N. Katano et al. / Phytochemistry 58 (2001) 53–58
57
Japan) and 20 unit catalase (Sigma, USA) (Kochs and
Grisebach, 1987). After 5 min, the incubation was initi-
ated by addition of 250 nmol NADPH and continued as
described above. As a comparative experiment, heat-
inactivated glucose oxidase was used.
were 170 mM and 12.47 pkat/mg protein and for
NADPH 18 mM and 8.33 pkat/mg protein, respectively.
Acknowledgements
We are grateful to Ms. K. Arakawa, Kyoto Analytical
Applications Center, Shimadzu Corporation for the
measurement of LC–MS spectrum. Financial support
by Japanese Bioindustry Association is gratefully
acknowledged.
3.7. Inhibition experiments
The P450 inhibitors, ketoconazole, miconazole,
metyrapone, ancymidol and cytochrome c were tested.
Metyrapone and cytochrome c were dissolved in H2O
and others were dissolved in DMSO. KCN was dis-
solved in 100 mM Tris–HCl buffer (pH 7.5). The con-
centrations of inhibitors were adjusted to 1 or 0.1 mM
with each solvent. The incubation mixture contained
250 nmol 7-deoxyloganin, 250 nmol NADPH, 175 ml
enzyme (24.95 pkat/mg protein) and 25 ml the solution
of each inhibitor in total volume of 250 ml and con-
tinued as described above.
References
Battersby, A.R., 1967. Biosynthesis of the indole and colchicum alka-
loids. Pure and Applied Chemistry 14, 117–136.
Battersby, A.R., Burnett, A.R., Parsons, P.G., 1970. Preparation and
isolation of deoxyloganin: its role as precursor of loganin and the
indole alkaloids. Journal of the Chemical Society Chemical Com-
munications, 826–827.
3.8. HPLC analysis
Battersby, A.R., Westcott, N.D., Glusenkamp, K.-H., Tiezte, L.-F.,
1981. Untersuchungen zur Biogenese der Indolalkaloide. Synthese
und Verfuetterung radioaktiv markierter Hydroxyloganin-Derivate.
Chemische Berichte 114, 3439–3447.
The amount of loganin was determined by reversed
phase HPLC using Bio-Sil C18 HL 90-5 S column (5
mm, 4.6Â250 mm, Bio-Rad, USA) in an oven at 40ꢀC,
with a CH3CN/H2O linear gradient solvent system,
from 10% MeCN to 30% MeCN in 40 min at a flow
rate of 1.0 ml/min, monitoring the absorption at 240
nm. The quantities were calculated from the peak area
at 240 nm recorded by Chromatopac C-RA4 (Shi-
madzu, Japan). Rt. of loganin, 12.8 min, Rt. of 7-deox-
yloganin, 32.8 min.
Bianco, A., Guiso, M., Iavarone, C., Passacantilli, P., 1978. Iridoids
XXV. New iridoid glucosides from Rubiaceae. Gazzetta Chimica
Italiana 108, 13–16.
Bock, K., Jensen, S.J., Nielsen, B.J., 1976. Secogalioside, an iridoid
glucoside from Galium album Mill. and carbon-13NMR spectra of
some secoiridoid glucosides. Acta Chemica Scandinavica Ser. B 30,
743–748.
Bolwell, G.P., Bozak, K., Zimmerlin, A., 1994. Plant cytochrome
P450. Phytochemistry 37, 1491–1506.
Bouwmeester, H.J., Konings, C.J.M., Gershenzon, J., Karp, F., Cro-
teau, R., 1999. Cytochrome P-450 dependent (+)-limonene-6-
hydroxylation in fruits of caraway (Carum carvi). Phytochemistry
50, 243–248.
3.9. Identification of the reaction product
Bradford, M.M., 1976. A rapid and sensitive method for the quanti-
tation of microgram quantities of protein utilizing the principle of
protein-dye binding. Analytical Biochemistry 72, 248–254.
De Carolis, E., De Luca, V., 1994. 2-Oxoglutarate-dependent dioxy-
genase and related enzymes: biochemical characterization. Phy-
tochemistry 36, 1093–1107.
The identification of the product was carried out on
the basis of UV and MS spectra obtained with Shimadzu
SPDM-6A HPLC-photodiode array system (Shimadzu,
Japan) as well as Shimadzu high performance liquid chro-
matograph–mass spectrometer LCMS-QP8000. The
HPLC condition for the LC–MS system were as follows:
column, Shim-pack VP-ODS (5 mm, 2.0Â150 mm); sol-
vent, CH3CN/H2O; linear gradient solvent system, from
10% MeCN to 30% MeCN in 30 min at a flow rate of
0.2 ml/min, monitoring the absorption at 240 nm oven
temp., 40ꢀC, Rt. of loganin, 9.5 min. Negative FAB–MS
m/z; 389 ([MÀH]À).
Fujita, M., Oba, K., Uritani, I., 1982. Properties of a mixed function
oxygenase catalyzing ipomeamarone 15-hydroxylation in micro-
somes from cut-injured and Ceratocystis fimbriata-infected sweet
potato root tissues. Plant Physiology 70, 573–578.
Funk, C., Croteau, R., 1993. Introduction and characterization of a
cytochrome P-450-dependent camphor hydroxylase in tissue cul-
tures of common sage (Salvia officinalis). Plant Physiology 101,
1231–1237.
Hallahan, D.L., Dawson, G.W., West, J.M., Wallsgrove, R.M., 1992.
Cytochrome P-450 catalyzed monoterpene hydroxylation in Nepeta
mussinii. Plant Physiology and Biochemistry 30, 435–443.
Hashimoto, T., Yamada, Y., 1986. Hyoscyamine 6b-hydroxylase, a 2-
oxoglutarate-dependent dioxygenase, in alkaloid-producing root
cultures. Plant Physiology 81, 619–625.
3.10. Determination of kinetic parameters
The apparent Michaelis constant (Km) and maximal
reaction velocity (Vmax) for 7-deoxyloganin was deter-
mined by Lineweaver–Burk plots at 0.04, 0.08, 0.16,
0.32 mM, using 250 nmol NADPH and those for
NADPH at 0.02, 0.04, 0.08, 0.16 mM, using 250 nmol 7-
deoxyloganin. Km and Vmax values for 7-deoxyloganin
Inoue, K., Takeda, Y., Tanahashi, T., Inouye, H., 1981. On the pos-
sibility of the intermediacy of 10-hydroxyloganin in the biosynthesis
of secologanin. Chemical Pharmaceutical Bulletin 29, 981–990.
Inoue, K., Tanahashi, T., Inouye, H., Kuwajima, H., Takaishi, K.,
1989. Intermediacy of 6-hydroxyloganin in the ring cleavage course
of loganin to secologanin. Phytochemistry 28, 2971–2979.