UDP-glucose:Isoflavone 7-O-glucosyltransferase from roots of chick pea (Cicer arietinum L.)

ARCHIVES OF BIOCHEMISTRY Vol.

212, No. 1, November,

AND BIOPHYSICS pp. 98-104, 1981

UDP-GlucoseAsoflavone

7OGlucosyltransferase Pea (Cicer arietinum

JOHANNES Lehrstuhl

fiir

Biochemie Received

&r

KijSTER Pjtanzen,

April

AND

Universit&

from

of Chick

L.)’

WOLFGANG Miinsier,

‘7, 1981, and in revised

Roots

form

BARZ

D&$00

M&x&r,

June

29, 1981

West Germany

A glucosyltransferase which catalyzes the glucosylation of isoflavones in position 7 using uridine diphosphate glucose as glucosyl donor has been purified about 120-fold from 4-day-old roots of chick pea (Cicer arietinum L). The soluble enzyme showed a pH optimum of 8.5-9.0 and a molecular weight of 50,600. The K, for uridine diphosphate glucose was 200 hi and for the isoflavones biochanin A and formononetin, 12 and 24 PM, respectively. While the aforementioned I’-methoxy isoflavones were the best substrates, the I’-hydroxy isoflavones genistein and daidzein were poor substrates. The enzyme was unable to catalyze the glucosylation of hydroxy substitutes isoflavanones, flavones, flavanones, tlavonols, coumarins, cinnamic acids, and benzoic acids.

Higher plants possessa pronounced capability for the glucosylation of a wide variety of secondary plant constituents. Of interest to the plant biochemist are the variation of the glycosides within the type of sugar moiety, nature of linkage, and linkage position (1). A number of studies have been carried out on glucosyltransferases for simple phenols (2,3), aromatic or phenolic acids (4-7), coumarins (8), cyanogenic glucosides and glucosinolates (9, lo), as well as flavonoids and anthocyanidins (11-21). In most cases the purified enzymes showed a pronounced specificity with respect to glycosyl moiety, type of substrate, and position of linkage. While the enzymology of flavonoid and anthocyanidin glycoside biosynthesis has well been elucidated (22), less information is available on the enzymes involved in isoflavone glycoside formation and metabolism. Thus, an isoflavone 4’-O-methyltransferase (23) and a set of isoflavone 70-glucoside-specific glucohydrolases (24) have been purified from Cicer arietinum. These glucohydrolases are mainly local1 This work was schungsgemeinschaft Naturstoffe/Zellkulturen).

ized in the cortex and rhizodermal cells of the roots (25), where the bulk of the isoflavone glucosides of biochanin A and formononetin (see Fig. 1) are also found. In this paper we report the partial purification and properties of a glucosyltransferase from C. arietinum roots catalyzing the 7-O-glucosylation of the endogenous isoflavone constituents with UDP-Glc as glucose donor. MATERIALS

O603-9861/81/136&%07$02.00/0

METHODS

Chemicals. UDP-[U-‘4Clglucose (sp. radioact, 240 mCi/mmol) was bought from Amersham Buchler, Braunschweig, West Germany, and [G-3H] biochanin A (42.3 mCi/mmol) stemmed from previous work (26). UDP-glucose, ADP-glucose, CDP-glucose, TDPglucose, UDP-xylose, UDP-glucuronic acid, and reference proteins were purchased from Sigma Chem-

FIG. 1. Chemical formulas of the most efficient substrates of the isoflavone 7-O-glucosyltransferase and their respective products. Biochanin A (R = OH) and formononetin (R = H). The I’-hydroxy isoflavones mentioned in the test are genistein (R = OH) and daidzein (R = H).

supported by the Deutsche For(Forschergruppe SekundHre

Copyright0 1981by AcademicPress.Inc. All rights of reproductionin anyform reserved.

AND

98

Cicev arietinum

L. ISOFLAVONE

7-0-GLUCOSYLTRANSFERASE

ical Company, St. Louis, Missouri, and BoehringerMannheim, Mannheim, West Germany. Samples of flavonoids, phenolics, biochanin A 7-O-glucoside,, and formononetin ‘I-0-glucoside were from the institute’s collection or previous studies (24). Chromatography. Silica gel thin-layer plates with fluorescence indicator GF;, (Merck, Darmstadt, W. Germany) were developed with the following solvent systems (v/v): Si n-butanol:acetic acidwater (4:l:l); S2 benzene:methyl ethyl ketone:methanol (7:1:3); Ss ethyl acetate:methyl ethyl ketone:formic acidwater (5:3:1:1); Sq ethyl acetate:water:acetic acid (8:l:l); Ss benzene:methanol (82); Sg benzene:ethyl acetate:methanol (1:4:5); S7 chloroform:methanol:methyl ethyl ketone (12:2:1); S8 chloroform:methanol:methyl ethyl ketone (6:2:1). Radioactivity measurement. Radioactive compounds were located on thin-layer plates by scanning (LB 2723 scanner, Berthold-Frieseke, Waldbad, W. Germany), eluted from the gel and measured by scintillation spectrophotometry (Betazint BF 5000, Berthold) using a toluene scintillator (5 g PPO’ in 1 liter toluene). Protein estimatiun. The protein content of all fractions was determined by the method of Bradford (27). P&t material. Commercially available seeds of C. arietinum were germinated on wet filter paper under running tap water at 20°C for 4 days and only the roots were taken for enzyme extraction. Enzyme purificatia. All purification steps were carried out at 4°C unless otherwise mentioned. All buffers contained 40 mM 2-mercaptoethanol. Root material (250 g) was homogenized in a Waring Blendor with 125 g wet Polyclar AT and 800 ml 0.2 M Tris/ HCl buffer, pH 8.5. The homogenate was squeezed through a linen cloth and the effluent centrifuged for 30 min at 30,OOOg.To the supernatant (1030 ml) was added solid ammonium sulfate to 40% saturation. After stirring for 1 h protein was collected by centrifugation for 20 min at 20,OOOg.The precipitate was dissolved in 40 ml 0.2 M Tris/HCl buffer, pH 8.5, and the solution desalted by filtration through a Sephadex G-26 column (4.5 X 40 cm) which had been equilibrated and was developed with 0.02~ Tris/HCl buffer, pH 8.5. The column eluate (350 ml) was applied to a DEAE-Sephacel column (2.5 x 62 cm) previously equilibrated with 0.02 M Tris/HCl buffer, pH ’ Abbreviations used: PPO, 2,5-diphenyloxazol; DEAE, diethylaminoethyl; SDS, sodium dodecylsulfate; TLC, thin-layer chromatography; DTE, Dithioerythritol.

PURIFICATION

99

8.5. After extensive washing with the equilibration buffer, protein was eluted with a linear gradient (O0.5 M NaCl in 0.02 M Tris/HCl buffer, pH 8.5, total volume 800 ml) at a flow rate of 40 ml/h and a fraction volume of 6 ml each. Fractions 175 to 220 with the highest glucosyltransferase activity were pooled, concentrated by ultrafiltration, and desalted on Sephadex PD-10 columns with 0.02 M Tris/HCl buffer, pH 8.5. The eluate of this column (30 ml) was applied to a second DEAE-Sephacel column (2.5 X 41 cm). This time, protein was eluted with a linear gradient (O-O.4 rdNaC1 in Tris/HCl buffer, pH 8.5, total volume 600 ml) at a flow rate of 40 ml/h and a fraction size of 4 ml each. Fractions 105 to 125 were pooled and again concentrated by ultrafiltration. The concentrate of these fractions, which showed the highest specific glucosyltransferase activity, was further chromatographed on an Ultrogel AcA 34 column (2.6 X 80 cm) with 0.2 IUTris/HCl buffer, pH 8.5, at a flow rate of 20 ml/h and a fraction volume of 2.4 ml each. Fractions 120 to 150 of this column were also pooled, and, after concentration to 10 ml by ultrafiltration, this solution was used to determine the properties of the glucosyltransferase. For storage purposes this solution was frozen at -20°C in l-ml samples after the addition of 20% (v/v) glycerol. Assay of enzyme activity. The standard assay mixture for glucosyltransferase activity contained 200 nmol of UDP-[U-i4Clglucose (0.05 pCi), 20 nmol biochanin A (dissolved in 10 ~1 of methanol), 8 pmol 2mercaptoethanol, 0.2 M Tris/HCl buffer, pH 8.5, and 10 to 100 ~1 enzyme solution (depending on the state of purification) in a total volume of 200 ~1. The reaction was started by the addition of protein and terminated, after incubation at 30°C for 40 min, by adding 150 ~1 of a methanolic solution of biochanin A ‘I-0-glucoside. The resulting mixture was applied to a thin-layer plate and the plate devleoped in Sg. The bands of biochanin A 7-0-glucoside and UDP[U-“Clglucose were assayed for radioactivity. Enzyme assays for determination of glycosyl donor specificity contained, in a total volume of 200 ~1, 5 nmol [G-3Hjbiochanin A (0.085 &i, dissolved in 10 ~1 of methanol), 200 nmol glycosyl nucleoside, 8 amol 2-mercaptoethanol, 0.2 M Tris/HCl buffer, pH 8.5, and 20 rl of enzyme solution. Incubation conditions and radioactivity measurements were essentially the same as described for the standard assay. Enzyme assays for determination of acceptor specificity were carried out as described for the standard procedure with 20 nmol phenolic substrate and 200 nmol UDP-[U-‘4Clglucose (0.5 &i). The thin-layer plates for these assays were developed with Ss except for biochanin A and formononetin, where Ss was used. Determination of mdecular weight. The molecular weight of the glucosyltransferase was determined by

KCSTER AND BARZ

100

gel chromatography on an Ultrogel AcA 34 column (2.6 X 30 cm) with 0.2 M Tris/HCl buffer containing 40 mM 2-mercaptoethanol according to Andrews (23). Reference proteins (5 mg each of chymotrypsinogen (MW W,OOO), egg albumin (MW 45,000), and bovine serum albumin (MW 67,000)) were separately chromatographed at a flow rate of 20 ml/h and a fraction volume of 2.4 ml. The void volume of the column was determined with dextran blue (MW 2 X 106). After gel chromatography the reference proteins were determined by the Bradford method (27) and the glucosyltransferase was assayed for by the standard procedure. Ultracentrifugation was performed according to Martin and Ames (37) using cytochrome c (MW 12,300), horse myoglobin (MW 17,300), and catalase (MW 240,000), respectively, as reference proteins. Gel ekztrophoresis. SDS-gel electrophoresis was performed after each purification step according to the method by Weber and Osborn (29). Isolelectic focusing.The determination of the isoelectric point of the glucosyltransferase was performed according to the procedure by Vesterberg (30) in a pH range of 3.5 to 10. RESULTS

Purification

of the Glucosyltransferase

The present studies have shown that the roots of chick pea plants (C. arietinum) between Days 3 and 6 after germination are a good source of the isoflavone 7-Oglucosyltransferase. As previously determined, this is the period of maximum accumulation of isoflavone glucosides (31). The enzyme was found to be readily soluble because the supernatant solutions of a protein extract after centrifugation at 30,0009 and at llO,OOOg, respectively, showed essentially identical enzyme activity, with the microsomal precipitate being devoid of any enzyme activity. The purification of the enzyme (Table I) has led to a preparation with a specific activity about 120 times greater than that of the crude homogenate. The introductory fractionation with ammonium sulfate to 40% saturation considerably separated the glucosyltransferase from the isoflavone 7-0-glucoside-specific glucosidase, which mainly precipitated between 40 to 65% saturation. The best enrichment of the enzyme was obtained by two subsequent chromatography steps on DEAE-

Sephacel. The second DEAE-Sephacel column chromatography resulted in two activity peaks (Fig. 2) with very different specific activities (Table I). Only fraction I was carried through the following purification procedure. The enzyme preparation from peak II was not purified any further because it showed a low specific enzyme activity with biochanin A (Table I) and no other phenolic substrate with a better specificity than the isoflavone could be found. As a final step of purification, chromatography on Ultrogel AcA 34 was chosen (Fig. 3) due to its more rapid flow in comparison to Sephadex G-200. This final protein preparation was used for the experiments described below. When subjected to SDS-gel electrophoresis this final preparation still exhibited several protein bands after staining with Coomassie blue R 250. The number of these bands could not be lowered upon further purification steps (Sephadex G-200, hydroxylapatite). Alkaline disc electrophoresis could not be applied because the bulk of the enzyme protein did not move from the side of the cathode. Staining for glycoprotein according to Racusen (33) showed negative results. Properties

of the Pur$ed

Enzyme

pH Optimum. The optimum pH for the glucosylation of the isoflavones determined using several different buffer systems was about 8.5-9.0 (Fig. 4). Protein and time linearity. The rate of isoflavone glucosylation as catalyzed by 3.5 pg of the partially purified enzyme was linear from at least 50 min and proportional to the amount of protein added up to at least 10.5 pg. Ident$catian of Product Enzymatically formed biochanin A glucoside was shown by TLC (solvents So-Ss) to be a homogeneous product which cochromatographed with the known biochanin A ‘I-0-glucoside (24). Upon spectrophotometric studies with diagnostic reagents (32) the product showed the ab-

Cicer

arietinum

L. ISOFLAVONE

7-0-GLUCOSYLTRANSFERASE TABLE

101

PURIFICATION

I

PURIFICATION OF UDP-GLUCOSLQ~SOFLAVONE 7-O-GLUCOSYLTRANSFERASE~

Fraction Crude homogenate Sephadex G-25 column after (NH,),SOI fraction DEAE-Sephacel column I DEAE-Sephacel column II, fraction I Fraction II Ultrogel AcA 34 from fraction I

Total protein bg)

Total activity (nkat)

Yield (96)

Specific activity (&at&z)

1498

17.4

166

11.6

1

534

30.4

175

56.96

4.9

42

11.5

66

275.15

23.7

7.6 27.5

4.5 4.1

26 24

587.4 149.6

50 13

2.1

2.9

17

1388.63

129

Enrichment

a Conditions for column chromatography are described under Materials and Methods. 2-Mercaptoethanol was routinely added to the protein preparations to avoid rapid enzyme denaturation.

sorption values essentially as described for an isoflavone 7-0-glucoside. Enzymatically formed biochanin A [U-14Cl@;lucoside was completely hydrolyzed by a sample of the previously described isoflavone 7-Oglucoside-specific glucosidase (24) with more than 99% of the liberated radioactivity being found in glucose (TLC, Ss). Enzymatically produced samples of formononetin 7-0-glucoside were similarly characterized by cochromatography with \

FIG. 2. Separation of two glucosyltransferases (fractions I and II) by DEAE-Sephacel chromatography. Column chromatography and assay for glucosyltransferase activity (0 ~ 0) and protein (A---A) were carried out as described under Materials and Methods.

reference material and enzymic hydrolysis by the aforementioned specific glucosidase. Due to lack of sufficient amount of material the enzymatically produced samples of genistein glucoside and daidzein glucoside (see Fig. 1) were not characterized with respect to the position of glucose linkage. Isoelectric

point

The isoflavone 7Gglucosyltransferase was shown to possess an isoelectric point at pH 5.4. Molecular weight. With the use of several reference proteins and an Ultrogel AcA 34 column the molecular weight of

JJ

r’ ::

.:

: 20 ” ‘0° “’ “’ ‘roC”On FIG. 3. Chromatography of isoflavone 7-0glucosyltransferase on Ultrogel AcA 34. Glucosyltransferaae activity (0 0) and protein (x - - - x) were measured as described under Materials and Methods.

102

K&TER

the isoflavone 7-O-glucosyltransferase was found to be 50,000. Upon ultracentrifugation in a linear sucrose gradient a molecular weight of 42,000 + 6000 was determined with cytochrome c, myoglobin, and catalase as reference proteins [37]. Enzyme stability. Upon storage at +4”C crude protein preparations lost some 50% of the enzyme activity within 5 days. This loss of activity could partially be reduced by the addition of 40 mM 2-mercaptoethan01 (50% loss of total enzyme activity in 9 days) which was therefore routinely used during all purification steps. DTE appeared to be an even better stabilizer leading to 85% (5 mM) or 150% (10 mM) of the orginal total enzyme activity. With increased purity the enzyme preparations became more stable so that the eluate from the final column (Table I) could be stored at -20°C in 20% glycerol for several months without any decrease in enzyme activity. Optimal enzyme stability was determined to be in the range of the pH optimum of 8.5-9.0. Addition of 2-mercaptoethanol at a concentration of up to 60 mM almost doubles the conversion of biochanin A to its 7-O-glucoside. Glucosyl donor spec$city. Various glucosyl donors such as UDP-Glc, ADP-Glc, CDP-Glc, and TDP-Glc, as well as the glycosyl donors UDP-glucuronic acid and UDP-xylose were tested with the purfied isoflavone glucosyltransferase. UDP-Glc was found to be the only active glucosyl donor and its apparent K, value of 200 PM was measured at a biochanin A concentration of 0.1 IrIM. Acceptor spec$icity. The apparent K,,, and maximum velocity values for the isoflavones biochanin A and formononetin measured by the method of Lineweaver and Burk were found to be 12.6 and 24 PM and 1615 and 1040 pkat/kg protein, respectively. These values were determined at a UDPG concentration of 0.2 mM. The isoflavones genistein and daidzein are comparatively poor substrates. This is indicated by the ratio of the relative velocity values for biochanin A, formononetin, genistein, and daidzein which were found to be 100:80:8.3:15.6, respectively. 6.7Dihydroxy-4’-methoxyisoflavone (texasin)

AND BARZ

7

8

9

10

PH

FIG. 4. Effect of pH on the isoflavone ‘I-O-glucosyltransferase activity. Biochanin A and UDP{U‘%lglucose were incubated with enzyme as derived from the Ultrogel AcA 34 column under the conditions mentioned in Materials and Methods with the following buffers: 0.5 M KH2P04-K2HP04 (x x), 0.5 M Tris/HCl (0 0), and 0.5 M glycine-NaOH (A-A).

and 4’,8-dimethoxy-5,7-dihydroxyisoflavone were not glucosylated by the glucosyltransferase. Other plant phenolics such as numerous isoflavanones, flavones, flavanones, chalcones, and flavonols with both a phloroglucinol and a resorcinol type of substitution pattern in ring A and/or a 4’-hydroxy substituent in ring B were not glucosylated by the purified enzyme preparation. Various mono- or poly-hydroxysubstituted coumarins, cinnamic acids, and benzoic acids were also shown not to serve as substrates for the glucosyltransferase. In these studies the sensitivity of the applied assay allowed the determination of a minimum conversion of substrate in the range of 0.6 nM (i.e., 250 cpm). DISCUSSION

The data in this study demonstrate the isolation from C. arietinum of a glucosyltransferase which as compared to other

Cicer arietinum

L. ISOFLAVONE

7-0-GLUCOSYLTRANSFERASE

glucosyltransferases (1) is remarkably specific, because the enzyme catalyzes the transfer of glucose exclusively to the 7hydroxyl group of an isoflavone. The 4’methoxy isoflavones, which are the main phenolic constituents in chick pea roots (34) were found to be by far the best substrates yielding low K, values. These data together with the observations obtained with the isoflavone 4’-O-methyltransferase (23) seem to indicate that glucosylation of the 4’-methoxy isoflavones biochanin A and formononetin is the final reaction in the biosynthesis of the isoflavone glucosides as compared to the 4’methylation of the 7-0-glucosides of genistein and daidzein. Although the enzyme has not been purified to homogeneity the results clearly indicate that the purification procedure employed has led to a protein preparation with a single glucosyltransferase only. Furthermore, other enzyme species of comparable specificity for a 4’-methoxy isoflavone could not be found in the root extracts. The enzyme is best isolated from root tissue and its activity is exceptionally high in young roots at times of intensive isoflavone biosynthesis ((31), unpublished). With regard to K, values, alkaline pH optimum and a molecular weight in the range of 42,000-50,000 the data obtained for the isoflavone 7-0-glucosyltransferase are in agreement with several other plant glucosyltransferases (1, 11, 35, 36). The early precipitation of the glucosyltransferase with ammonium sulfate (O-35% saturation) has greatly facilitated the separation of this enzyme from the previously described isoflavone 7-0-glucoside glucosidases (24). Such precipitation behavior of the glucosyltransferase is in contrast to most other glucosyltransferases (11, 15) which require much higher ammonium sulfate concentrations for precipitation. Further studies on the cellular compartmentation and tissue localization as well as on the metabolic interplay between the isoflavone 7-0-glucosyltransferase and the above-mentioned specific glucosidases in C. arietinum root tissue will lead to a better understanding of isoflavone and is-

103

PURIFICATION

oflavone glucoside metabolism in this organ (25, 31). Another main question to be answered is whether the isoflavone aglycones and the isoflavone glucosides are permanently interconverted or whether they show independent rates of turnover. REFERENCES 1. HOESEL, W. (1981) in The Biochemistry of Plants (Conn, E. E., and Stumpf, P. K., eds.), Vol. 7, Academic Press, New York, in press. 2. PRIDHAM, J. B. (1964) Phytochemistry 3,493-497. 3. TABATA, M., IKEDA, F., HIRAOKA, N., AND KoNOSHIMA, M. (1976) Phytochemistry 15, 12251229. 4. KLEINHOFS, A., HASKINS, F. A., AND GORZ, H. J. (1967) Phytochemistry 6, 1313-1318. J. J. (1977) C. R. Hebd. Seances Acad. 5. MACHEIX,

Sci. Ser. D 284, 33-36. 6. STRACK, D. (1980) Z. Naturforsch. 35~. 204-208. 7. MICHALCZUK, L., AND BANDURSKI, R. S. (1980) Biochem. Biophys. Res. Commun. 93,588-592. 8. IBRAHIM, R. K., AND BOULAY, B. (1980) Plant Sci. Lett. 18, 177-184. 9. REAY, P. F., ANDCONN, E. E. (1974) J. Biol. Chem. 249, 5826-5830. 10. MATSUO, M., AND UNDERHILL, E. W. (1971) Phytochemistry 10.2279-2286. 11. SURER, A., ORTMANN, R., AND GRISEBACH, H. (1972) Biochim. Biophys. Acta 258, 71-87. 12. SUTPER, A., AND GRISEBACH, H. (1973) Biochim.

Biophys. Acta 309, 289-295. 13. FRITSCH,

H., AND GRISEBACH,

H. (1975)

Phyto-

chemistry 14, 2437-2442. 14. SALEH, N. A. M., POULTON, J. E., ANDGRISEBACH, H. (1976) Phytochemistry 15, 1865-1868. 15. SALEH, N. A. M., FRITSCH, H., WITKOP, P., AND GRISEBACH, H. Planta 133.41-45. 16. POULTON, J. E., AND KAUER, M. (1977) Planta 136, 53-59. 17. KHO, K. F. F., KAMSTEEG, J., AND VAN BREDERODE, J. (1978) Z. Pflanzenphysiol. 88,449-464. 18. KAMSTEEG, J., VAN BREDERODE, J., AND VAN NIGHTERECHT, G. (1978) B&hem. Genet. 16, 1045-1058. J. G., AND MCCLURE, 19. BLUME, D. E., JAWORSKI, J. W. (1979) Planta 146, 199-202. 20. SHUTE, J. L., JOURDAN, P. S., AND MANSELL, R. L. (1979) Z. Naturforsch. 34c, 738-741. 21. DOONER, H. K. (1979) Phytochemistry 18, 749751. 22. HAHLBROCK, K., AND GRISEBACH, H. (1979) Recent Advan. Phytochem. 12, 221-248. 23. WENGENMAYER, H., EBEL, J., ANDGRISEBACH, H. (1974) Eur. J. B&hem. 50,135-143. W., AND BARZ, W. (1975) Eur. J. Biochem. 24. H&EL, 57,607-616.

104

KOSTER

25. GIERSE, H. -D., AND BARZ, W. (1976) Protoplasma 88, l-6. 26. BERLIN, J., KISS, P., MILLER-ENOCH, D., GIERSE, D., BARZ, W., AND JANISTYN, B. (1974) 2. Nuturforsch. 29c, 374-383. 27. BRADFORD, M. M. (1976) Ad Biochem. 72,249254. 28. ANDREWS, P. (1964) B&hem. J. 91,222-233. 29. WEBER, K., AND OSBORN, M. (1969) J. BioL Chem. 244,4406-4412. 30. VESTERBERG, 0. (1971) in Methods in Enzymology (Jakoby, W. B., ed.), Vol. 22, pp. 389-412, Academic Press, New York.

AND

BARZ

31. BARZ, W. (19’75) Ber. Dtsch. Bot. Gee. 88,71-81. 32. MABRY, T. J., MARKHAM, K. R., AND THOMAS, M. B. (1970) in Systematic Identification of Flavonoids, Springer-Verlag. Berlin. 33. RACUSEN, D. (1979) Anal. B&hem. 99,474-476. 34. WONG, E., MORTIMER, P. I., AND GEISSMAN, T. A. (1965) Phytochemistry 4.89-95. 35. HRAZDINA, G., ALSCHER-HERMAN, R., AND KISH, V. M. (1980) Phytochemistry 19.1355-1359. 36. HRAZDINA, G., WAGNER, G. J., AND SIEGELMAN. H. W. (1978) Phytochemist7y 17,53-56. 37. MARTIN, R. G., AND AMES, B. N. (1960) J. BioL

Chem. 236,1372-1379.