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Position-specific glucosylation of salicyl alcohol with an enzyme preparation from Gardenia jasminoides cultured cells
Plant Science Letters, 33 (1984) 47--52
47
Elsevier Scientific Publishers Ireland Ltd.
POSITION-SPECIFIC GLUCOSYLATION OF SALICYL ALCOHOL WITH AN ENZYME PREPARATION FROM GARDENIA JASMINOIDES CULTURED CELLS
T. TERAO, H. OHASHI and H. MIZUKAMI*
Faculty of Pharmaceutical Sciences, Naga~ki University, Nagasahi 852 (Japan) (Received May 25th, 1983) (Revision received July 12th, 1983) (Accepted July 13th, 1983)
SUMMARY
An enzyme activity catalyzing position-specific glucosylation o f salicyl alcohol to form salicin was demonstrated in a partially purified enzyme preparation from cultured cells o f Gardenia jasminoides. The reaction proceeded linearly with respect t o time and protein concentration and had a pH o p t i m u m o f 9.0 and a temperature o p t i m u m o f 50°C. Normal MichaelisMenten kinetics were observed for the substrates salicyl alcohol (Km = 0.53 raM) and UDP-glucose (0.64 raM). Formation o f isosalicin was n o t detected with the present e n z y m e preparation. The new e n z y m e described here can be classified as UDP-glucose: salicyl alcohol phenyl-glucosyltransferase.
Key words: Salicin -- Salicyl alcohol -- Glucosyltransferase -- Positional specificity - Cell cultures - Gardenia jasminoides INTRODUCTION
Salicin (o-hydroxymethyl phenyl #5-D-glucoside) was the firstglucoside found in nature. Although it had been considered to be formed from salicyl alcohol, later experiments showed that salicylalcohol was not converted to salicin either in vivo [1,2] or in vitro [1], but that it was converted to isosalicin(o-hydroxybenzyl ~-D-glucoside). O n the basis of feeding experiments, Zenk [3] concluded that salicylalcohol is not a direct precursor of salicin in Salix purpurea. In a previous paper [4] we reported that when various cultured plant cells *To whom correspondence should be addressed. Abbreviations: HPLC, high performance liquid chromatography ; TLC, thin-layer chromatography. 0304-4211/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
48
were examined for their ability to glucosylate salicylalcohol salicin was formed in cultured cellsof G. jasminoides and Lithospermum ery throrhizon whereas isosalicinwas formed in cell cultures of other species.This result suggested that in cultured cells o f G. jasminoides and L. erythrorhizon salicyl alcohol may be a direct precursor of salicin and that two kinds of UDP-glucose:salicyl alcohol glucosyltransferase, each having a different position specificity, are present in higher plants. Here we describe a novel enzyme catalyzing a position~specific glucosylation of salicyl alcohol to form salicin in a cell-free preparation from G. jasminoides cell cultures. MATERIALS AND METHODS
Cell culture Cultured cells induced from leaves of G. ]asminoides were maintained in Murashige and Skoog's liquid medium [5] supplemented with 10 -6 M 2,4dichiorophenoxyacetic acid and 10-6 M kinetin at about 26°C under dim light with transfer intervals of 2 weeks.
~nzyme preparation All steps of enzyme preparation were carried out at about 4°C. Typically, wet cells (10 g) harvested during the lineargrowth phase were homogenized in a Nissei Ace Homogenizer at 18 000 mv./min for 3 min with polyvinylpyrrolidone (5 g) in 30 ml of 0.2 M glycine-NaOH buffer (pH 9.0) containing 10 m M 2-mercaptoethanol. The slurry was passed through cheesecloth and centrifuged at 10 000 × g for 20 min. Enough solid ammonium sulfate to bring the solution to 75% of saturation was added to the supernatant fluid. The resulting precipitate was dissolved in the glycine-NaOH buffer (pH 9.0) and was dialyzed against the same buffer overnight. The dialysate was used as the enzyme preparation. The enzyme preparation lost 20% of the initial activity after a week and 50% after 3 weeks when stored in the frozen state at -20°C. Protein content was determined by the method of Bradford [6] with bovine serum albumin as a standard.
Assay of glucosyltransferase activity The assay system contained 3.3 mM UDP-[U-14C]glucose (0.05 #Ci), 3.3 mM salicyl alcohol, 10 mM 2-mercaptoethanol, 200 mM glycine-NaOH (pH 9.0), and enzyme in a final volume of 0.3 ml. The reaction was allowed to proceed for 60 min at 30°C and was stopped by adding 0.2 ml of ethanol containing 15 #mol of salicin as carrier. The mixture was centrifuged at 3500 × g for 5 rain and 0.1 ml of the supernatant fluid was analyzed by thin-layer chromatography (TLC) on Silica gel 60F (Merck) by using ethyl acetate/methanol (9:1,v/v) for development. The band corresponding to salicin was detected under UV-light (254 nm), scraped off, and counted in a scintillation counter.
49
Chemicals The sources of chemicals were as follows: UDP-glucose from Sigma (St. Louis, MO), UDP-[U-~4C]glucose from The Radiochemical Center, (Amersham, U.K.) and protein assay reagent from Bio-Rad (Richmond, CA). All other chemicals were of reagent grade. RESULTS
Identification of the reaction product For identificationof the reaction product, the reaction mixture was extracted with n-butanol. The butanol extract was chromatographed on Silicagel 60F with solvent systems: (I)ethyl acetate/methanol (9: 1, v/v);(If) ethyl acetate/methanol/water (65:22:13, v/v) or (III) ethyl acetate/i-propanol/water (65:24:11, v/v), which showed that the radioactivity was associated with the position of salicinand not with the position of isosalicin. The incorporation of radioactivity into salicin was also detected by high performance liquid chromatography (HPLC) analysis as shown in Fig. 1, whereas the formation of isosalicinwas again not detected. Finally, a large scale incubation was carrried out to identify the reaction product. In a total vol. of 1.0 ml were incubated: 10 m M UDP-[U-~4C]glucose (0.4 ~Ci); 10 m M salicylalcohol; 10 m M 2-mercaptoethanol; 100 m M
a >(
~a "-'6 ._> 4
e~
e~
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I
2
4
6
8
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Retention time (rain) Fig. 1. Identification by HPLC of the product formed in the reaction catalyzed by the glucosyltransferase. An aliquot of the butanol extract of the reaction mixture was injected into a Radiai-pak A (Waters) column. Separation was carried out with methanol/ water ( 2 5 : 7 5 , v/v) at a flow rate of 1.5 ml/min. (a) Fractions were collected every 30 s and were analyzed for radioactivity. (b) Chromatogram of reference compounds: (1) UDP--giucose (2) salicin (3) isosalicin (4) sallcyl alcohol; the compounds were detected through their absorbance at 270 nm.
50 Tris--HCl (pH 7.5) and 10 mg of enzyme. The reaction t o o k place for 3 h at 37°C. At the end o f the incubation period salicin (15 rag) was added to the mixture as carrier; the mixture was then extracted with n-butanol. Radioactive salicin with a constant spec. act. o f 2.1 × 103 d.p.m./#mol was obtained from the butanol extract by preparative TLC on Silica gel 6 0 F with solvent system I followed b y repeated recrystallization. This indicates formation o f salicin from salicyl alcohol and UDP~lucose in about a 13% yield.
Properties o[ the glucosy ltransferase The enzyme catalyzed formation o f salicin was linear for a b o u t 60 min and the rate of reaction was linear up to 600 pg o f protein under standard assay conditions. The enzyme exhibited normal Michaelis-Menten kinetics with both substrates. The apparent Kin-values were found to be 0.53 mM for salicyl alcohol and 0.64 mM for UDP-glucose. The enzyme activity showed a broad pH proftle ranging from pH 5.5 to 10.5. The o p t i m u m pH was 9.0 (glycine-NaOH) with half maximum activities at pH 7.5 (Tris--HCl) and 10.2 (glycine-NaOH). The temperature o p t i m u m for the reaction was found to be 50°C although 90% of the activity was lost when the enzyme preparation was treated at 50°C for 5 min, as shown in Fig. 2. The whole enzyme activity was found in the supernatant fraction when the crude cell extract obtained b y centrifugation o f the cell homogenate at 10 000 × g for 20 min was further centrifuged at 100 000 X g for 60 min. This indicates that the glucosyltransferase is a soluble enzyme. °~
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60
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Temperature ( C ) Fig. 2. Temperature dependence o f the glucosyltransferase. (a) The reactions were incubated at the indicated temperatures for 60 rain. (b) Aliquots o f the enzyme preparation were treated at the indicated temperatures for 5 rain, and were then added to the reaction mixtures which were incubated at 30°C for 60 rain.
51
A
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Time
(day)
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Fig. 3. T i m e course o f changes in the glycosyltransferase activity (• (c o ) of G. jasminoides cells.
• ) during growth
Figure 3 shows that the enzyme activity gradually increases during the exponential and linear phases o f growth and then rapidly decreases in the stationary phase. This result is consistent with the kinetics o f glucosylation o f salicyl alcohol in vivo by cultured cells o f G. jasminoides (Mizukami et al., unpublished). DISCUSSION
This is the first report o f the enzymatic formation o f salicin from salicyl alcohol and UDP-glucose. The enzyme catalyzing this reaction, namely UDPglucose : salicyl alcohol phenyl-glucosyltransferase, is shown to be a soluble enzyme although some gtucosyltransferases are reported to be b o u n d to Golgi membranes [7] or cell membranes [8] in the case of plant sterol glucosyltransferases or to be localized in cell particles [ 9] in the case of the phenol glucosyltransferase of mollusks. The pH o p t i m u m of this enzyme is high when compared with other plant glucosyltransferases so far reported [ 10--15] and is rather similar to that of the phenol glucosyltransferase of mollusks [9]. It is of interest that a detectable a m o u n t of isosalicin was not formed with the present enzyme preparation although isosalicin as well as salicin was formed in a I : 2 ratio when salicyl alcohol was administered to G. jasminoides cell cultures [4]. It m a y be that the localization and/or optimal reaction conditions of an enzyme catalyzing the formation of isosalicin from salicyl alcohol (UDP-glucose : salicyl alcohol benzyl-glucosyltransferase) are quite different from those of the phenyl-glucosyltransferase or that salicyl alcohol is n o t a direct precursor of isosalicin in cultured cells of G. jasminoides.
52 Our remits clearly demonstrate the presence of a UDP-glucose: salicyl alcohol phenyl-glucosyltransferase which has a clear specificity for the phenolic position of salicyl alcohol. The significance o f the presence of this enzyme is n o t clear because salicin is not endogeneously synthesized in G. jasminoides cultured cells, b u t the enzyme may play a role similar to t h a t of the UDP-glucuronyltransferase of vertebrates. ACKNOWLEDGEMENTS We wish to thank Dr. S.L. Nickel, University of Toledo, for reviewing the manuscript. This work was supported by a Grant-in-Aid for Scientific Research (57771464) from the Ministry of Education, Science and Culture o f Japan. REFERENCES 1 J.B. Pridham and M.J. Saltmarah, Biochem. J., 87 (1963) 218. 2 M. Tabata, F. Ikeda, N. Hiraoka and M. Konoshima, Phytochemktry, 15 (1976) 1225. 3 M.H. Zenk, Phytochemistry, 6 (1967) 245. 4 H. Mizukami, T. Terao, H. Miura and H. Ohaahi, Phytochemktry, 22 (1983) 679. 5 T. Muraahige and F. Skoog, Physiol. Plant., 15 (1962) 473. 6 M.M. Bradford, Anal. Biochem., 72 (1976) 248. 7 H.E. Hopp, P.A. Romero, G.R. Dale<) and R. Pont Lezica, Phytochemistry, 17 (1978) 1049. 8 9 10 11 12 13 14 15
M.-A. Hartmann-BouiUon, B. Walter and E. Lazar, Plant Cell Rep., 1 (1981) 56. G.J. Dutton, Arch. Biochem. Biophys., 116 (1966) 399. T. Yamaha and C.E. Cardini,Arch. Biochem. Biophys., 86 (1960) 127. A. Sutter,R. Ortmann and H. Grisebach, Biochim. Biophys. Acta, 258 (1972) 71. A. Sutter and H. Grisebach, Biochirn.Biophys. Acta, 309 (1973) 289. R.K. Ibrahim and H. Grkebach, Arch. Biochem. Biophys., 176 (1976) 700. T. Yoahikawa and T. Furuya, Phytochemistry, 18 (1979) 239. R.K. Ibrahim and B. Boulay, Plant Sci. Lett., 18 (1980) 177.
47
Elsevier Scientific Publishers Ireland Ltd.
POSITION-SPECIFIC GLUCOSYLATION OF SALICYL ALCOHOL WITH AN ENZYME PREPARATION FROM GARDENIA JASMINOIDES CULTURED CELLS
T. TERAO, H. OHASHI and H. MIZUKAMI*
Faculty of Pharmaceutical Sciences, Naga~ki University, Nagasahi 852 (Japan) (Received May 25th, 1983) (Revision received July 12th, 1983) (Accepted July 13th, 1983)
SUMMARY
An enzyme activity catalyzing position-specific glucosylation o f salicyl alcohol to form salicin was demonstrated in a partially purified enzyme preparation from cultured cells o f Gardenia jasminoides. The reaction proceeded linearly with respect t o time and protein concentration and had a pH o p t i m u m o f 9.0 and a temperature o p t i m u m o f 50°C. Normal MichaelisMenten kinetics were observed for the substrates salicyl alcohol (Km = 0.53 raM) and UDP-glucose (0.64 raM). Formation o f isosalicin was n o t detected with the present e n z y m e preparation. The new e n z y m e described here can be classified as UDP-glucose: salicyl alcohol phenyl-glucosyltransferase.
Key words: Salicin -- Salicyl alcohol -- Glucosyltransferase -- Positional specificity - Cell cultures - Gardenia jasminoides INTRODUCTION
Salicin (o-hydroxymethyl phenyl #5-D-glucoside) was the firstglucoside found in nature. Although it had been considered to be formed from salicyl alcohol, later experiments showed that salicylalcohol was not converted to salicin either in vivo [1,2] or in vitro [1], but that it was converted to isosalicin(o-hydroxybenzyl ~-D-glucoside). O n the basis of feeding experiments, Zenk [3] concluded that salicylalcohol is not a direct precursor of salicin in Salix purpurea. In a previous paper [4] we reported that when various cultured plant cells *To whom correspondence should be addressed. Abbreviations: HPLC, high performance liquid chromatography ; TLC, thin-layer chromatography. 0304-4211/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
48
were examined for their ability to glucosylate salicylalcohol salicin was formed in cultured cellsof G. jasminoides and Lithospermum ery throrhizon whereas isosalicinwas formed in cell cultures of other species.This result suggested that in cultured cells o f G. jasminoides and L. erythrorhizon salicyl alcohol may be a direct precursor of salicin and that two kinds of UDP-glucose:salicyl alcohol glucosyltransferase, each having a different position specificity, are present in higher plants. Here we describe a novel enzyme catalyzing a position~specific glucosylation of salicyl alcohol to form salicin in a cell-free preparation from G. jasminoides cell cultures. MATERIALS AND METHODS
Cell culture Cultured cells induced from leaves of G. ]asminoides were maintained in Murashige and Skoog's liquid medium [5] supplemented with 10 -6 M 2,4dichiorophenoxyacetic acid and 10-6 M kinetin at about 26°C under dim light with transfer intervals of 2 weeks.
~nzyme preparation All steps of enzyme preparation were carried out at about 4°C. Typically, wet cells (10 g) harvested during the lineargrowth phase were homogenized in a Nissei Ace Homogenizer at 18 000 mv./min for 3 min with polyvinylpyrrolidone (5 g) in 30 ml of 0.2 M glycine-NaOH buffer (pH 9.0) containing 10 m M 2-mercaptoethanol. The slurry was passed through cheesecloth and centrifuged at 10 000 × g for 20 min. Enough solid ammonium sulfate to bring the solution to 75% of saturation was added to the supernatant fluid. The resulting precipitate was dissolved in the glycine-NaOH buffer (pH 9.0) and was dialyzed against the same buffer overnight. The dialysate was used as the enzyme preparation. The enzyme preparation lost 20% of the initial activity after a week and 50% after 3 weeks when stored in the frozen state at -20°C. Protein content was determined by the method of Bradford [6] with bovine serum albumin as a standard.
Assay of glucosyltransferase activity The assay system contained 3.3 mM UDP-[U-14C]glucose (0.05 #Ci), 3.3 mM salicyl alcohol, 10 mM 2-mercaptoethanol, 200 mM glycine-NaOH (pH 9.0), and enzyme in a final volume of 0.3 ml. The reaction was allowed to proceed for 60 min at 30°C and was stopped by adding 0.2 ml of ethanol containing 15 #mol of salicin as carrier. The mixture was centrifuged at 3500 × g for 5 rain and 0.1 ml of the supernatant fluid was analyzed by thin-layer chromatography (TLC) on Silica gel 60F (Merck) by using ethyl acetate/methanol (9:1,v/v) for development. The band corresponding to salicin was detected under UV-light (254 nm), scraped off, and counted in a scintillation counter.
49
Chemicals The sources of chemicals were as follows: UDP-glucose from Sigma (St. Louis, MO), UDP-[U-~4C]glucose from The Radiochemical Center, (Amersham, U.K.) and protein assay reagent from Bio-Rad (Richmond, CA). All other chemicals were of reagent grade. RESULTS
Identification of the reaction product For identificationof the reaction product, the reaction mixture was extracted with n-butanol. The butanol extract was chromatographed on Silicagel 60F with solvent systems: (I)ethyl acetate/methanol (9: 1, v/v);(If) ethyl acetate/methanol/water (65:22:13, v/v) or (III) ethyl acetate/i-propanol/water (65:24:11, v/v), which showed that the radioactivity was associated with the position of salicinand not with the position of isosalicin. The incorporation of radioactivity into salicin was also detected by high performance liquid chromatography (HPLC) analysis as shown in Fig. 1, whereas the formation of isosalicinwas again not detected. Finally, a large scale incubation was carrried out to identify the reaction product. In a total vol. of 1.0 ml were incubated: 10 m M UDP-[U-~4C]glucose (0.4 ~Ci); 10 m M salicylalcohol; 10 m M 2-mercaptoethanol; 100 m M
a >(
~a "-'6 ._> 4
e~
e~
b I
I
I
I
2
4
6
8
I
Retention time (rain) Fig. 1. Identification by HPLC of the product formed in the reaction catalyzed by the glucosyltransferase. An aliquot of the butanol extract of the reaction mixture was injected into a Radiai-pak A (Waters) column. Separation was carried out with methanol/ water ( 2 5 : 7 5 , v/v) at a flow rate of 1.5 ml/min. (a) Fractions were collected every 30 s and were analyzed for radioactivity. (b) Chromatogram of reference compounds: (1) UDP--giucose (2) salicin (3) isosalicin (4) sallcyl alcohol; the compounds were detected through their absorbance at 270 nm.
50 Tris--HCl (pH 7.5) and 10 mg of enzyme. The reaction t o o k place for 3 h at 37°C. At the end o f the incubation period salicin (15 rag) was added to the mixture as carrier; the mixture was then extracted with n-butanol. Radioactive salicin with a constant spec. act. o f 2.1 × 103 d.p.m./#mol was obtained from the butanol extract by preparative TLC on Silica gel 6 0 F with solvent system I followed b y repeated recrystallization. This indicates formation o f salicin from salicyl alcohol and UDP~lucose in about a 13% yield.
Properties o[ the glucosy ltransferase The enzyme catalyzed formation o f salicin was linear for a b o u t 60 min and the rate of reaction was linear up to 600 pg o f protein under standard assay conditions. The enzyme exhibited normal Michaelis-Menten kinetics with both substrates. The apparent Kin-values were found to be 0.53 mM for salicyl alcohol and 0.64 mM for UDP-glucose. The enzyme activity showed a broad pH proftle ranging from pH 5.5 to 10.5. The o p t i m u m pH was 9.0 (glycine-NaOH) with half maximum activities at pH 7.5 (Tris--HCl) and 10.2 (glycine-NaOH). The temperature o p t i m u m for the reaction was found to be 50°C although 90% of the activity was lost when the enzyme preparation was treated at 50°C for 5 min, as shown in Fig. 2. The whole enzyme activity was found in the supernatant fraction when the crude cell extract obtained b y centrifugation o f the cell homogenate at 10 000 × g for 20 min was further centrifuged at 100 000 X g for 60 min. This indicates that the glucosyltransferase is a soluble enzyme. °~
150
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30
40
50
?
J
t
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30
40
N e"
J'~'"?~? 50
60
70
Temperature ( C ) Fig. 2. Temperature dependence o f the glucosyltransferase. (a) The reactions were incubated at the indicated temperatures for 60 rain. (b) Aliquots o f the enzyme preparation were treated at the indicated temperatures for 5 rain, and were then added to the reaction mixtures which were incubated at 30°C for 60 rain.
51
A
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_rex
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l
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16
Time
(day)
I
20
I
24
Fig. 3. T i m e course o f changes in the glycosyltransferase activity (• (c o ) of G. jasminoides cells.
• ) during growth
Figure 3 shows that the enzyme activity gradually increases during the exponential and linear phases o f growth and then rapidly decreases in the stationary phase. This result is consistent with the kinetics o f glucosylation o f salicyl alcohol in vivo by cultured cells o f G. jasminoides (Mizukami et al., unpublished). DISCUSSION
This is the first report o f the enzymatic formation o f salicin from salicyl alcohol and UDP-glucose. The enzyme catalyzing this reaction, namely UDPglucose : salicyl alcohol phenyl-glucosyltransferase, is shown to be a soluble enzyme although some gtucosyltransferases are reported to be b o u n d to Golgi membranes [7] or cell membranes [8] in the case of plant sterol glucosyltransferases or to be localized in cell particles [ 9] in the case of the phenol glucosyltransferase of mollusks. The pH o p t i m u m of this enzyme is high when compared with other plant glucosyltransferases so far reported [ 10--15] and is rather similar to that of the phenol glucosyltransferase of mollusks [9]. It is of interest that a detectable a m o u n t of isosalicin was not formed with the present enzyme preparation although isosalicin as well as salicin was formed in a I : 2 ratio when salicyl alcohol was administered to G. jasminoides cell cultures [4]. It m a y be that the localization and/or optimal reaction conditions of an enzyme catalyzing the formation of isosalicin from salicyl alcohol (UDP-glucose : salicyl alcohol benzyl-glucosyltransferase) are quite different from those of the phenyl-glucosyltransferase or that salicyl alcohol is n o t a direct precursor of isosalicin in cultured cells of G. jasminoides.
52 Our remits clearly demonstrate the presence of a UDP-glucose: salicyl alcohol phenyl-glucosyltransferase which has a clear specificity for the phenolic position of salicyl alcohol. The significance o f the presence of this enzyme is n o t clear because salicin is not endogeneously synthesized in G. jasminoides cultured cells, b u t the enzyme may play a role similar to t h a t of the UDP-glucuronyltransferase of vertebrates. ACKNOWLEDGEMENTS We wish to thank Dr. S.L. Nickel, University of Toledo, for reviewing the manuscript. This work was supported by a Grant-in-Aid for Scientific Research (57771464) from the Ministry of Education, Science and Culture o f Japan. REFERENCES 1 J.B. Pridham and M.J. Saltmarah, Biochem. J., 87 (1963) 218. 2 M. Tabata, F. Ikeda, N. Hiraoka and M. Konoshima, Phytochemktry, 15 (1976) 1225. 3 M.H. Zenk, Phytochemistry, 6 (1967) 245. 4 H. Mizukami, T. Terao, H. Miura and H. Ohaahi, Phytochemktry, 22 (1983) 679. 5 T. Muraahige and F. Skoog, Physiol. Plant., 15 (1962) 473. 6 M.M. Bradford, Anal. Biochem., 72 (1976) 248. 7 H.E. Hopp, P.A. Romero, G.R. Dale<) and R. Pont Lezica, Phytochemistry, 17 (1978) 1049. 8 9 10 11 12 13 14 15
M.-A. Hartmann-BouiUon, B. Walter and E. Lazar, Plant Cell Rep., 1 (1981) 56. G.J. Dutton, Arch. Biochem. Biophys., 116 (1966) 399. T. Yamaha and C.E. Cardini,Arch. Biochem. Biophys., 86 (1960) 127. A. Sutter,R. Ortmann and H. Grisebach, Biochim. Biophys. Acta, 258 (1972) 71. A. Sutter and H. Grisebach, Biochirn.Biophys. Acta, 309 (1973) 289. R.K. Ibrahim and H. Grkebach, Arch. Biochem. Biophys., 176 (1976) 700. T. Yoahikawa and T. Furuya, Phytochemistry, 18 (1979) 239. R.K. Ibrahim and B. Boulay, Plant Sci. Lett., 18 (1980) 177.