0-glucoside by cochromatography with authentic material on silica gel plates, paper, and polyamide sheets. No additional product was observed under the conditions described. In the case of malonylated isorhamnetin 3-0-glucoside (Fig. 1) no substrate labeled in the aglycon portion was available. However, as was observed by uv absorption after large-scale incubations, the flavonoid glucoside accumulated as a product of the enzymatic reaction here also. Therefore, the enzyme activity present in extracts of parsley plants hydrolyzed malonic hemiesters of flavonoid glucosides to malonic acid and the respective flavonoid glucoside. Crude enzyme was extracted from the tissue and stored in buffer E at 4°C. Under these conditions only minimal loss of activity was measured over several weeks, while repeated freezing and thawing reduced the enzyme activity considerably. Addition of 2-mercaptoethanol or dithiothreitol to crude extracts had no effect on

ACYLHYDROLASES

1

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2

FIG. 1. Malonic acid hemiesters serving as substrates for e&erases isolated from parsley plants, (1) apigenin 7-0-(6-0-malonylglucoside) and (2) malonylated isorhamnetin 3-0-glucoside.

the stability of esterases. When crude cell homogenates were subjected to centrifugation on a 15 to 45% sucrose gradient, as was described previously for flavonoid glycoside-specific malonyltransferases (28), esterase activity was recovered from the top layer of the gradient. The pH optimum of ester cleavage was determined in 200 mM potassium acetate buffer, pH 4.5 to 6.0, and in 200 InM potassium phosphate buffer, pH 6.0 to 8.25. Usually, enzyme extracts in buffer E were dialyzed and diluted 1:20with the desired buffer. Optimal hydrolysis was observed over a broad pH range from pH 7.25 to 7.75 with both malonylated isorhamnetin 3-0-glucoside and malonylated apigenin ‘7-0-glucoside, and activity was reduced only by about 30% at pH 5. Therefore, standard incubations were carried out at pH 7.5 in either phosphate buffer or buffer B. Relative Distribution of Malmyltransferase Activities and of Esterase Activity in Parsley Plants Specific activities of esterases and of malonyl-CoA:flavone/flavonol 7-O-glucoside malonyltransferase (MAT-7), as well as of malonyl-Coh:flavonol 3-0-glucoside malonyltransferase (MAT-3) (28), were determined in parsley plants which had been grown in growth chambers and harvested at different time intervals after planting. Root, stem, cotyledon, and leaf were harvested separately as early as possible. Results are summarized in Tables I

and II. Specific esterase activity in crude extracts of stem and leaves increased with the age of the plant. This increase was most prominent when the leaves unfolded. Very young leaf buds, on the other hand, contained no esterase activity. Similarly, in roots no esterase activity was measurable until 27 days after planting (Table I). Both young roots and leaf buds, however, contained considerable activities of flavonoid glucoside-specific malonyltransferases, which were absent from leaves, cotyledon, or stem (Table II). In leaf buds, specific activities of the malonyltransferases measured initially exceeded those activities known to occur in uv-irradiated parsley cell cultures (28) by a factor of about 350 for MAT-7 and by a factor of about 1000 for MAT-3. These activities decreased rapidly with the age of the leaf and could not be detected any more in larger unfolded leaves. In crude leaf extracts from l&dayold seedlings, some esterase activity was present already, which presumably led to liberation of malonic acid from the maionic acid conjugates under standard assay conditions for malonyltransferases. With respect to esterase activity, commercially available mature parsley plants contained only about one-third the specific esterase activity, as compared to crude extracts of plants grown in the growth chambers. Pur@icatim of Esterases Crude extracts were prepared from mature parsley leaves and stems by grinding

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MATERN

TABLE SPECIFIC

ESTERASE

I

IN CRUDE EXTRACTS OF PARSLEY PLANTS

ACTIVITY

Esterase

activity

Time after planting Plant organ Root Stem Cotyledon Leaf bud Leaf

9

13

0 30 30

0 28 29

14

0 (No leaves)

Note. Seedlings of similar ‘7-O-(6-0-malonylglucoside) conditions was determined.

15

16

0 70 67 2

0 152 35 6

II

SPECIFIC MALONYLTRANSFERASE

ACTIVITIES

IN

CRUDE EXTRACTS OF PARSLEY PLANTS

Malonyltransferase (MAT-7”/MAT-3b) Time after planting Plant organ Root Stem Cotyledon Leaf bud Leaf

14 1305 o/o o/o 350/1290

(days)

(months) 18

22

27

5

13 133 37

239 240

30

0 134 26 (Unfolded) 51

96

140

size were harvested at different time intervals after planting. [1”‘,3’“-“C]Apigenin served as a substrate, and [1,3-“C]malonic acid released under standard assay

the tissue with quartz sand in buffer E. From these extracts, four esterases, all of which liberated malonic acid from the

TABLE

(rkat/kg)

18

activities (pkat/kg) (days) 22

26 o/o

o/o o/o (Unfolded) 66/33 20/10

716

Note. Seedlings of similar size were harvested at different time intervals after planting. Apigenin ‘70-glucoside (MAT-7) or isorhamnetin 3-0-glucoside (MAT-3) and [1,3-“C]malonyl-CoA were used as substrates, and transferase activities were determined, as described previously (28). Results are not corrected for esterase activity already present in extracts of l&day-old seedlings. a Malonyl-CoA:flavone/flavonol ‘I-0-glucoside malonyltransferase. b Malonyl-CoA:flavonol 3-0-glucoside malonyltransferase.

malonylated flavonoid glucosides in the standard assay, were purified to apparent homogeneity by conventional procedures (Table III). Initial purification included MnClz precipitation of contaminants, precipitation with ammonium sulfate, chromatography on DEAE-cellulose and ConASepharose. At this stage of purification, no distinction between individual esterases was possible. After ammonium sulfate precipitation, the apparent specific and total esterase activity increased about 2.5fold, indicating that in crude extracts the activity of these esterases was inhibited. Chromatography on DEAE-cellulose was carried out in buffer B, using a linear gradient of buffer concentrations. Under these conditions, esterase activity was eluted from the column over a broad concentration range between approximately 170 and 500 mM salt. This step in the purification procedure was inefficient, since only a minor increase in specific activity was achieved, while a considerable loss of total activity was observed. Loss of activity was at least in part due to loss of protein during concentration of large volumes of enzyme extracts. Initially, concentration was carried out employing Diaflo ultrafilter membranes. With increasing purity of the enzymes, however, loss of enzyme activity in the Diaflo cell was noticed, similar to results obtained previously with malonyl-

ACYLHYDROLASES

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III

PURIFICATION OF ESTERASES FROM 5.8 kg OF PARSLEY LEAVES AND STEMS

Purification

step

Crude extract MnCl,-precipitation (50 mM) (NH,),S04-fractionation (30-60% saturation) DEAE-cellulose column ConA-Sepharose column Isoelectric focussing esterase I (pZ = 3.8) esterase II (pZ = 3.9) esterase III (pZ = 4.0) esterase IV (pZ = 4.05) Acrylex P-100 column esterase I esterase II esterase III esterase IV Phenyl-Sepharose column esterase I esterase II esterase III esterase IV

Volume (ml)

Protein (mg)

13,000 13,520 230 400 2.6

1,960 1,713 519 54 8.1

Yield (%I

58

100

514 623 1,378

234 30 10

1.05 3.36 2.5 4.5

0.18 0.87 0.7 1.35

2,400 1,800 1,060 930

5.7 4.1 3.4 3.3

0.05 0.27 0.39 0.14

1,620” 4,000c 14,740” 33,030”

1.7 1.0 2.5 1.0

0.03 0.05 0.07 0.03

Note. [l”‘J”‘-“‘C]Apigenin 7-0-(6-0-malonylglucoside) was used as substrate, leased under standard assay conditions was determined. ’ Fluorimetric assay with a-naphthyl propionate as substrate. d Fluorimetric assay with a-naphthyi acetate as substrate.

transferases (28). Therefore, concentration of enzyme extracts was accomplished usually by placing the enzyme in a dialysis tube embedded in crystalline sucrose. Minor losses of esterase activity occurred here also. Subsequently, esterase activity was bound to ConA-Sepharose in buffer G and eluted with the same buffer to which 5% (w/v) methyl cu-D-mannoside had been added. Isoelectric focusing separated the esterase activity into four fractions with p1’s of 3.8 (esterase I), 3.9 (esterase II), 4.0 (esterase III), and 4.05 (esterase IV). During this separation, approximately 60% of the total esterase activity was lost, although pH of the enzyme solutions was adjusted immediately following elution to pH 7 by addition of a zoo-mM Tris solution. After isoelectric focusing, all further enzyme determinations were also carried out fluorimetrically with a-naphthyl acetate

Specific activity (&at/kg)

700 370 308 820

0.4 1.4 0.7 1.1

7,800d 2,180d 1,380d 990d

and [1,3-“Clmalonic

acid re-

or cr-naphthyl propionate as a substrate. At this stage of purification, individual esterase solutions contained only minor impurities as was determined by gel filtration experiments. Molecular weight determination of esterases I through IV on Acrylex P-100 in buffer H revealed that all four esterases possessed molecular weights of approximately 35,000. After additional chromatography on phenyl-sepharose, SDS-polyacrylamide gel electrophoresis (Fig. 2) showed only one protein band for esterase I, corresponding to a molecular weight of 36,000, while esterases II, III, and IV showed one band each, equivalent to a molecular weight of 18,000. Substrate Specificities and Inhibition of Esterases When pure esterases were assayed with the substrates a-naphthyl propionate and

268

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MATERN

MW x

10

-3

25

Reference I

II

III

IV

proteins

FIG. 2. Separation of the four e&erases purified from parsley plants on a 14% SDS-polyacrylamide gel, numbered I through IV (from left to right). The molecular weight markers were bovine serum albumin (S?,OOO),ovalbumin (45,000), and ehymotrypsinogen (25,000). Gel electrophoresis was carried out according to LHmmli (37).

a-naphthyl acetate, it became apparent that all four esterases were unspecific with respect to these substrates and the maionic hemiesters of flavonoid glucosides mentioned above. On the other hand, (Ynaphthyl myristate did not serve as a substrate. Minor differences in substrate specificities, however, were observed (Table III). Esterases II, III, and IV were more active toward a-naphthyl propionate than toward a-naphthyl acetate as a substrate, while esterase I exerted only moderate activity toward a-naphthyl propionate, similar to its activity toward malonylated tlavonoid glucosides. Under the conditions described, all four esterases cleaved CYnaphthyl acetate faster than the malonic hemiesters, ranging from a factor of 1.2 for esterase IV to a factor of approximately 10 for esterase I. During these experiments, synthetic substrates were used at a concentration of 260 to 300 PM, which should suffice for saturation of all known plant esterases according to the literature. Since malonylated flavonoid glucosides were available in only small quantities, 2.6

PM concentration was used and enzymatic conversion of substrate was limited to 20% by appropriate enzyme dilutions. Inhibition studies were carried out with 4-chloromercuribenzoic acid (PCMB) and diisopropyl fluorophosphate (DFP), two inhibitors used in previous esterase studies (31), at concentrations of lop3 and 1O-4 M. Esterase activities were determined fluorimetrically with a-naphthyl acetate as substrate. While no esterase was inhibited by low4 M DFP, lop3 M PCMB did inhibit esterases II, III, and IV. DISCUSSION

Four esterases which liberate malonic acid from malonylated apigenin 7-O-glucoside or malonylated isorhamnetin 3-0glucoside were isolated from parsley plants. Separation of multiple enzyme forms, numbered I through IV according to their increasing pI values, was accomplished by isoelectric focusing. Additional chromatography on phenyl-Sepharose columns led to esterase proteins apparently

ACYLHYDROLASES

homogeneous in SDS-polyacrylamide gel electrophoresis. An overall purification factor of approximately 40 was calculated for all four enzymes, while recovery of total esterase activity was only 0.16% relative to the activity observed after ammonium sulfate precipitation. It should be noted, however, that the enzymes became labile during purification, which makes interpretation of purification factors difficult. Molecular weights of approximately 36,000 for all four esterases appear unusually small, when compared to esterases from other plants (34, 40-42), although a galactolipase from rice bran (49) and an acetylesterase from a fungus (43) of similarly small size have been reported recently. Little information is available concerning the subunit composition of other esterases, but trimer- and tetramer-composition has been suggested (4, 44). In parsley, esterase I consists of a single polypeptide chain, while esterases II, III, and IV are composed of two subunits each of 18,000 molecular weight. Since the esterases bind to ConA-Sepharose, these esterases are probably glycoproteins. All four esterases were not inhibited by of diisopropyl fluo1O-4 M concentration rophosphate, indicating that these esterases are most likely not serine-type esterases. On the other hand, 1O-3 M 4-chloromercuribenzoic acid inhibited those esterases composed of subunits, but not esterase I. According to Holmes and Masters (31), therefore, esterase I represents an acetylesterase, while esterases II, III, and IV are arylesterases. This classification, however, may be misleading with respect to substrate specificities of parsley esterases, since the natural substrates which we isolated fall into neither group. None of the isolated esterases was specific for dicarboxylic acid hemiesters. Nevertheless, differences were observed in affinities of the individual esterases to malonic hemiesters, cr-naphthyl acetate or cr-naphthyl propionate, while a-naphthyl myristate was not accepted. Only esterase I was similarly active toward either maionic hemiesters or cY-naphthyl esters. The

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other esterases accepted more readily the a-naphthyl esters under the conditions described, which may support their classification as being arylesterases. To our knowledge, this is the first report on isolated plant acylhydrolases accepting hemiesters of dicarboxylic acids as substrates. Similar to other enzymes involved in flavonoid biosynthesis (45), high activities of flavonoid glycoside-specific malonyltransferases were measured in leaf buds, exceeding the activities reported from parsley cell cultures (28) considerably. These malonyltransferase activities decreased rapidly with tissue age. In this respect, the malonyltransferases are typical enzymes of flavonoid biosynthesis. Esterase activities developed later, and the relative distribution of malonyltransferases and acylhydrolases suggests that both enzyme functions are coordinated in parsley plants. Subcellular localization of acylhydrolases has not yet been elucidated. However, since malonylated flavonoid glycosides were isolated from mature plants (30) and were shown to accumulate in vacuoles (29), most likely the acylhydrolases are to be found in the cytoplasm. Moreover, since the malonyltransferases are active only in embryonal tissue, our results suggest that in very young parsley leaves flavonoids are transported into the vacuole immediately following synthesis in the cytoplasm and prior to formation of esterase activity. Several attempts have been undertaken to draw some conclusion about the physiological significance of unspecific esterases from their relative distribution in plants (34, 46, 47). In all of these reports, however, the natural substrate was not defined, leaving the physiological question unanswered. In parsley, the esterases I through IV do not serve any vital function in primary metabolism, since growing tissue cultures of parsley lacked this activity. In differentiated plants, on the other hand, ester cleavage of malonic hemiesters is certainly of some importance once the tissue is disrupted and vacuolar contents become available to the esterases. We assume that the esterases described in this

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report are involved in the transfer of maionic acid to other organic acceptors under such conditions. The synthesis of carboxylic esters by acetylesterases, for example, has been reported recently (48). Malonylated flavonoid glucosides which accumulate in large amounts in parsley vacuoles comprise compounds with a high transfer potential with respect to malonic acid. Upon wounding, malonic acid may contribute to the synthesis of wound-specific compounds such as suberins. ACKNOWLEDGMENTS Excellent technical assistance of Ch. Feser is greatly appreciated. The author is indebted to J. Chappell and M. Hahn from our Department for critical reading of the manuscript.

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