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Acylhydrolasesfromparsley(Petroselinumhortense).Relativedistributionandpropertiesoffouresteraseshydrolyzingmalonicacidhemiestersofflavonoidglucosides

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ARCHIVES OF BIOCHEMISTRY AND 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 MATERN

Znstitut fiir Biologic ZZ, Lehrstuhl fiir Biochemie ckr Pjlanzen, Universitiit F&burg, Schiinzlestr. 1, 07800 Freiburg, West Germany

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 dif- ferential inhibition by mercury com- pounds or phosphate esters. With the ex- ceptions of pectinesterase and an esterase involved in polyacetylene metabolism (9) all esterases so far isolated from plant sources are relatively nonspecific, but usu- ally show some preference with respect to

1 This work was supported by Deutsche For- schungsgemeinschaft and by Wissenschaftliche Ge- sellschaft Freiburg.

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 ester- ases from plant, fish, or mammalian sources. Phthalic acid esters are hydro- lyzed in fish and mammalian liver to the hemiesters prior to conjugation to gluc- uranic acid (10-12). Similarly, only the 12- ester 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 sub- strates, Levy and Ocken (16) demonstrated that charged esters in general were very poor substrates for aryl-, acetyl-, or car- boxy1 esterases. To date, no esterase from

261 0003-9861/83 $8.00 Copyright Q 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

262 ULRICH MATERN

a plant source has been shown to hydro- lyze hemiesters of dicarboxylic acids.

Numerous malonic acid hemiesters of p- glucosides have been isolated from plants (17-26). Such malonylglucosides are resis- tant to glucosidases like almond emulsin (27). From uv-irradiated parsley cell cul- tures about 85% of the total flavonoid gly- cosides were isolated as their respective malonic acid conjugates (20), and two fla- vonoid 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 hem- iesters (20). Recently, we have discovered that malonylated flavonoids are exclu- sively located within the vacuoles of pro- toplasts 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 cul- tures. In the plant, esterases must be lo- calized in a separate cell compartment from the malonylglucosides or be inactive in the presence of these glucosides. More- over, 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-gluco- sides or flavonol3-0-glucosides. These es- terases do not completely fit either one of the esterase categories suggested by Holmes and Masters (31), but are best de- scribed as an acetylesterase and three ar- ylesterases. Their subunit composition and their distribution, as well as the distri- bution of flavonoid glycoside-specific mal- onyltransferases in parsley plants, is dem- onstrated.

MATERIALS AND METHODS

Chemicals and materials. Analytical grade chemi- cals were purchased from Sigma, Munich (cy-naph- thy1 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-‘%]malonyl- coenzyme 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 puri- fication were from Pharmacia, Freiburg (Con A-Se- pharose, phenyl-Sepharose, Sephadex LH-20, am- pholytes Pharmalyte, pH 2.5-5), LKB Instruments GmbH, GrSifelfing (Ampholine PAGplates, pH 3.5- 9.5, 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 (DEAE- cellulose, 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 Tris- HCI, 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, contain- ing 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), em- ploying 500 gg of the flavonoid substrate, 1 PCi [1,3- ‘“C]malonyl-CoA and 500 ~1 of the malonyltransfer- ase preparation. The mixture (4 ml total in buffer A) was incubated for 2 h at 30°C. Malonylated glucosides

’ Abbreviations used: DFP, diisopropyl fluorophos- phate; PCMB, 4-chloromercuribenzoic acid, sodium salt; ~1, isoelectric point; SDS, sodium dodecyl sul- fate; MAT-7, malonyl-CoA:flavone/flavonol 7-O-glu- coside malonyltransferase; MAT-3, malonyl- CoA:flavonol3-0-glueoside malonyltransferase.

ACYLHYDROLASES FROM PARSLEY 263

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-glucosyltransfer- ase (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 50- ml aliquots of n-butanol. [2-“C]Apigenin 7-O-gluco- side was purified from the butanol phase by chro- matography on Sephadex LH-20 in methanol, yield- ing 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 malo- nyltransferase (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 malonyl- transferase 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 7- 0-(6-0-malonylglucoside) was purified from the bu- tanol phase by chromatography on Sephadex LH-20 in methanol, yielding 0.1 &i of product.

Cell cultures. For preparative isolation of malo- nyltransferases and glucosyltransferases, parsley cell cultures were grown and irradiated, as described pre- viously (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 seed- lings 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 malonyltransfer- ases were isolated from irradiated parsley cell cul- tures, 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, re- spectively. 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 7- 0-glucosyltransferase was supplied by W. Heller in our laboratory, and was obtained in the course of chalcone synthase purification from irradiated pars- ley cell cultures (33). Protein (extracted from 1 kg of cells) which was not bound to hydroxyapatite was precipitated with ammonium sulfate (80% satura- tion), dissolved in buffer D (10 ml), and dialyzed against buffer B prior to use.

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 con- taining high esterase activity were concentrated, di- alyzed against buffer G, and applied to a ConA-Se- pharose column. Esterase activity eluted with methyl a-D-mannoside (5% in buffer G) was subjected to iso- electric focusing either on Ampholine PAGplates, pH 3.5-9.5 or, on a preparative scale, in Ultrodex gel us- ing ampholytes Pharmalyte, pH 2.5-5, at 10 W con- stant 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 cal- ibrated Acrylex P 100 column in buffer H. Fractions containing high esterase activity were concentrated, ammonium sulphate was added to give a 25% satu- ration, and the enzymes were applied to a phenyl- Sepharose 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 malonyl- CoA and malonic acid in the incubation mixture, and therefore results are not corrected for esterase ac- tivity 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-meth- oxyethanol, 50 ~1 buffer B and 10 pl enzyme of ap- propriate dilution in buffer B. The mixture was in- cubated 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 sub- strates, and measuring the a-naphthol released in a Perkin-Elmer MPF-2A fluorescence spectrophotom- eter 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-

264 ULRLCH MATERN

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-naph- thy1 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-naph- thy1 acetate in Triton X-loo-phosphate buffer. So- lutions of PCMB and a-naphthyl myristate were son- icated. The latter substrate did not dissolve com- pletely, but rather formed a semistable suspension. Standard fluorimetric assay was carried out accord- ing to Thomas and Bingham (34), using 3- or 5-min incubation times at 25°C. For quantitative esterase determination with either the assay employing ra- dioactive substrates or the fluorimetric assay, en- zyme concentrations were chosen such that not more than 20% of the substrate was utilized.

Chromatography. Separation of enzymatic prod- ucts was achieved on Whatman 3MM paper in 10% acetic acid (malonylated products: R, about 0.18, ma- Ionic 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 chro- matography on silica gel in ethyl acetate/2-butan- one/formic acid/water, 5:3:0.&l, using authentic ma- terial as reference, and by paper chromatography and electrophoresis as described (39). Apigenin 7-O-glu- coside was identified as a product of the esterase re- action 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 al- bumin, 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 ac- tive toward malonic hemiesters. When ei-

ther [1”‘,3 “‘-14C]apigenin 7-O-(6-O-malo- nylglucoside) (Fig. 1) or the correspond- ingly 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 addi- tion of plant extracts. Boiled plant ex- tracts failed to catalyze this reaction. The enzyme activity was identified as an es- terase activity by the following experi- ments. Using substrates labeled in the ma- ionic acid portion, malonic acid was de- termined as one of the products by cochromatography with authentic malonic acid on silica gel plates, paper, and by elec- trophoresis (29). Rf value of malonic acid varied with the amount of material ap- plied, e.g., from Rf 0.39 to 0.55 on silica gel. Furthermore, hydrolysis in alkali, as de- scribed previously (39), resulted in a ra- dioactive product indistinguishable from the enzymatically prepared product. When instead a substrate labeled in the aglycon portion, [2-14C]apigenin 7-O-(6-O-malo- nylglucoside), was used in the incubation, the other reaction product was identified as apigenin 7-0-glucoside by cochroma- tography with authentic material on silica gel plates, paper, and polyamide sheets. No additional product was observed under the conditions described. In the case of mal- onylated isorhamnetin 3-0-glucoside (Fig. 1) no substrate labeled in the aglycon por- tion was available. However, as was ob- served by uv absorption after large-scale incubations, the flavonoid glucoside accu- mulated as a product of the enzymatic re- action here also. Therefore, the enzyme ac- tivity present in extracts of parsley plants hydrolyzed malonic hemiesters of flavo- noid glucosides to malonic acid and the re- spective flavonoid glucoside.

Crude enzyme was extracted from the tissue and stored in buffer E at 4°C. Under these conditions only minimal loss of ac- tivity was measured over several weeks, while repeated freezing and thawing re- duced the enzyme activity considerably. Addition of 2-mercaptoethanol or dithio- threitol to crude extracts had no effect on

ACYLHYDROLASES FROM PARSLEY 265

1 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 centrifu- gation on a 15 to 45% sucrose gradient, as was described previously for flavonoid gly- coside-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 1:20 with the desired buffer. Optimal hydrolysis was observed over a broad pH range from pH 7.25 to 7.75 with both malonylated iso- rhamnetin 3-0-glucoside and malonylated apigenin ‘7-0-glucoside, and activity was reduced only by about 30% at pH 5. There- fore, 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-gluco- side 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 har- vested at different time intervals after planting. Root, stem, cotyledon, and leaf were harvested separately as early as pos- sible. 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, con- tained 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, con- tained 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 mea- sured 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 rap- idly with the age of the leaf and could not be detected any more in larger unfolded leaves. In crude leaf extracts from l&day- old seedlings, some esterase activity was present already, which presumably led to liberation of malonic acid from the ma- ionic acid conjugates under standard as- say conditions for malonyltransferases. With respect to esterase activity, com- mercially available mature parsley plants contained only about one-third the specific esterase activity, as compared to crude ex- tracts of plants grown in the growth chambers.

Pur@icatim of Esterases Crude extracts were prepared from ma-

ture parsley leaves and stems by grinding

266 ULRICH MATERN

TABLE I

SPECIFIC ESTERASE ACTIVITY IN CRUDE EXTRACTS OF PARSLEY PLANTS

Esterase activity (rkat/kg)

Time after planting (days) (months) Plant

organ 9 13 14 15 16 18 22 27 5

Root 0 0 0 0 0 13 239 Stem 30 28 70 152 134 133 240 Cotyledon 30 29 67 35 26 37 Leaf bud 0 2 6 (Unfolded) Leaf (No leaves) 30 51 96 140

Note. Seedlings of similar size were harvested at different time intervals after planting. [1”‘,3’“-“C]Apigenin ‘7-O-(6-0-malonylglucoside) served as a substrate, and [1,3-“C]malonic acid released under standard assay conditions was determined.

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

TABLE II

SPECIFIC MALONYLTRANSFERASE ACTIVITIES IN

CRUDE EXTRACTS OF PARSLEY PLANTS

Malonyltransferase activities (MAT-7”/MAT-3b) (pkat/kg)

Plant organ

Time after planting (days)

14 18 22 26

Root Stem Cotyledon Leaf bud Leaf

1305 o/o o/o

350/1290

o/o o/o

(Unfolded) 66/33 20/10

o/o

716

Note. Seedlings of similar size were harvested at different time intervals after planting. Apigenin ‘7- 0-glucoside (MAT-7) or isorhamnetin 3-0-glucoside (MAT-3) and [1,3-“C]malonyl-CoA were used as sub- strates, and transferase activities were determined, as described previously (28). Results are not cor- rected for esterase activity already present in ex- tracts of l&day-old seedlings.

a Malonyl-CoA:flavone/flavonol ‘I-0-glucoside ma- lonyltransferase.

b Malonyl-CoA:flavonol 3-0-glucoside malonyl- transferase.

malonylated flavonoid glucosides in the standard assay, were purified to apparent homogeneity by conventional procedures (Table III). Initial purification included MnClz precipitation of contaminants, pre- cipitation with ammonium sulfate, chro- matography on DEAE-cellulose and ConA- Sepharose. At this stage of purification, no distinction between individual esterases was possible. After ammonium sulfate precipitation, the apparent specific and to- tal esterase activity increased about 2.5- fold, 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 gra- dient of buffer concentrations. Under these conditions, esterase activity was eluted from the column over a broad concentra- tion range between approximately 170 and 500 mM salt. This step in the purification procedure was inefficient, since only a mi- nor 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 dur- ing concentration of large volumes of en- zyme 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-

267 ACYLHYDROLASES FROM PARSLEY

TABLE III

PURIFICATION OF ESTERASES FROM 5.8 kg OF PARSLEY LEAVES AND STEMS

Volume Protein Specific activity Yield Purification step (ml) (mg) (&at/kg) (%I

Crude extract 13,000 1,960 58 100 MnCl,-precipitation (50 mM) 13,520 1,713 (NH,),S04-fractionation (30-60% saturation) 230 519 514 234 DEAE-cellulose column 400 54 623 30 ConA-Sepharose column 2.6 8.1 1,378 10 Isoelectric focussing

esterase I (pZ = 3.8) 1.05 0.18 2,400 0.4 esterase II (pZ = 3.9) 3.36 0.87 1,800 1.4 esterase III (pZ = 4.0) 2.5 0.7 1,060 0.7 esterase IV (pZ = 4.05) 4.5 1.35 930 1.1

Acrylex P-100 column esterase I 5.7 0.05 1,620” esterase II 4.1 0.27 4,000c esterase III 3.4 0.39 14,740” esterase IV 3.3 0.14 33,030”

Phenyl-Sepharose column esterase I 1.7 0.03 700 7,800d esterase II 1.0 0.05 370 2,180d esterase III 2.5 0.07 308 1,380d esterase IV 1.0 0.03 820 990d

Note. [l”‘J”‘-“‘C]Apigenin 7-0-(6-0-malonylglucoside) was used as substrate, and [1,3-“Clmalonic acid re- 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, concentra- tion of enzyme extracts was accomplished usually by placing the enzyme in a dialysis tube embedded in crystalline sucrose. Mi- nor 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). Dur- ing this separation, approximately 60% of the total esterase activity was lost, al- though 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 en- zyme determinations were also carried out fluorimetrically with a-naphthyl acetate

or cr-naphthyl propionate as a substrate. At this stage of purification, individual es- terase solutions contained only minor im- purities as was determined by gel filtra- tion experiments. Molecular weight deter- mination 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 ad- ditional chromatography on phenyl-se- pharose, SDS-polyacrylamide gel electro- phoresis (Fig. 2) showed only one protein band for esterase I, corresponding to a mo- lecular weight of 36,000, while esterases II, III, and IV showed one band each, equiv- alent 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 ULRICH 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 car- ried out according to LHmmli (37).

a-naphthyl acetate, it became apparent that all four esterases were unspecific with respect to these substrates and the ma- ionic hemiesters of flavonoid glucosides mentioned above. On the other hand, (Y- naphthyl myristate did not serve as a sub- strate. Minor differences in substrate specificities, however, were observed (Ta- ble 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 ac- tivity toward a-naphthyl propionate, sim- ilar to its activity toward malonylated tla- vonoid glucosides. Under the conditions described, all four esterases cleaved CY- naphthyl acetate faster than the malonic hemiesters, ranging from a factor of 1.2 for esterase IV to a factor of approxi- mately 10 for esterase I. During these ex- periments, 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 stud- ies (31), at concentrations of lop3 and 1O-4 M. Esterase activities were determined fluorimetrically with a-naphthyl acetate as substrate. While no esterase was inhib- ited by low4 M DFP, lop3 M PCMB did in- hibit esterases II, III, and IV.

DISCUSSION

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

ACYLHYDROLASES FROM PARSLEY 269

homogeneous in SDS-polyacrylamide gel electrophoresis. An overall purification factor of approximately 40 was calculated for all four enzymes, while recovery of to- tal esterase activity was only 0.16% rel- ative to the activity observed after am- monium sulfate precipitation. It should be noted, however, that the enzymes became labile during purification, which makes in- terpretation of purification factors diffi- cult.

Molecular weights of approximately 36,000 for all four esterases appear un- usually 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 sim- ilarly small size have been reported re- cently. Little information is available con- cerning the subunit composition of other esterases, but trimer- and tetramer-com- position 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 es- terases bind to ConA-Sepharose, these es- terases are probably glycoproteins.

All four esterases were not inhibited by 1O-4 M concentration of diisopropyl fluo- rophosphate, indicating that these es- terases 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 repre- sents an acetylesterase, while esterases II, III, and IV are arylesterases. This classi- fication, 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 spe- cific for dicarboxylic acid hemiesters. Nev- ertheless, differences were observed in af- finities 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 ma- ionic hemiesters or cY-naphthyl esters. The

other esterases accepted more readily the a-naphthyl esters under the conditions de- scribed, which may support their classi- fication as being arylesterases. To our knowledge, this is the first report on iso- lated plant acylhydrolases accepting hemiesters of dicarboxylic acids as sub- strates.

Similar to other enzymes involved in fla- vonoid biosynthesis (45), high activities of flavonoid glycoside-specific malonyltrans- ferases were measured in leaf buds, ex- ceeding the activities reported from pars- ley cell cultures (28) considerably. These malonyltransferase activities decreased rapidly with tissue age. In this respect, the malonyltransferases are typical enzymes of flavonoid biosynthesis. Esterase activ- ities developed later, and the relative dis- tribution of malonyltransferases and acyl- hydrolases 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 iso- lated 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 mal- onyltransferases are active only in em- bryonal 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 phys- iological significance of unspecific ester- ases from their relative distribution in plants (34, 46, 47). In all of these reports, however, the natural substrate was not de- fined, leaving the physiological question unanswered. In parsley, the esterases I through IV do not serve any vital function in primary metabolism, since growing tis- sue 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 tis- sue is disrupted and vacuolar contents be- come available to the esterases. We as- sume that the esterases described in this

270 ULRICH MATERN

report are involved in the transfer of ma- ionic acid to other organic acceptors under such conditions. The synthesis of carbox- ylic esters by acetylesterases, for example, has been reported recently (48). Malonyl- ated flavonoid glucosides which accumu- late in large amounts in parsley vacuoles comprise compounds with a high transfer potential with respect to malonic acid. Upon wounding, malonic acid may con- tribute 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 read- ing of the manuscript.

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