our laboratory [23]. More studies will be necessary to determine the possibility of cross-talk and ex-
change of intermediates between the MVA and MEP pathways in the formation of different ter-
penoid classes, in particular in relation to the differentational and physiological state of the cell.
Imbault et al. [24] showed that the inhibition of endogenous MVA production by pravastatin
treatment blocked the alkaloid biosynthesis; sup- plying such cells with exogenous MVA allowed
accumulation of alkaloids, but did not result in incorporation of MVA. Recently, it was suggested
that isoprenylated proteins are involved in the regulation of secologanin biosynthesis. These
proteins may thus form the link between the MVA and the MEP pathway in alkaloid biosynthesis
[25]. The MVA pathway thus seems to play an essential role in the biosynthesis of terpenoid-in-
dole alkaloids, either in a direct or an indirect way.
Therefore, in continuation of our studies on the early steps of isoprenoid biosynthesis [26] atten-
tion was focused on MK. MK was studied in C. roseus plants and suspension cultured cells grown
under standard conditions and on a medium known to induce the accumulation of terpenoid-
indole alkaloids [27].
2. Materials and methods
2
.
1
. Chemicals and enzymes Polyvinylpolypyrrolidone
PVPP, phospho-
enolpyruvate PEP, bovine serum albumin BSA and the mixed solution of pyruvate kinaselactate
dehydrogenase PKLDH rabbit muscle enzymes in ammonium sulphate or glycerol were obtained
from Sigma. Sucrose, KCl, MgCl
2
·6H
2
O, 1,4- dithiothreitol DTT, ATP and NADH were ob-
tained from Merck. KF, Tris Ultrapure and mevalonolactone were obtained from J.T. Baker,
GibcoBRL and Fluka, respectively. Mevalonolac- tone was hydrolyzed to the free acid according to
Popja´k [28].
2
.
2
. Biological materials
2
.
2
.
1
. Plants C. roseus roseus L. G. Don plants were grown
in the greenhouse. Growth conditions were: tem- perature 24°C, humidity 60 and in summer nor-
mal daylight.
2
.
2
.
2
. Suspension cultured cells Cell suspensions of C. roseus were grown on MS
medium [29] without growth regulators and con- taining 3 sucrose. The cultures were kept under
continuous light ca. 1200 lx at 100 rpm and 25°C, and were subcultured weekly by weighing 5
g cells fresh weight fr. wt. in 50 ml medium. C. roseus cells grown for 14 days on MS medium
were transferred to MS medium or IM 2 medium [27] by weighing 20 g cells fr. wt in 500 ml
medium. Due to practical constraints the experi- ment was performed in batches.
2
.
3
. Sampling for MK acti6ity
2
.
3
.
1
. Plants Two plants were collected and subdivided in
flowers and buds, young leaves up to 3 cm long, completely elongated leaves, stem, roots and
fruits. The flowers and buds from each plant were pooled because of the small quantities. Extracts
were prepared and the MK activity was deter- mined by means of the radiochemical assay see
below.
2
.
3
.
2
. Cell suspensions
:
effects of media and inoculum size
At the days indicated in Fig. 2, cells from two flasks were harvested, frozen separately in liquid
nitrogen and stored at − 80°C until further use. This was done for the three growth conditions
mentioned above. Determinations were made of accumulation of fr. wt. and dry wt. after lyophili-
sation and MK activity in the acetone precipitate by means of spectrophotometrical assay see be-
low. In addition, the alkaloid accumulation was monitored in the samples collected at the days
indicated in Fig. 3.
2
.
3
.
3
. Variation during the day For the determination of the variation of MK
activity during the day, two flasks cultured under standard growth conditions were harvested every 4
h during the exponential phase of the growth cycle; the cells were frozen in liquid nitrogen and
stored at − 80°C until further use. Determinations were made of fr. wt. and dry wt. accumulation and
MK activity in the crude extract by means of a radiochemical assay see below.
2
.
4
. Enzyme extraction Crude extracts were prepared by grinding the
frozen biomass in a precooled − 20°C mortar in the
presence of
sea-sand and
polyvinyl- polypyrrolidone 10 wt.fr. wt. each and in the
presence of homogenisation buffer HB in a ratio of 2 ml buffer to 1 g fr. wt. The HB consisted of
0.1 M Tris – HCl, pH 7.5, 0.1 M sucrose, 50 mM KCl and 10 mM 1,4-dithiothreitol. All manipula-
tions were performed at 4°C and the materials, extracts and buffers were kept on ice. The crude
extracts were centrifuged at 3500 × g for 30 min. at 4°C, and the supernatants were filtered over Mira-
cloth to remove any floating particles. Where indi- cated, the proteins were precipitated with cold
acetone − 20°C to 60 vv, and collected by centrifugation at 3500 × g for 30 min. at 4°C. The
protein precipitate was resuspended in 1:1 water diluted HB, and subsequently centrifuged at 13 000
g for 5 min. at 4°C. The protein concentrations were determined by the method of Peterson [30].
2
.
5
. Enzyme assays MK activity was measured by means of a spec-
trophotometrical assay and a radiochemical assay, both principally according to Popja´k [28].
2
.
5
.
1
. Spectrophotometrical assay Enzyme solution was incubated at 30°C with
reaction mixture to determine base-line activities, e.g. ATPases and NADH oxidases. Subsequently,
MVA was added to start the measurement of MK activity. The decrease in absorption at 340 nm was
monitored for both the base line and MK activities for at least 2 min, when stable. Final concentrations
in a 0.5 ml final volume were: 3.3 mM MVA R,S, 2.3 mM ATP, 3.5 mM MgCl
2
, 3.5 mM KF, 0.3 mM phosphoenolpyruvate, 0.14 mM NADH, 5.75 U
LDH, 4 U PK, and 3.5 mM 1,4-dithiothreitol in 34.5 mM Tris – HCl, pH 7.5.
2
.
5
.
2
. Radiochemical assay Enzyme solution 10 ml was incubated at 30°C
with MVA in a reaction mixture giving final con- centrations of 0.5 mM MVA R,S 59.24 MBq
mmol [2-
14
C]MVA, 2 mM ATP, 2 mM MgCl
2
, 2 mM KF and 50 mM Tris – HCl, pH 7.5, in a total
volume of 20 ml. The reaction was started by addition of MVA and stopped after 15 min
by addition of 5 ml 72 TCA. The samples were applied to Silica F
254
TLC plates after centri- fugation at 16 000 × g for 2 min. The plates
were developed
in ethanol – ammonia – water
80:12.5:7.5. The radioactivity on the TLC plate was visualised and quantified using a beta-scope.
MK activity was calculated from the sum of the radioactivity in the phosphorylated products. R
f
values were 0.79, 0.18, 0, and 0.05 for MVA, 5 - phosphomevalonate, 5 - diphosphomevalonate
and IPP, respectively.
2
.
6
. Alkaloid determination The alkaloid extraction was performed as previ-
ously described [31]. Freeze-dried cell material 50 mg was extracted twice with 5 ml dichloromethane
and the extracts were combined. To reach extrac- tion of tryptamine, 0.5 ml 1 M NaOH was added
to the remaining biomass and the extraction was repeated with 5 ml dichloromethane. Medium, 2
ml, was extracted with 4 ml dichloromethane; 3 ml extract were collected. The solvent was evaporated
and the residues were dissolved in 0.5 ml HPLC eluent 5.52 g NaH
2
PO
4
.H
2
O in 800 ml water, 150 ml acetonitrile, and 50 ml 2-methoxy-ethanol, pH
set to 3.9 using concentrated phosphoric acid. The alkaloids were identified by HPLC analysis using
photodiode-array detection [32]. Retention times: ajmalicine 21 min., vindolinine 8.3 min., catha-
ranthine 17.8 min, and tabersonine 25.4 min.
2
.
7
. Statistical analysis The data for the determination of the variation
of MK activity during the day, were grouped according to the time of harvesting the cells, yield-
ing six groups with harvesting times at 4, 8, 12, 16, 20 and 24 h, respectively. The variances were
determined to be equal according to the Bartlett’s test. The statistical significance between means was
assessed by the Kruskal – Wallis test and the Dun- can’s multiple range test.
3. Results and discussion
