Specificity and properties of UDP-galactose:tomatidine galactosyltransferase from tomato leaves

Plant Science 136 (1998) 139 – 148

Specificity and properties of UDP-galactose:tomatidine galactosyltransferase from tomato leaves Jan Zimowski * Institute of Biochemistry, Warsaw Uni6ersity, al. Z: wirki i Wigury 93, 02 -089 Warsaw, Poland Received 2 October 1997; received in revised form 25 May 1998; accepted 26 May 1998

Abstract The specificity of galactosyltransferase involved in the biosynthesis of tomatidine monogalactoside in tomato leaves was studied using a wide spectrum of 3-OH steroids (i.e. steroidal alkaloids of spirosolane and solanidane type, steroidal sapogenins, typical sterols, androstane, pregnane and cholesterol derivatives as well as triterpenic alcohols) as sugar acceptors. The highest activity was found with tomatidine, but some other structurally-related compounds such as solasodine, nuatigenin, isonuatigenin, hecogenin, tigogenin, diosgenin were also glycosylated, however, at lower rates. UDP-galactose appeared to be the best sugar donor. The enzyme preparation was also able to utilize UDP-glucose as a sugar source for tomatidine glucosylation, however, at distinctly lower rate. Kinetic data showed apparent Km values of 2.48 mM for UDP-galactose and 0.83 mM for tomatidine. The investigated galactosyltransferase was stimulated by 2-mercaptoethanol and inhibited in the presence of UDP, UTP, divalent metal ions such as Cu2 + , Zn2 + , Hg2 + , Triton X-100, high ionic strength and N-ethylmaleimide. Divalent metals ions such as Mg2 + , Ca2 + , Mn2 + and chelating agents (EDTA, EGTA) had no significant effect on the enzyme activity. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lycopersicon esculentum; Solanaceae; Galactosylation of 3b-hydroxysteroids; UDP-Galactose-dependentgalactosyltransferase; 3b-D-Galactosides of steroids; Tomatidine 3b-D-galactoside

1. Introduction Steroidal alkaloids are nitrogen-containing secondary plant metabolites found in numerous Solanaceous plants and generally occurring as 3-O-glycosidic derivatives [1]. Steroidal glycoalka* Tel.: +48-22-8232046; Fax: + 48-22-8232046.

loids have been shown to act as cholinesterase inhibitors and are reported to exhibit a range of toxic effects in animals and man [2–4]. Their postulated role in plants is self-defense against pests [5–8]. Although numerous investigations have been concerned on chemical structure, occurrence and biological activities of steroidal glycoalkaloids, until now very little is known about

0168-9452/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 9 8 ) 0 0 1 0 9 - 5

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J. Zimowski / Plant Science 136 (1998) 139–148

Fig. 1. Biosynthetic pathway of a-tomatine.

their biosynthesis, especially at the enzymatic level. The biosynthesis of an aglycon moiety was partly elucidated in experiments with labeled precursors [9–11]. However, still there is a little information involving the biosynthesis of sugar side chain of steroidal glycoalkaloids. There are only a few reports [12 – 23] on glycosyltransferases which glycosylate steroidal alkaloids in the potato, the tomato and the eggplant. Most of them [12–18] refer to the UDP-glucose:solanidine glucosyltransferase derived from different parts of the potato plant. This enzyme catalyzes the synthesis of g-chaconine (solanidine monoglucoside), a postulated intermediate in the biosynthesis of a-chaconine—one of two major steroidal glycoalkaloids in the potato. This glucosyltransferase was partly purified and characterized [13,15,16]. Recently, cDNA encoding this enzyme has been isolated by functional expression in yeast [24]. The ability of crude enzyme preparations from the potato [14,16,18], the eggplant [19] and the tomato [22,23] to synthesize the monogalactoside of steroidal alkaloid in the presence of UDPgalactose and solanidine, solasodine and tomatidine, respectively, has been described. Recently, in our laboratory, galactosyltransferase catalyzing the synthesis of tomatidine galactoside has been partly purified [23]. In this paper we present further evidence that the soluble galactosyltransferase which catalyzes the formation of tomatidine monogalactoside is a tomatidine-

UDP-galactose-specific enzyme involved in the initiation of sugar chain synthesis of atomatine—the main glycoalkaloid of the tomato (Fig. 1).

2. Material and methods

2.1. Plant material Tomato (Lycopersicon esculentum Mill., cv. Kora) seeds were sown in pots containing garden soil and germinated at 26°C. After 1 week seedlings were transferred into a greenhouse and grown under 16/8 h day/night cycles and watered twice a week. In all experiments fresh leaves of 6-week-old plants were used as a source of enzyme preparations.

2.2. Chemicals and materials UDP-[14C]galactose and UDP-[3H]galactose were obtained from Amersham. Nucleotide sugars, sterols and its derivatives, some steroid sapogenins (tigogenin, diosgenin, hecogenin, smilagenin, sarsasapogenin), steroid alkaloids (demissidine, solanidine, tomatidine, solasodine), lanosterol, DEAE-Sephacel and Sephadex G-100 were obtained from Sigma. Nuatigenin and isonuatigenin were gifts from Dr M. Kalinowska (Department of Biochemistry, University of Warsaw),

J. Zimowski / Plant Science 136 (1998) 139–148

ruscogenin, chlorogenin and kogagenin from Dr P.K. Kintia (Institute of Ecological Genetics, Kishinev) and b-amyrin from Dr B. Wil*komirski (Institute of Botany, University of Warsaw). The Silica gel 60 TLC-plates were purchased from Merck.

2.3. Enzyme preparations Leaves of 6-week-old tomato plants (100 g of fresh wt.) were homogenized with 200 ml of icecold 0.02 M Tris–HCl buffer, pH 7.3, containing 10 mM 2-mercaptoethanol. The homogenate was filtered through cheesecloth and centrifuged at 105 000× g (1.5 h). The supernatant (the soluble protein fraction) was added dropwise to a 10-fold amount of cold (−20°C) acetone and acetone powders were obtained, as previously described [14].

2.4. Galactosyltransferase purification Gel filtration was carried out on a Sephadex G-100 column (1× 40 cm) equilibrated with 0.05 M Tris–HCl buffer, pH 7.3, containing 10 mM 2-mercaptoethanol (buffer A). The soluble protein fraction (2.5 mg of acetone powder from the 105 000×g supernatant) was suspended in the above buffer (10 mg/ml) and centrifuged at 20 000×g for 10 min. The supernatant was applied onto the column and eluted with the same buffer at a flow rate of 0.4 ml/min. Fractions exhibiting the highest galactosyltransferase activity were combined (12 – 20 ml) and then applied onto a DEAE-Sephacel ion-exchange column (1 × 3 cm), previously equilibrated with buffer A at a flow rate of 0.4 ml/min, for 4 h. The unbound proteins were washed out from the column with 30 ml of 0.15 M Tris – HCl buffer, pH 7.3, containing 10 mM 2-mercaptoethanol and then the column was developed with 20 ml of 0.25 M Tris –HCl buffer, pH 7.3, containing 10 mM 2mercaptoethanol (at a flow rate of 0.4 ml/min). This fraction was dialyzed overnight against buffer A and galactosyltransferase activity was assayed. The specific activities of crude soluble protein fraction, after Sephadex G-100 and DEAE-Sephacel, were 0.065, 1.04, 9.92 pkat/mg

141

protein, respectively. This partly purified enzyme preparation was rather unstable, it lost about 70% of galactosyltransferase activity during 4 days of storage at 4°C.

2.5. Glycosyltransferase assays The standard reaction mixture contained in a total volume of 0.52 ml: 0.5 ml of the dialyzed fraction eluted from the column; 2.5 mmol of Tris–HCl (pH 7.3); 5 mmol of 2-mercaptoethanol; 48 mM of tomatidine (or another sugar acceptor) in 0.01 ml of ethanol; and UDP-[14C]galactose (2.2×105 dpm; 711 nM) or UDP-[3H]galactose (4.4×105 dpm; 20.4 nM) in 0.01 ml of 50% ethanol. In some cases the incubation mixture contained 1 mM of [3H]tigogenin (3.5× 105 dpm) in 0.01 ml of ethanol as a sugar acceptor and 7.7 mM of nucleotide sugar in 0.01 ml of 50% ethanol as a sugar donor. During purification specific activity of galactosyltransferase was measured at saturating concentration of tomatidine (48 mM) and at non-saturating concentration of UDP[3H]galactose (3 mM). In order to determine the kinetic parameters the enzyme activity was measured with varying concentrations of UDP-galactose (0.02–20 mM) at a fixed concentration of tomatidine (48 mM) or with varying concentrations of tomatidine (0.1–48 mM) at a fixed concentration of UDP-galactose (3.0 mM). Once the sugar nucleotide was added, the reaction was run at 30°C for 30 min and then stopped by an addition of 1 ml methanol and heating for 3 min on a boiling water bath. Subsequently samples were extracted with 4 ml of n-butanol saturated with 0.1% aqueous ammonia and butanolic extracts were washed several times with 0.1% aqueous ammonia saturated with n-butanol. The samples were air-dried and radioactivity was counted in a liquid scintillation counter (see [14]) or they were applied on silica gel TLC-plates and developed in chloroform:methanol (85:15 v/v; solvent A) or chloroform:methanol:NH3(aq) (65:25:2 v/v; solvent B) as the solvent system. The chromatographic mobilities of radioactive products were compared with those of the following authentic reference compounds: for solvent A—tigogenin 3-O-b-D-monoglucopyranoside

J. Zimowski / Plant Science 136 (1998) 139–148

142

Fig. 2. Chemical structures of some steroid substrates used for enzyme specificity studies.

(Rf = 0.40), tigogenin 3-O-b-D-monogalactopyranoside (Rf = 0.36), tomatidine 3-O-b-D-monoglucopyranoside (Rf = 0.23), tomatidine 3-O-b-Dmonogalactopyranoside (Rf =0.16), nuatigenin monogalactopyranoside (Rf =0.31) and for solvent B—solasodine 3-O-b-D-monogalactopyranoside (Rf =0.51). After autoradiography, the silica gel with the radioactive compounds were scraped out and then these compounds were eluted with methanol containing 0.1% of 28% NH3(aq). The samples were air-dried and counted as above.

2.6. Other methods Labeled [3H]tigogenin was obtained by oxidation of tigogenin with CrO3 under mild conditions [25] and subsequent reduction of the resulting 3-oxo derivative with NaB[3H]4 (specific activity=296 GBq/mmol). The radioactive product was diluted with an authentic cold tigogenin and crystallized twice from ethanol. The obtained [3H]tigogenin had a specific activity of 11.7 GBq/ mmol and was radiochemically pure when analyzed by TLC/autoradiography. Unlabelled steroidal monoglycosides i.e. 3-O-b-D-monoglucopyranoside of tigogenin and tomatidine, 3-O-b-

D-monogalactopyranoside of tomatidine, solasodine and nuatigenin were obtained by the modified Koenigs–Knorr method using CdCO3 as the catalyst [26]. Crude reaction products were purified on silica gel columns using CHCl3CH3OH gradient (0–40% CH3OH) for elution.

3. Results and discussion As we have previously shown, the purification of soluble protein fraction from tomato leaves by gel filtration on Sephadex G-100 and DEAE-Sephacel results in a ca. 150-fold increase of specific activity and a ca. 3.0-fold increase of total activity of galactosyltransferase catalyzing the synthesis of tomatidine monogalactoside in the presence of UDP-galactose and tomatidine [23]. The partially purified UDP-galactose:tomatidine galactosyltransferase exhibits an apparent Km = 2.48 mM for UDP-galactose and 0.83 mM for tomatidine and an apparent Vmax = 11.36 pkat/mg protein. This enzyme preparation was used to study substrate specificity of the investigated galactosyltransferase towards the sugar moiety acceptor in

J. Zimowski / Plant Science 136 (1998) 139–148

the presence of UDP-galactose (Fig. 2). As potential sugar acceptors, steroidal alkaloids of spirosolane and solanidane type, steroidal sapogenins, typical sterols and their oxidized derivatives, triterpenic alcohols and C19 or C21 steroids (e.g. androstane and pregnane derivatives) were tested. In all cases, when a perceptible incorporation of [14C]galactose into n-butanol extracts was determined, TLC analysis with subsequent autoradiography showed the presence of a single labeled product with chromatographic mobility expected for the monogalactoside of a given steroid. With tigogenin, tomatidine, nuatigenin and solasodine as substrate, the corresponding products had chromatographic mobilities identical to those of authentic standards of tigogenin 3-O-b-D-monogalactopyranoside, tomatidine 3-O-b-D-monogalactopyranoside, nuatigenin 3-O-b-D-monogalactopyranoside, solasodine 3-O-b-D-monogalactopyranoside. Of the 29 compounds tested (Table 1), tomatidine was the best sugar acceptor. Another steroidal alkaloid of spirosolane type, solasodine, was a somewhat less effective substrate. This galactosyltransferase was also able to galactosylate some steroidal sapogenins e.g. nuatigenin, hecogenin, tigogenin, diosgenin, isonuatigenin (D5 or 5a-H steroidal sapogenins) and, to same extent, C-22 oxidized cholesterol derivatives, e.g. cholest-5-en-3b,22S-diol, cholest-5-en3b,22R-diol, cholest-5-en-3b-ol-22-on. In contrast, solanidine and demissidine (steroidal alkaloids of solanidane type), smilagenin and sarsasapogenin (5b-H steroidal sapogenins), sito-, stigma-, campesterol (typical sterols), androstenolone and pregnenolone (androstane and pregnane derivatives), as well as other cholesterol derivatives e.g. 25-hydroxy-, 19-hydroxy-, 20a-hydroxy-, 7-oxo- and tiocholesterol, lanosterol and b-amyrin (triterpenic alcohols) practically failed to be utilize as sugar residue acceptor in the galactosylation process. Data presented in Table 1 suggest that the galactosylation rate can be influenced by several structural factors such as the steroidal ring system conformation (compare D5 or 5a-H to 5b-H compounds), the structure of the molecule fragment bound to the ring D of steroidal nucleus (compare steroidal alkaloids of spirosolane type to steroidal

143

alkaloids of solanidane type or typical sterols or steroidal saponins), the presence of double bound at C-5 (compare tigogenin to diosgenin) and substitution pattern at C-22 (see C-22 oxidized cholesterol derivatives). It is noteworthy that enzyme preparations at different purification steps (i.e. crude soluble protein fraction from acetone powder, enzyme preparation after chromatography on Sephadex G-100 and partly purified enzyme preparation after Sephadex G-100 and DEAE-Sephacel) have shown practically identical relative ratio of galactosylation of tomatidine, nuatigenin, and tigogenin. These results suggest that the same enzyme utilizes all these substrates, however, the presence of several isoenzymes (e.g. enzymes catalyzing galactosylation of steroids) with very similar physical properties can not be ruled out. The above results indicate that tomatidine, the aglycon of a-tomatine occurring in the tomato, is probably the native substrate of the investigated galactosyltransferase. To determine the specificity of the enzyme with respect to the sugar donor, the effect of an 400fold excess of various non-labeled (cold) sugar nucleotides on the formation of tomatidine or tigogenin [3H]monogalactoside in the presence of UDP-[3H]galactose was tested. Results presented in Table 2 indicate that only cold UDP-galactose effectively decreases the synthesis of labeled tomatidine or tigogenin monogalactoside (ca. 85% inhibition). A slight inhibitory effect (15–25%) was observed in the case of UDP-glucose, UDP-xylose and UDP-mannose added to the incubation mixture. ADP-glucose, TDP-glucose, GDP-glucose and UDP-glucuronic acid had no effect on the incorporation of radioactivity from UDP[3H]galactose into both monogalactosides. These results indicate that cold UDP-galactose, UDPglucose, UDP-xylose and UDP-mannose may compete with UDP-[3H]galactose for the active site of the galactosyltransferase or decrease enzyme activity in another manner. It means that these sugar nucleotides can act as potential sugar donors or inhibitors of the investigated enzyme. Additionally, the decrease of tomatidine or tigogenin galactosylation in the presence of the above mentioned sugar nucleotides and saturating concentration of sugar acceptor cannot be due to

J. Zimowski / Plant Science 136 (1998) 139–148

144

Table 1 Specificity of the soluble galactosyltransferase from tomato leaves for some galactosyl moiety acceptorsa Acceptor

Steroid sapogenins Nuatigenin (22S,25Sepoxy-furost-5-ene3b,26-diol) Isonuatigenin (25Rspirost-5-ene-3b,25bdiol) Hecogenin (25R-5aspirostan-3b-ol-12-on) Diosgenin (25R-spirost-5en-3b-ol) Tigogenin (25R-5aspirostan-3b-ol) Ruscogenin (25R-spirost5-ene-1b,3b-diol) Smilagenin (25R-5bspirostan-3b-ol) Sarsasapogenin (25S-5bspirostan-3b-ol) Chlorogenin (25R-5aspirostane-3b,6a-diol) Kogagenin (25S-5bspirostane-1b,2b,3a,5btetraol) Sterols and their deri6ati6es Sitosterol (stigmast-5-en3b-ol) Stigmasterol (stigmasta5,22-dien-3b-ol) Cholesterol (cholest-5-en3b-ol) 25-Hydroxycholesterol (cholest-5-en-3b,25-diol) Cholest-5-en-3b,22R-diol Cholest-5-en-3b,22S-diol Cholest-5-en-3b-ol-22-on

Acceptor

[14C]Galactoside formation pmol/sample Relative activity (%)

Steroid alkaloids (a) Spirosolane type Tomatidine (25S5a,22bN-spirosolanin3b-ol) Solasodine (25R-22aNspirosol-5-enin-3b-ol) (b) Solanidane type Solanidine (22S,25Ssolanid-5-enin-3b-ol) Demissidine (22S,25S-5asolanidanin-3b-ol)

Table 1 (Continued)

36.2

29.8

100.0

82.3

0.5

1.4

2.0

5.5

26.1

72.1

28.9

79.8

21.0

58.0

15.5

42.8

25.4

70.2

3.9

10.8

0.3

0.8

0.0

0.0

3.5

9.7

0.0

0.0

0.2

0.6

0.2

0.6

0.3

0.8

0.3

0.8

0.8 3.1 0.8

2.2 8.6 2.2

Cholest-5-en-3b,20a-diol Cholest-5-en-3b,19-diol Cholest-5-en-3b-ol-7-on Tiocholesterol (cholest-5en-3b-thiol)

[14C]Galactoside formation pmol/sample Relative activity (%) 0.2 0.6 0.0 0.0 0.2 0.6 0.0 0.0

Androstane or pregnane deri6ati6es Androstenolone (androst0.0 5-en-3b-ol-17-on) Pregnenolone (pregn-5-en- 0.5 3b-ol-20-on) Triterpenic alcohols Lanosterol (4,4%,14trimethyl-5a-cholest-8en-3b-ol) b-Amyrin (olean-12-en3b-ol)

0.0 1.4

0.0

0.0

0.0

0.0

a The structure of some steroid substrates used are given in Fig. 2.

competition for the aglycon by other enzymes present in incubation mixture. Results presented in Table 3 clearly indicate that among sugar nucleotides tested, UDP-galactose and UDP-glucose were the only ones which were utilized for the glycosylation of [3H]tigogenin, however at very different rates. The ratio of the [3H]tigogenin glucoside to [3H]tigogenin galactoside formation was as 1:52. In our previous studies [23], we have shown that partly purified enzyme preparations from tomato leaves were able to synthesize tomatidine monoglucoside (in the presence of UDP[14C]glucose) and its monogalactoside (in the presence of UDP-[14C]galactose). In that case the ratio of the tomatidine monoglucoside to tomatidine monogalactoside formation was also very low (1:62.5). As we have shown previously [23], the crude soluble protein fraction was able to synthesize two types of tomatidine glycosides–glucoside and galactoside and the ratio of tomatidine glucoside formation (in the presence of UDP-glucose) to tomatidine galactoside formation (in the presence of UDP-galactose) was 1:2.5. As it fol-

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Table 2 Tomatidine and tigogenin [3H]galactoside formation in the presence of some sugar nucleotides by enzyme preparation from tomato leaves Unlabelled sugar nucleotide added (8.0 mM)

Biosynthesis of steroidal [3H]galactoside in the presence of UDP-[3H]galactose and Tomatidine

None UDP-galactose UDP-glucose UDP-xylose UDP-mannose UDP-glucuronic acid ADP-glucose TDP-glucose CDP-glucose GDP-glucose

pmol/sample

Relative activity (%)

pmol/sample

Relative activity (%)

1.31 0.18 1.06 1.13 0.99 1.30 1.32 1.29 1.31 1.30

100 14 81 86 76 99 101 98 100 99

0.89 0.13 0.74 0.79 0.68 0.87 0.88 0.90 0.89 0.87

100 15 74 79 76 98 99 101 100 98

lows from the present paper, the partly purified enzyme exhibited only traces of activity with UDP-glucose. These results indicate that in the crude enzyme preparation the synthesis of tomatidine glucoside and galactoside is catalyzed by at least two separate glycosyltransferases: the first specific towards UDP-glucose and the second to UDP-galactose. The presence of glucosyltransTable 3 The formation of [3H]tigogenin glycoside in the presence of some nucleotide sugar by enzyme preparation from tomato leaves Nucleotide sugar added (7.7 mM)

UDP-galactose UDP-glucose UDP-xylose UDP-mannose UDP-glucuronic acid ADP-glucose TDP-glucose CDP-glucose GDP-glucose a

Formation of [3H]tigogenin glycosidesa pmol/sample

Relative activity (%)

38.93 0.74 0.01 0.00 0.02

100.0 1.9 B0.1 0.0 B0.1

0.01 0.02 0.01 0.01

B0.1 B0.1 B0.1 B0.1

Incubations were carried out for 2 h.

Tigogenin

ferase activity in the partly purified enzyme preparation can be explained either by the ability of UDP-galactose:tomatidine galactosyltransferase to utilize UDP-glucose at a limited rate or by the contamination with the UDP-glucose specific enzyme which has not been separated completely from the galactosyltransferase during purification procedure. Long-term (24 h) incubations of the investigated enzyme preparation in the presence of tomatidine and labeled UDP-glucose or UDPgalactose have shown practically no decrease of the radioactivity incorporated into tomatidine glucoside or tomatidine galactoside. It means that neither glucosidase nor galactosidase is present in the partly purified enzyme preparation in appreciable amounts. The very low activity of the partially purified enzyme with UDP-glucose cannot be explained by rapid hydrolysis of tomatidine glucoside formed in contrast to tomatidine galactoside formed in the presence of UDP-galactose. On the other hand, the lack of the [3H]tigogenin glycoside formation in the presence of UDP-xylose or UDP-mannose (see Table 3) and slight inhibition of tigogenin and tomatidine galactosylation in the presence of the labeled UDP-galactose and the above mentioned sugar nucleotides (see Table 2) suggest that UDP-xylose or UDPmannose can act as inhibitors of galactosyltrans-

J. Zimowski / Plant Science 136 (1998) 139–148

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Table 4 Some properties of UDP-galactose:tomatidine galactosyltransferase from Lycopersicon esculentum leaves

Activator Inhibitor

No effect

Effector

Concentration (mM)

Relative activity (%)

None 2-Mercaptoethanol UMP; UDP; UTP Oxidized UDP (UDP-2%,3%-dialdehyde) Zn2+; Hg2+; Cu2+ N-Ethylmaleimide 0.1% Triton X-100 NaCl, NaF Iodoacetic acid Mg2+, Ca2+, Mn2+, EDTA, EGTA

–

100 120 88; 8; 9 55 1; 54; 34 57 22 9; 42 7 98 – 101

ferase activity. A similar effect of these sugar nucleotides have been observed in the case of some other plant glycosyltransferases, e.g. UDPglucose:sterols glucosyltransferase from oat [26] and UDP-glucose:solanidine glucosyltransferase from the eggplant [25]. Some other properties of investigated UDPgalactose:tomatidine galactosyltransferase are presented in Table 4. Divalent metal ions, such as Mg2 + , Ca2 + or Mn2 + , and some divalent metal chelators, i.e. EDTA or EGTA, appeared no effect on the galactosyltransferase activity. These results indicate that UDP-galactose:tomatidine galactosyltransferase, like other plant glycosyltransferases catalyzing the glycosylation of steroidal alkaloids, have no requirement for metal cofactors [13,16,18,21]. On the other hand, heavy metal ions (Hg2 + , Zn2 + , Cu2 + ) exert a strong inhibitory effect. The galactosyltransferase activity was slightly stimulated by 10 mM 2-mercaptoethanol and was inhibited in the presence of 1 mM N-ethylmaleimide. However, this inhibitory effect has been almost completely reversed by an addition of 10 mM 2-mercaptoethanol. This observation suggests that investigated enzyme requires reduced cysteine residues for its full activity. Tomatidine galactosylation was strongly inhibited in the presence of periodate oxidized UDP, i.e. UDP-2%,3%-dialdehyde, UTP and UDP. Inhibition of UDP-sugar-dependent plant glycosyltransferases by UDP as an reaction product has been frequently reported [27 – 30]. A similar effect of oxidized UDP has also been observed for other

0.1 0.01 1.0 1.0 1.0 – 500.0 1.0 0.01 – 0.1

glycosyltransferases acting on steroidal substrates [21,31,32]. The galactosyltransferase from the tomato was also sensitive to increased ionic strength of the incubation medium. The biosynthesis of tomatidine galactoside was strongly inhibited by 0.5 M NaF and NaCl (ca. 60 and 90% inhibition, respectively). A similar effect of ionic strength on steroidal alkaloid monoglycoside formation was reported for UDP-glucose:solanidine glucosyltransferase [16] and UDP-galactose:solanidine glycosyltransferases [18] from the potato and UDP-glucose:solasodine glucosyltransferase from the eggplant [25]. Tomatidine galactosylation was also strongly inhibited by 0.1% Triton X-100 (ca. 80% inhibition) when partly purified enzyme preparation has been used. This result somewhat differs from that obtained for crude enzyme preparation (only ca. 30% inhibition of tomatidine galactoside formation) [23]. It should be mentioned, however, that the total activity of tomatidine galactosyltransferase in the presence of Triton X-100 was comparable for both enzyme preparations and amounted ca. 1.6 pmol/h per mg acetone powder. On the other hand, in the absence of this detergent the partly purified enzyme showed a 3.5-fold rise in total activity as compared to the crude enzyme [23]. Observed increase of total activity during purification of the crude enzyme preparation seems to be a result of UDP-glucose 4%epimerase elimination influencing the tomatidine galactoside formation by decreasing of UDPgalactose concentration in the incubation

J. Zimowski / Plant Science 136 (1998) 139–148

medium. Observed difference in inhibition rate of tomatidine galactoside formation by crude and partly purified enzyme preparations in the presence of Triton X-100 seems to be a result of UDP-glucose 4%-epimerase activity present only in crude enzyme preparation. The investigated UDP-galactose:tomatidine galactosyltransferase shares some properties with other glycosyltransferases acting on steroidal alkaloids [13–21]. All these glycosyltransferases do not require divalent metal cofactors and exhibit the highest activity within alkaline pH range (7.0 – 9.0). In all cases, the major part of the glycosyltransferase activity is present in soluble protein fraction (supernatant 105 000×g). This suggests that all these enzymes are either soluble proteins or they are loosely bound to peripheric part of unidentified membranes. The apparent molecular masses of these glycosyltransferases are also similar and has been reported as ca. 50 kDa for tomatidine galactosyltransferase from the tomato [23] and 55 kDa for solasodine glucosyltransferase from the eggplant [21]. The apparent molecular masses ranging from 38 to 50 kDa have been shown for solanidine glucosyltransferase [13 – 15] and 40 – 50 kDa for solanidine galactosyltransferase [15,16] from the potato. However, these glycosyltransferases, e.g. solanidine glycosyltransferase from the potato, tomatidine galactosyltransferase from the tomato and solasodine glucosyltransferase from the eggplant, differ in their substrate specificities. Enzymes from the tomato and the eggplant are specific for spirosolane alkaloids while enzymes from the potato are highly active with spirosolane as well as solanidane alkaloids [13 – 15]. On the other hand, the partly purified galactosyltransferase from the tomato utilizes UDP-galactose and UDP-glucose (however, at a very different rate) for spirosolane alkaloids glycosylation in contrast to partially purified glucosyltransferase from the eggplant which is completely unable to synthesize monogalactosides of solasodine and tomatidine [21].

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