Planta

Planta (1988) 173:532-543

9 Springer-Verlag 1988

Chalcone synthases from spinach (Spinacia oleracea L.) I. Purification, peptide patterns, and immunological properties of different forms L. Beerhues and R. Wiermann* Botanisches Institut der Westf~ilischen Wilhelms-Universitfit, Schlossgarten 3, D-4400 Miinster, Federal Republic of Germany

Abstract. The two chalcone-synthase forms from leaves of Spinacia oleracea L. were purified to apparent homogeneity. Antibodieg were raised against both proteins in rabbits. The specificity of the antibodies was tested using immunotitration, immunoblotting, and immunoelectrophoresis techniques. The antibodies exhibited exclusive specificity for chalcone synthase and did not discriminate between the two antigens. The homodimeric chalcone synthases had the same subunit molecular weight but differed in their apparent native molecular weights. The peptide maps indicated extensive homology between the proteins. Chalcone-synthase activity was not detected in isolated spinach chloroplasts. Both enzyme forms were present in spinach cell-suspension cultures in which they were induced by light. Key words: Spinacia (chalcone synthase) - Chalcone synthase (immunology, peptides) - Enzyme (different forms).

Introduction Flavonoids, mostly in the conjugated form, occur universally in vascular plants (Harborne 1986). They constitute one of the most abundant classes of phenolic compounds in higher plants and serve important functions as flower pigments, antimicro* To whom correspondence should be addressed Abbreviations: D E A E =diethylaminoethyl; DTE = 1,4-dithioerythritol; E D T A = ethylenediaminetetraacetic acid; HPLC=high-performance liquid chromatography; I g G = i m munoglobulin G; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Parts of the results were presented at the 14th International Botanical Congress at Berlin in July 1987

bial agents (phytoalexins), UV-protective compounds (Kreuzaler et al. 1983), and play a role in plant resistance to insects (Hedin and Waage 1986). Chalcones, or isomeric flavanones, are central intermediates in the formation of all flavonoids (Hahlbrock 1981). The formation of the C-15 flavonoid skeleton from the condensation of the activated precursors 4-coumaroyl-CoA and malonylCoA is catalysed by chalcone synthase (Kreuzaler and Hahlbrock 1972; Heller and Hahlbrock 1980; Sfitfeld and Wiermann 1980), an enzyme which plays a key role in the regulation of flavonoid biosynthesis during differentiation (Spribille and Forkmann 1981, 1982; Hinderer et al. 1983; Mol et al. 1983). In cell-suspension cultures of parsley and bean the enzyme is induced by UV-B light (Hahlbrock et al. 1976) and an elicitor (Lawton et al. 1983), respectively. The induction is the result of a rapid transient increase in the rate of synthesis of chalcone-synthase m R N A (Kreuzaler etal. 1983; Ryder et al. 1984). Both UV light and the elicitor selectively induce enzymes involved in flavonoid biosynthesis (Ebel and Hahlbrock 1977; Robbins et al. 1985). A UV-B receptor, a blue-light receptor, and phytochrome participate in the regulation of light induction of flavonoid synthesis (DuellPfaff and Wellmann 1982; Oelm/,iller and Mohr 1985; Bruns et al. 1986). Chalcone synthase has been demonstrated in a variety of plants including spinach (Beerhues and Wiermann 1985). In homogenates of young spinach leaves two forms of chalcone synthase were resolved and partially characterized. Multiple forms have also been reported for the key enzyme of general phenylpropanoid metabolism, phenylalanine ammonia-lyase (Boudet et al. 1971; Nishizawa et al. 1979; Bolwell et al. 1985).

L. Beerhues and R. Wiermann: Chalcone synthases from spinach. I.

In the first part of the present work we report the production and characterization of antibodies as well as structural and additional enzymological properties of the two chalcone-synthase forms from spinach. The second part will deal with immunofluorescence and immunogold localization of chalcone synthase. Material and methods Plant material. Spinach was grown in the Mfinster (FRG) botanical garden or in a greenhouse (illumination for 10 h at 18 ~ C under a light field composed of Osram L 36 W/77 Fhiora and Osram L 36 W/20 lamps (Osram, Miinchen, FRG); 13 ~ C during the dark period).

Enzyme assay and kinetic studies. Chalcone-synthase activity was determined as described previously (Beerhues and Wiermann 1985) except that the incubation buffer was adjusted to pH 7.8 and contained 0.375 retool.1-1 dithioerythritol (DTE) to give a final concentration of 0.5 retool.1-1 in the reaction mixture. For kinectic experiments carried out in duplicate the incubation time was restricted to 10 min. The Km values of the two substrates were determined as follows: for malonylCoA, the concentration was varied between 0.25 and 6.0 gmol. 1-1 while that of 4-coumaroyl-CoA was kept constant at 10 gmol. 1-1 ; for 4-coumaroyl-CoA, the concentration was varied between 0.05 and 0.95 gmol.1-1 while the reaction mixture contained 20 lamol" 1-1 malonyl-CoA.

Chloroplast isolation. Leaves were harvested at the end of the dark period to minimize their starch content. All steps were performed at 0 4 ~ C. A 6-g sample of deribbed leaves was cut into small strips, mixed with 40 ml of 50 mmol- 1-1 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes), pH 7.8, containing 330 mmol-1-1 sorbitol, 2 mmol-1- ~ ethylenediaminetetraacetic acid (EDTA), 2 mmol-1-1 MgCI2, and 0.1% (w/v) bovine serum albumin (isolation medium), and homogenized for 3 s with an Ultra-Turrax (Janke and Kunkel, Staufen i.Br. FRG). The homogenate was filtered through four layers of nylon cloth and centrifuged at 750.g for 1 rain. The pellet was resuspended in 30 ml of isolation medium, and underlayered with 14 ml (3.5 cm high) of 50 retool.1 -~ Hepes, pH 7.8, containing 40% (v/v) Percoll (Pharmacia, Freiburg, FRG), 330 mmol. 1-1 sorbitol, and 0.1% (w/v) bovine serum albumin (BSA; Mills and Joy 1980). After centrifugation at 2500.g for 1 min in a swinging-bucket rotor the supernatant was removed by vacuum suction. The pellet was taken up in a small volume of 0.1 tool-1-1 potassium-phosphate buffer, pH 8.0, containing 2.5 retool. 1- ~ DTE and was centrifuged at 27 500.g for 20 rain. The resulting supernatant and pellet were designated as stroma and membrane fractions, respectively. Diethylaminoethyl (DEAE) ion-exchange chromatography. This procedure was performed as described previously (Beerhues and Wiermann 1985) or by high-performance liquid chromatography (HPLC; Bio-Gel DEAE-5-PW; Bio-Rad, Munich, FRG). The latter process was performed at room temperature. The buffer comprised 50retool-1 1 imidazole, pH 6.5, and 2.5 retool-1- 1 DTE. Following equilibration the protein sample was applied by single or multiple injections. Unbound components were removed (15 min; flow rate: I ml-min-1). Bound proteins were ehited by a linear salt gradient of 0-500 retool. 1-1 NaC1 (70 min; flow rate: i ml-min-1). Fractions of 1 ml were collected.

533 It should be noted that a satisfactory separation of the two chalcone-synthase proteins was occasionally not attainable. When this occurred the 55-80% ammonium-sulfate-precipitated fraction of the crude leaf extract used for the chromatographic procedure was found to contain substances which moved slowly through the ion-exchange medium. Their removal from the column needed four gel volumes of buffer instead of the two normally used. Subsequent gradient elution did not resolve the two activities. The nature of these compounds is unknown except that they lack protein character as shown by the use of a protein-sensitive dye.

Molecular-weight determination by gel filtration. A Trisacryl AcA 34 column (LKB, Gr/ifelfing, F R G ; 90 cm long, 1.5 cm i.d.) was calibrated with dextran blue (Serva, Heidelberg, FRG) and the marker proteins catalase (220000; Sigma, Deisenhofen, FRG), aldolase (158 000; Boehringer, Mannheim, FRG), transferrin (81000; Serva), bovine serum albumin (66000; Biomol, Ilvesheim, FRG), ovalbumin (43000; Serva), ~-chymotrypsin (25000; Sigma) and myoglobin (17000; Serva). Elution was carried out with 0.1 mol.1-1 potassium-phosphate buffer, pH 6.8, at a flow rate of 4 cm. h-1. Fractions of 15 drops were collected. The molecular weights were plotted against the ehition volume:void volume ratios. Protein determination. Protein concentration was measured according to the method of Bradford 1976, modified by Read and Northcote t981. Ovalbumin was employed as standard protein. Cell culture. The cell-suspension culture of Spinacia oleracea was a kind gift of Professor Hartmann, Braunschweig, FRG, and was propagated as described previously (Wink et al. 1980). For harvest, 60 g cells were collected by suction filtration and homogenized in liquid nitrogen. The powder was suspended in a mixture of 300 ml of 0.1 mol.1-1 potassium-phosphate buffer, pH 6.8, containing 2.5 mmol.1-1 DTE, 4 0 m m o l . l -~ ascorbic acid, 1 mmol. 1 1 E D T A and 5 mmol. 1-1 thiourea and 15 g Polyclar AT (Serva). After stirring for 15 min and centrifugation (10000.g, 5min) the supernatant was mixed with (NH4)2SO4. Protein precipitating between 55% and 80% saturation was passed through Sephadex G-25 (Pharmacia) equilibrated with 50 mmol-1 1 imidazole, pH 6.5, and 2.5 retool.1-1 DTE. An aliquot of the protein fraction containing a quantity of about 20 mg protein was applied by multiple injections to a D E A E - H P L C column. The subsequent procedure is described above. Purification procedure. The following buffers were used: A: 0.1 tool.1-1 potassium-phosphate buffer, pH 6.8, containing 2.5mmol.1-1 DTE, 1 mmol-1 i E D T A (Ozeki et al. 1985), 40 mmol.1-1 ascorbic acid (Siitfeld et al./978) and 5 retool.1 a thiourea (van Driessche et al. 1984); B: 50 retool.1 1 potassium-phosphate buffer, pH 6.8, 2.5 retool.1 i DTE; C: 50mmot-1-1 imidazole, pH6.5, 2.5mmol.1-1 D T E ; D: 25mmol.1-1 piperazine, pH5.35, 2.5mmol-t -1 DTE; E: 25 retool. 1-1 piperazine, pH 5.15, 2.5 mmol-1 1 D T E ; F: polybuffer 74 (Pharmacia), 1:10 dilution, pH 4.0, 2.5 mmol.1-1 DTE; G: polybuffer 74, 1:10 dilution, pH 3.8, 2.5 mmol.1-1 DTE; H: 0.1 tool.1-1 sodium-phosphate buffer, pH 12.0; I: 10 mmol.1 -~ sodium-phosphate buffer, pH 6.5; K: 0.4 mol.l 1 sodium-phosphate buffer, pH 6.5. All steps were carried out at 0-4 ~ C. A 500-g sample of young leaves ( 1 - 4 c m in length; Beerhues and Wiermann 1985) was collected from the greenhouse. Portions of 50 g were homogenized with 250 ml buffer A and 12.5 g Polyclar AT for 1.5 min in a Waring blendor,

534

L. Beerhues and R. Wiermann : Chalcone synthases from spinach. I.

filtered through nylon cloth, and centrifuged at 10000.g for 10 rain. The pellets of 55-80% (NH4)2SO4-precipitated protein were resuspended in buffer A containing (NH4)2SO4 to 80% saturation, and combined. After centrifugation for 20 rain at 27500-g the protein pellet was dissolved in 28 ml of buffer B and loaded in equal amounts onto two TrisacryI AcA 34 columns (LKB; 90 cm long, 2.6 cm i.d.) equilibrated with buffer B. Following elution with the same buffer (flow rate= 4 cm-h 1) the fractions containing enzyme activity were combined and applied to a hydroxyapatite column (LKB ; 21 cm long, 3.0 cm i.d.) equilibrated with buffer B. Unbound protein was removed by washing with two gel volumes of the same buffer. Bound protein was eluted with a linear gradient from 50-300 mmol. 1- ~ potassium phosphate, and 2.5 mmol'1-1 DTE using five bed volumes of buffer (flow r a t e = 10 c m . h - 1). The fractions containing chalcone synthase were combined and passed through a column of Sephadex G-25 (Pharmacia; 52 cm long, 5.0 cm i.d.) equilibrated with buffer C. The protein fraction was subjected to ion-exchange chromatography on a column of DEAETrisacryl M (LKB; 15 cm long, 2.6 cm i.d.) equilibrated with buffer C. After removal of unbound protein by washing with two bed volumes of the same buffer the chalcone synthases were eluted in a linear salt gradient from 0-300 mmol. 1- 1 NaC1 in six bed volumes of buffer (flow rate = 10 cm. h-1). Fractions containing chalcone-synthase activity AI and AII were pooled separately. Buffer exchange was performed by passing the protein solutions through columns of Sephadex G-25 (20 cm long, 5.0 cm i.d.) one of which had been equilibrated with buffer D for AI, the other with buffer E for AII. The AI-protein fraction was chromatofocused on a column (PB E 94; Pharmacia; 50 cm long, 0.9 cm i.d.) equilibrated with buffer D, and that containing AII activity on a similar column equilibrated with buffer E. Elution of AI was carried out with buffer F, that of AII with buffer G (flow rate: 24 cm-h-~). Aiiquots of the fractions containing enzyme activity were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to check for protein constituents. Each aliquot comprised 5 gg protein, and was concentrated under a stream of nitrogen when necessary. Fractions containing only AI or AII protein were combined. Removal of polybuffer substances was performed as described in 'Biologics' (Vol. 10 (3); Calbiochem, Frankfurt a. M., FRG). The protein solutions were diluted 9:10 with buffer H to give a 10 mmol.1-1 phosphate concentration and a pH of about 6.5. They were applied to hydroxyapatite columns (LKB; 1.5cm long, 0.9cm i.d. in case of AI; 3.0cm long. 0.9 cm i.d. for AII) equilibrated with buffer I, and washed with two bed volumes of the same buffer. The chalcone-synthase proteins, free from polybuffer, were eluted with small volumes of buffer K and stored at - 20 ~ C.

Polyacrylamide gel electrophoresis. All runs were performed with slab gels. To determine the native molecular weights the method of Andersson and Mikaelsson (1972) was slightly modified. The gel contained a linear polyacrylamide gradient of 4-30% (w/v) total monomer concentration. The protein samples were made to 10% (v/v) glycerol. Electrophoresis was carried out at 150 V and 4 ~ C for 17 h. The marker proteins bovine serum albumin (Behring, Marburg, FRG) and ovalbumin were applied in 0.4 tool-1 1 sodium-phosphate buffer, pH 6.5, and a mixture of 90 mmol- 1- i Tris, 80 mmol-t- i borate, 3 mmol. 1-1 EDTA, pH 8.3. For P A G E under denaturing conditions the method of Laemmli (1970) was used. The linear polyacrylamide gradients are given in the appropriate figures. The molecular-weight markers (LMW-markers ; Pharmacia) were rabbit muscle phosphorylase (97400), bovine serum albumin (67000), ovalbumin (43 000), carbonic anhydrase (30 000), soybean trypsin inhibitor

(20100), and ~-lactalbumin (14400). Proteins were stained with Serva Blue G (Serva).

Preparation ofantisera. Antibodies were raised in six-month-old rabbits. Prior to immunization the rabbits were bled for preimmune sera. Aliquots of 75/ag of AI protein and 100 ~g of AII protein were used per rabbit to initiate immunization. The protein solutions were diluted with distilled water to 1 ml, emulsified in the same volume of complete Frennd's adjuvant, and injected subcutaneously at four sites on the back of the animals. First and second booster injections with 55 and 45 gg of AI or 80 and 50 gg of AII protein were carried out intramuscularly after 9 and 21 weeks using incomplete Freund's adjuvant. Blood, collected on the 14th day after the booster injections from an ear vein, was maintained at room temperature for 2 h prior to centrifugation at 5000.g for 5 rain. The crude sera were stored at - 2 0 ~ C. Isolation of IgG fractions from crude sera. All steps except HPLC were performed at 0-4 ~ C. For partial purification of the IgG fractions, 10 ml of each crude serum were used. Following addition of solid (NH4)zSO4 to 45 % saturation and centrifugation (30 000.g, 10 min) the resulting pellet was washed three times in 1.75 mol. 1-1 (NH4)2SO4 solution, pH 7.3. It was then dissolved in a small volume of 10 retool-l-1 potassium-phosphate buffer, pH 6.8, and passed through a column of Sephadex G-25 equilibrated with distilled water. The protein fraction was stirred for I h and centrifuged at 30000.g for 10 rain. The supernatant was applied to a column of Sephadex G-25 equilibrated with 10 mmol-1-x potassium-phosphate buffer, pH 6.8, containing 0.01 mmol-1-1 CaC12. Further purification was achieved by HPLC on hydroxyapatite (Biol-Gel HPHT; BioRad) following the instructions of the producer. Not more than 50 mg protein were applied per run. After removal of unbound substances, the IgG fraction was etuted in a linear gradient of 10 to 300 m m o l - t - i potassium-phosphate buffer, pH 6.8, containing 0.01 mmol.1-1 CaC12 (30 min; flow rate: 1 ml.min 1). It was concentrated by (NH4)2SO4-precipitation (45% saturation) and transferred through Sephadex G-25 into phosphate-buffered saline (PBS; 10 m m o l . l - 1 sodium-phosphate buffer, pH 7.4, containing 150 mmol-1-1 NaC1). The volume of the protein fraction was adjusted to that of the crude serum employed. The fraction was stored at - 2 0 ~ C.

Immunotitration. The two chalcone-synthase proteins AI and AII were separated by D E A E ion-exchange chromatography as described above. The E D T A was dissolved in 50 mmol. 1- I imidazole, pH 6.5, and added to the enzyme solutions to give a final E D T A concentration of 3 mmol-l-1. Aliquots of the crude sera and the purified IgG fractions were serially diluted with PBS (from 1:2 to 1:512). Fifty gl of enzyme solution were mixed with 50 gl of either the original or a diluted antibody solution. After 20 rain incubation at room temperature, 50 gl PBS containing 6% (w/v) polyethyleneglycol 8 000 (Hudson and Hay 1980) were added. Following incubation at 4 ~ C overnight the mixtures were centrifuged at the same temperature for 10 min at 8700.g (Microfuge; Beckmann, Munich, FRG). One hundred gl of the supernatant were used to determine the non-precipitated enzyme activity. After addition of 10/zl of 4-coumaroyl-CoA and 5 txl of [2-14C]malonyl-CoA solution the assay procedure was as described above. In the control tests, PBS lacking immunoglobulins was employed. Enzyme activities in the supernatants of incubation mixtures containing antibodies were calculated as percentage of those which were measured in the supernatants of the control assays.

Immunoelectrophoresis. Rocket-immunoelectrophoresis

followed the method of Weeke (1973). Glass plates were thinly

L. Beerhues and R. Wiermann: Chalcone synthases from spinach. I. precoated with 1% (w/v) agar-agar (Serva). Two percent (w/v) agarose (Serva) in water was mixed at 55~ in a 1:1 ratio with 50 mmol-1-1 barbituric-acid buffer, pH 8.6, containing 4 mmol-1-t E D T A and 0.4% (w/v) NAN3. Crude immune serum (1-4%, v/v) was added and the mixture applied to the precoated glass plates. When the layer had set, sample wells were made using a punch. Each well was filled with 5 gl antigen solution prepared from a crude extract of spinach which had been concentrated by (NHg)2SO,-precipitation. The protein pellet was dissolved in the above barbituric-acid buffer diluted 1 : 1 with distilled water. The antigen preparation was then subjected to serial dilution so that decreasing protein quantities could be applied to the sample wells. The 1 : 1-diluted barbituricacid buffer served as reservoir buffer. After electrophoresis at 80 V and 4 ~ C for 15 h the gel layer was dried, washed several times with 150 mmol-1 1 NaC1, then with distilled water and dried again. Precipitates were made visible by staining with Serva Blue G.

Immunoblotting. Immunoblotting was performed according to

535

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Beerhues et al. (1988).

Dot-blot analyses. If a preceding electrophoresis of proteins was not necessary small volumes of a non-denatured protein solution were dotted under vacuum suction directly onto nitrocellulose. Subsequent immunostaining was as described by Beerhues et al. (1988).

Peptide patterns. Prior to protein cleavage (Lischwe and Ochs

MP

AI

All

MP

Fig. 1. Analysis by SDS-PAGE of the purified spinach chalcone-synthase proteins in a 7.5-15% gradient gel. Aliquots of 10 gg AI and AII protein were applied. M P = m a r k e r proteins

1982) the purified chalcone-synthase proteins were subjected to SDS-PAGE. Twenty gg of AI and AII protein were run in a 7.5 15% gradient gel and stained by soaking the gel in 4 mol.1-1 sodium acetate (Higgins and Dahmus 1979). The gel slices containing the proteins (10-2 mm) were excised and transferred into beakers. Cleavage was performed for 30 min at room temperature in 15 mmol.1-1 N-chlorosuccinimide. After cleavage, the gel slices were homogenized in a small mortar with sample buffer. The volumes of the resulting homogehates ranged from 200 to 240 gl. The gel slices or the homogenates were stored at - 2 0 ~ C. The peptides were separated by SDS-PAGE using a linear polyacrylamide gradient of 10-20% and applying half the volumes of the homogenates. For stacking gel and sample buffer, equimolar mixtures of 2-amino-2(hydroxymethyl)-l,3-propanediol (Tris) and imidazole were used to facilitate the electroelution of the peptides (Rittenhouse and Marcus 1984). After electrophoresis the patterns were either stained by silver (Marshall 1984) or immunoblotted (Beerhues et al. 1988).

Results

Purification to homogeneity of the two spinach chalcone-synthase proteins. After fractionated ammonium-sulfate precipitation, molecular sieving, and adsorption chromatography, which did not differentiate the two activities, AI and AII were separated by ion-exchange chromatography and purified to homogeneity by chromatofocusing (Table 1). The elution profiles have, in part, been documented previously (Beerhues and Wiermann 1985). After chromatofocusing, the purity of AI and AII in the individual fractions containing enzyme activity was electrophoretically monitored. This step was indispensible because some of the

Fig. 2. Molecular-weight determination by non-denaturing electrophoresis: 10 gg AI protein, 10 gg AII protein, 20 gg bovine serum albumin (BSA), and 20 ~tg ovalbumin (OA) were applied. The sample buffers were 0.4 mol.1 1 sodium-phosphate buffer, pH 6.5 (SPB), and a mixture of 90mmol.1-1 Tris, 80 mmol.1-1 borate, 3 mmol-1 1 EDTA, pH 8.3 (TBB)

536

L. Beerhues and R. Wiermann: Chalcone synthases from spinach. I.

Table l. Purification to homogeneity of the two chalcone-synthase proteins from leaves of spinach Fraction

Crude

Volume (ml) extract

55-80% (NH4)2SO4-fractionation

Total activitya (pkat)

Total protein (mg)

Specific activity Purification (gkat-kg- 1) (-fold)

Recovery (%)

2 415.0

2 330.9

12100.00

0.193

1.0

100.00

28.0

1576.7

1 120.00

1.408

7.3

67.64

320.0

462.6

691.00

0.670

3.5

19.85

210.0

174.4

134.4

1.298

6.7

7.48

31.0 35.0

8.6 39.2

4.3 15.4

2.000 2.546

10.4 13.2

0.37 1.68

12.8 12.8

4.1 6.9

15.769 13.800

81.7 71.5

0.18 0.30

Molecular sieving (Trisacryl AcA 34) Adsorption chromatography (Hydroxyapatite) Anion-exchange chromatography (DEAE-Trisacryl M) AI AII Chromatofocusing AI AII

0.26 0.50

Activities were calculated on the basis of cpm values

fractions contained contaminating protein. The combined fractions were concentrated, and polybuffers were removed, on hydroxyapatite. Ultrafiltration was impractical for this step since an extensive loss of protein occurred. Aliquots of the final AI and AII preparations were subjected to SDSPAGE (Fig. 1). A single band was detectable in each case, indicating apparent homogeneity of both the AI and AII proteins. The subunits exhibited identical molecular weights of about 41000. The chalcone synthases of different purification stages gave predominantly naringenin in the enzyme assay besides low amounts of non-flavonoid release products.

Molecular weights. The molecular weights of the enzyme proteins were estimated by gel filtration and non-denaturing gel electrophoresis. In the latter case, aliquots of the homogeneous proteins were used immediately following purification. Relative to bovine serum albumin and its oligomers, the molecular mass of AI was about 88000, that of AII 78000 (Fig. 2). In contrast to AII, the AI protein exhibited additional bands with molecular weights of about 155000, 200000, and 230000. No bands were observed in the molecular-weight range of the chalcone-synthase subunit (41000). The migration of the marker proteins which were applied in 0.4 mol.1-1 sodium-phosphate buffer, pH 6.5, or a mixture of 90 mmol-1-1 Tris, 80 mmol.1-1 borate, 3 mmol'1-1 EDTA, pH 8.3, was similar, indicating that the sodium-phosphate buffer in

which AI and AII were present did not affect the molecular-weight estimation. Molecular masses of 48 000_+1000 and 62 000 ___2 000 were obtained following the passage of AI and AII, respectively, through a calibrated gel-filtration column.

Substrate affinities. Previous investigations (Beerhues and Wiermann 1985) had shown that, of the activated hydroxycinnamic acids, 4-coumaroyl-CoA was metabolised with the highest rate. Thus, it was used for kinetic experiments along with malonyl-CoA. Both chalcone-synthase forms exhibited similar K m values for 4-coumaroyl-CoA (AI = 0.8 gmol. 1-1 ; AII = 1.0 gmol" 1-1) and malonyl-CoA (AI = 2.0 gmol" 1-1 ;AII = 1.9 gmo1.1-1). Stability of the enzyme activities. The chalcone-synthase enzymes were relatively unstable. In crude extracts maintained at 0~ the loss in activities amounted to about 50% within 4 h. The partially purified proteins lost half their catalytic activity after 4-5 d at 4 ~ C. Following freezing for 1 h in the absence of glycerol, only 5% of the original activity of each enzyme remained. Storage at - 2 0 ~ C in the presence of 20% (v/v) glycerol resulted in preparations which were stable for four weeks, whereafter a steady loss of activity occurred. Chalcone synthase was not detected enzymologically in chloroplasts. Chalcone-synthase activity was not present in stromal or membrane fractions obtained

L, Beerhuesand R, Wiermann: Chalc0ne synthases from spinach, I,

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from osmotically shocked chloroplasts. Percoll, through which the chloroplasts were centrifuged during isolation, did not inhibit chalcone-synthase activity.

Occurrence of the two enzyme forms in a spinach cell-suspension culture. Chalcone-synthase heterogeneity was also studied in spinach ceil-suspension cultures. The growth of cultures routinely illuminated for 16 h per day was followed by determination of cell fresh weight and dry weight (data not shown), and by measuring the content of extractable protein (Fig. 3). In relation to the latter, highest chalcone-synthase activity occurred in the early stationary phase after about 18 d growth (Fig. 3). Cells from this stage contained both AI and AII activities9 In cultures maintained in the dark for 18 d, AI activity was not detected and AII activity was low. In cells maintained in the dark for 17 d and subsequently iliuminated for 24 h, both enzyme forms were present.

Antibody production.

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Antibodies were raised against both chalcone-synthase proteins in rabbits. Immunotitration showed that specific antibodies were present in the anti-AI sera and the anti-AII sera. A relatively strong inhibition or unspecific precipitation of the enzyme activities occurred in the presence of the preimmune sera. The enzyme activities in the supernatants of the titration mixtures containing the preimmune sera were sigzfificantly lower than the enzyme activities in the supernatants of the controls. This was also observed

Fig. 3, Chalcone-synthase activity in spinach ceilsuspension cultures as a function of culture growth

after the low-molecular-weight constituents had been removed from the sera by passing them through Sephadex G-25, and when pH, DTE or E D T A concentrations were altered and various concentrations of bovine serum albumin were added to the titration mixtures. Therefore, immunotitration was performed once again after the IgG fractions had been isolated from the crude sera (Fig. 4). With AI preimnmne IgG an inhibition or unspecific precipitation of the enzyme activities did not occur. With AII preimmune IgG a reduction of the enzyme activities was still present. It was, however, markedly lower than with the corresponding crude serum. Significant cross-reactions between the antibodies and the respective heterologous antigens were observed.

Proof of exchfsive specificity for chalcone synthase. Evidence for the exclusive specificity of the IgG preparations for chalcone synthase was provided using two methods, one of which was immunoblotting following SDS-PAGE. Comparison of the protein patterns in the gel prior to, and on nitrocellulose after blotting as well as staining of the blotted gel indicated a satisfactory transfer. When crude extract from leaves was subjected to SDSPAGE and subsequent immunoblotting, both antiAI and anti-AII IgGs stained only one protein band which exhibited a molecular weight corresponding to that of a chalcone-synthase subunit (Fig. 5). Incubation with the preimmune IgG fractions failed to produce any immunoreaction. Immunoblotting was also performed with the sepa-

538

L. Bcerhues and R. Wiermann: Chalcone synthases from spinach. 1.

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DILUTION OF THE [ g G - F R A C T I O N S (-FOLD}

Fig. 4. Immunotitrations of the spinach chalcone-synthase activities. The amounts of enzyme activity remaining in the supernatants after addition of decreasing quantities of preimmune AI IgG (pAl), anti-AI lgG (aAI), preimmune AII IgG (pAll), or anti-AII IgG (aAII) were determined. Immunoglobulins were omitted from the controls

Fig. 5. Immunoblotting of crude spinach leaf extract (CE). The SDS-PAGE was performed in a 9-16.5% gradient gel. A 400-Ftg sample of protein was applied per lane. The blots were either stained with amido black or incubated with anti-AI IgG (aAh, preimmune AI IgG (pAh, antiAll IgG (aAIl), or preimmune All IgG (pAil). MP= marker proteins

rate e n z y m e proteins by applying, after c h r o m a t o g r a p h y , aliquots o f the fractions c o n t a i n i n g similarly high a m o u n t s o f A I a n d A I I activity. I m m u nostaining showed t h a t b o t h proteins were recognized by b o t h I g G p r e p a r a t i o n s . F u r t h e r p r o o f o f exclusive specificity was p r o vided by i l n m u n o e l e c t r o p h o r e s i s . W i t h a m m o n i u m - s u l f a t e - c o n c e n t r a t e d crude leaf extract, b o t h a n t i - A I a n d a n t i - A I I sera p r o d u c e d only a single precipitation arc. A 1% (v/v) c o n c e n t r a t i o n o f

a n t i - A I s e r u m in a g a r o s e gel yielded a clear imm u n o r e a c t i o n . A similar result with a n t i - A I I serum, however, required a s e r u m c o n c e n t r a t i o n f o u r times greater (data n o t shown).

Immunological relationship between the two enzyme .forms. Decreasing a m o u n t s o f h o m o g e n e o u s A I a n d A I I p r o t e i n were applied to nitrocellulose and i m m u n o s t a i n e d with the various I g G fractions (Fig. 6). T h e staining o f A I by anti-AI or a n t i - A I I

L. Beerhues and R. Wiermann: Chalcone synthases from spinach. I.

539

Fig. 6. Estimation of the immunological relationship between the two spinach chalcone~synthase forms. Decreasing amounts (2.0, 1.0, 0.5, 0.25, 0.125, 0.063, 0.032, 0.016, 0.008, 0.004 ~tg; top to bottom) of AI and AI1 protein were dotted onto nitrocellulose. The strips were incubated with anti-AI IgG (aA1), antiAII IgG (aAIi), preimmune AI IgG (pAl), or preimmune AII IgG (pAil)

Fig. 8, Immunoblotting of the peptide maps of the spinach chalcone-synthase proteins. The quantities employed arose from 8 btg uncleaved protein. The blot was incubated with anti-AII IgG

0.13 gg AII protein. Negligible reactions occurred in the presence of the preimmune IgG preparations.

Peptide mapping. The proteins were chemically

Fig. 7. Peptide maps of the two chalcone-synthase proteins from spinach leaves and the chalcone synthase from tulip anthers resulting from cleavage with N-chlorosuccinimide/urea. The quantities applied were equivalent to 10 gg uncleaved AI and AII protein and to approx. 5 btg uncleaved tulip protein. M P = marker proteins

IgG was saturated by about 0.13 gg AI protein. Half this protein amount was stained to a lesser extent but was still clearly detectable. For AII, the anti-AII IgG response saturated at about 0.06 btg AII protein, and the anti-A! IgG response at about

fragmentated with N-chlorosuccinimide/urea in order to determine whether the two enzyme forms differ in their primary structures. The resulting peptides were electrophoresed and stained with silver (Fig. 7). The peptide patterns were composed of numerous well-separated protein fragments. The peptide maps of AI and AII were found to have the bulk of fragments in common. Three principal components with molecular weights of about 31 000, 15500, and 12500 were present in both patterns. A fragment with an approximate molecular weight of 30000 was also contained in both maps but occurred in different quantities. Several minor peptides in the molecular-weight range of 20000 to 26 500 did not coincide between the two maps. A residue of uncleaved protein (41 000) appeared in both maps. The weak bands with molecular weights of 53000 and 64000 were probably due to keratins (Ochs 1983).

540 Homogeneous chalcone-synthase protein from tulip anthers was also subjected to fragmentation (Fig. 7). This peptide map contained three main fragments with molecular weights of 30 000, 16 000, and 12 800 which were very similar to those of the main peptides derived from AI and AII. With the exception of the Mr-24000 component the minor fragments in the tulip pattern did not coincide with peptides in the map of AI or AII. Residual uncleaved tulip protein, of which the subunit molecular weight was found to be identical to that of the spinach proteins, was also detected. As an alternative to unspecific staining the peptides were transferred after electrophoresis to nitrocellulose and treated with anti-AI or anti-AII IgG. The incubations resulted in largely identical patterns of immunodetected fragments except that the maps were stained more intensely by anti-AII IgG (Fig. 8). The immunostained peptide patterns of AI and AII protein were similar. Of the three main bands mentioned above, the Mr-12500 component exhibited only poor antigenicity. The similarly strong staining in both maps at the position approximately corresponding to Mr 30000 indicated that probably two fragments with this molecular weight were present. A minor fragment presumably occurred in both the AI and AII maps and accounted for the staining. A major fragment was probably restricted to the map of AI and did not stain. In both patterns, the peptides with approximate molecular weights of 36500, 35500, 27000, 19 500, and the residual uncleaved protein (41 000) were also immunostained. In the map of AII the minor components with molecular weights of 21 500 and 16 500 were additionally detected.

Discussion

The two spinach leaf chalcone-synthase forms exhibited similar Km values for the preferred hydroxycinnamic-acid substrate, 4-coumaroyl-CoA (Beerhues and Wiermann 1985), and malonylCoA. The substrate affinities ofchalcone synthases from other species are similar (Siitfeld et al. 1978; Hinderer and Seitz 1985; Hrazdina etal. 1986; Knogge et al. 1986). In spinach cell-suspension cultures, light induced both enzyme forms. The lightmediated induction of chalcone synthase has been intensely studied in cell-suspension cultures of parsley (Kreuzaler et al. 1983). In accordance with previously reported data (Hrazdina et al. 1980), chalcone synthase was not enzymologically detected in Percoll-purified spinach leaf chloroplasts. The literature contains c o n -

L. Beerhuesand R. Wiermann:Chalconesynthasesfrom spinach. I. flicting reports which indicate that enzyme activity is (Ranjeva etal. 1977; Charriere-Ladreix etal. 1981) or is not (Hrazdina et al. 1980; Fuisting and Weissenb6ck 1980) associated with chloroplasts from various species. It is possible that, under some extraction conditions, endoplasmic-reticulure vesicles or enzymes may bind to the outside of the chloroplast envelope (Stafford 1981). Molecular-weight estimations using SDS- and non-denaturing gel electrophoresis indicated that both enzyme proteins are dimers, each of which consists of identical subunits with a relative molecular mass of 41 000. All non-spinach chalcone synthases investigated to date also exhibit a similar subunit mass and the dimeric structure (Kreuzaler etal. 1979; Ozeki etal. 1985; Hrazdina etal. 1986). Previously published divergent data (Kehrel and Wiermann 1985) should be corrected. As the enzyme forms are composed of subunits with identical molecular masses, it is possible that the discrepancy in the apparent dimeric masses (AI = 88000; AII=78000) may be due to different spatial conformations of the two proteins. The formation of oligomers during the non-denaturing electrophoresis by AI protein exclusively may also be attributed to this heterology. Molecular-weight estimation by gel filtration confirmed the discrepancy in the apparent molecular masses. However, the values obtained were significantly lower for both enzyme forms, and the AII protein exhibited a larger mass than the AI protein. Similarly divergent data in molecular-weight estimation have already been obtained for other proteins (Weinstein and Griffith 1987). It has been suggested that molecular-weight estimation by gel filtration, based on the determination of elution positions alone, is not reliable (Le Maire et al. 1987). The observation that the subunits of the two spinach chalconesynthase forms have identical molecular weights argues against the possibility that one form is the product of proteolytic degradation of the other. The non-denaturing electrophoresis demonstrated that the two enzyme forms are not interconvertible. This was also confirmed by anion-exchange rechromatography of the individual chalcone-synthase activities. Multiple forms of an enzyme can arise from differences in primary structure. Comparison of peptide maps can provide valuable information as to which amino-acid sequences differ. The two forms of chalcone synthase were fragmentated with N-chlorosuccinimide/urea, a reagent which selectively cleaves tryptophanyl peptide bonds (Shechter et al. 1976; Lischwe and Sung 1977). The peptide maps of AI and AII showed a great struc-

L. Beerhues and R. Wiermann: Chalcone synthases from spinach. I.

541

tural homology between the proteins. Nevertheless, a few peptides were distinctly unique to the

Ozeki et al. 1985; Hrazdina et al. 1986). The spinach proteins were also successfully employed for

map of either AI or AII. Thus, there are some

raising antibodies. The exclusive gpecifieity for

regions in the amino-acid sequences which are heterologous. As it is not known whether these restricted differences in the primary structures are genetically determined or arise from modifications at the level of transcription or translation, the two forms of chalcone synthase in spinach cannot, at present, be unequivocally referred to as isoenzymes (IUPAC-IUB CBN 1977). If one of the two spinach chalcone-synthase forms was the product of post-translational modification of the original enzyme amino-acid sequence, then the subunits of the two proteins would not exhibit identical sizes following SDS-gel electrophoresis. In parsley cell cultures post-translational modification of the chalcone-synthase amino-acid sequence does not occur (Reimold et al. 1983). Similarly, in mustard seedling cotyledons, no light-mediated post-translational activation of an inactive pro-enzyme was observed (BrOdenfeldt and Mohr 1986). In soybean cells, there is some indication that more than one chalcone-synthase m R N A is present (Grab et al. 1985), indicating that either pre-translational modification occurs or that different alleles or genes are present. Two different chalcone-synthase genes have recently been detected in Petunia hybrida (Reif et al. ]985). The genes are non-allelic and differ in the length of their introns, of which each gene probably contains only one. In the present study, chalcone synthase from tulip anthers was also subjected to cleavage. One major and one minor peptide in the tulip map coincided with components in the patterns of AI or AII. The principal fragments in the three maps exhibited very similar molecular weights. The positions of tryptophan residues in the amino-acid sequence of the chalcone synthase from parsley cultures (Reimold et al. 1983) imply that the peptide map of this protein might also contain main fragments with similar molecular weights. The data indicate that chalcone-synthase proteins from various sources may share a certain basic structure. Both chalcone-synthase forms were purified to apparent homogeneity. The low yields obtained reflect the relatively poor stability which the enzymes have in common with chalcone synthases from other sources (Kreuzaler and Hahlbrock 1975; Whitehead and Dixon 1983; Hinderer and Seitz 1985). Despite this handicap generally encountered during studies on chalcone synthase, a few purifications of the enzyme to homogeneity followed by antibody production have been reported (Kreuzaler etal. 1979; Kehrel and Wiermann 1985;

chalcone synthase of the isolated IgG fractions was proved by immunoblotting and immunoelectrophoresis. Immunotitration and immunoblotting demonstrated that there were strong cross-reactions between the antibodies and the respective heterologous antigens. Therefore, the homogeneous enzyme proteins were used for dot-blot analyses in order to assess quantitatively the interactions between the antibodies and the homologous and heterologous antigens. Both antibodies recognized both chalcone-synthase proteins to a similar degree. This finding was inferred by the similarity of the peptide maps but was expected to occur less drastically. It was confirmed by immunoblotting of the peptide patterns which were demonstrated to share the majority of antigenic fragments. A major component of Mr 30000 was probably unique in the pattern of AI but seemed to lack antigenicity. The close immunological relationship of the two chalcone-synthase forms prevented a sparate immunocytochemical localization. However, the location of overall chalconesynthase protein was determined at both the tissue and the subcellular level and is presented in a subsequent communication (Beerhues et al. 1988). The financial support from the Deutsche Forschungsgemeinschaft, the Minister fiir Wissenschaft und Forschung des Landes Nordrhein-Westfalen, and the Fonds der Chemischen Industrie is gratefully acknowledged. We thank Dr. M. Steup (Mtinster) for helpful suggestions and R. and Dr. J. Hokum (Miinster) for revising the English.

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