Purification and Properties of ent-Kaurene - Plant Physiology

Plant Physiol. (1 995) 109: 1239-1 245

Purification and Properties of ent-Kaurene Synthase B from Immature Seeds of Pumpkin'

Tamio Saito, Hiroshi Abe, Hisakazu Yamane, Akira Sakurai, Noboru Murofushi, Koji Takio, Nobutaka Takahashi, and Yuji Kamiya*

Frontier Research Program (T.S., N.T., Y.K.) and The lnstitute of Physical and Chemical Research (RIKEN) (H.A., AS., K.T.), Wako-shi, Saitama, 351-01 Japan; and Biotechnology Research Center (H.Y.) and

Department of Applied Biological Chemistry (N.M), The University of Tokyo, Bunkyo-ku, Tokyo, 11 3 Japan

enf-Kaurene synthase B (KSB) was purified 291-fold from a crude enzyme preparation from endosperm of pumpkin (Cucurbifa maxima L.). Separation of ent-kaurene synthase A and KSB was achieved by hydrophobic interaction chromatography. The fractions containing KSB activity were further purified by diethylaminoethyl, phenyl, and hydroxyapatite column chromatography. Using sodium dodecyl phosphate-polyacrylamide gel electrophoresis, the purest enzyme preparation showed a major band at an apparent molecular m a s of 81 kD. The amount of protein in this band was correlated with KSB activity after diethylaminoethyl and hydroxyapatite chromatography. The N terminus of the 81-kD protein was blocked. Therefore, the protein was partially digested with protease and the amino acid sequences of the resulting major peptide fragments were analyzed. A polyclonal antibody was raised against a synthetic peptide based on the longest peptide fragment combined with a keyhole limpet hemocyanin. The antibody recognized only the 81 -kD denatured protein and not the native KSB. The properties of KSB were examined using the phenyl-purified enzyme preparation. The K,,, value for copalyl pyrophosphate was 0.35 p ~ , and the optimal pH was 6.8 to 7.5. The KSB activity required divalent cations such as Mg2+, Mn2+, and Co2+, whereas Cu2+, Ca2+, and BaZ+ inhibited the activity.

ent-Kaurene is an important intermediate in GA biosynthesis and is synthesized from GGPP via CPP (Fig. 1). These steps are catalyzed by KSA and KSB, respectively (Coolbaugh, 1983). Other terpenoids, such as carotenoids and phytol, also have GGPP as a precursor, whereas CPP is a precursor of macrocyclic diterpenes. Hence, KSA and KSB are important enzymes in the early stage of GA biosynthesis (Coolbaugh, 1983; Chung and Coolbaugh, 1986; Graebe, 1987). Duncan and West (1981) separated KSA and KSB from wild cucumber (Mavah macrocarpus L.) using a quaternalyaminoethyl column and suggested that the conversion of GGPP to ent-kaurene is catalyzed by the two different enzymes. These authors showed that KSA and KSB associated with each other during ent-kaurene synthesis and that KSB preferentially utilized endogenous CPP

This study was performed through special coordination funds of the Science and Technology Agency of the Japanese govern- ment, and the Shorai Foundation for Science and Technology.

* Corresponding author; e-mail [email protected]; fax 81-48 -462-4691.

produced by KSA rather than exogenous CPP. Both KSA and KSB were suggested to be localized in plastids (Aach et al., 1995). Sun et al. (1992) cloned the GAZ locus of Arabi- dopsis by genomic subtraction. Recently, Sun and Kamiya (1994) showed that the GA1 locus encodes KSA. A putative KSA has also been cloned from maize by transposon tag- ging (Bensen et al., 1995). Castor bean (Xicinus communis) produces kaurene and the related diterpenes beyerene, trachylobane, and sandaracopimaradiene, which are formed by alternative cyclization of the common intermediate CPP, and KSB was partially purified (Spickett et al., 1994). In this paper, we present the purification and characterization of KSB from endosperm of pumpkin (Cucwbita maxima L.) seeds.

MATERIALS AND METHODS

Plant Materials and Chemicals

Seeds of pumpkin (Cucuvbita maxima L. cv Riesenmelone gelb vernetzt) were obtained from van Waveren Pflanzen- zucht (Rosdorf, Germany) through Professor Jan Graebe. Plants were cultivated in a field in Saitama, Japan, and immature fruits were harvested between the middle of June and early July 1992 when the cotyledons had reached about half of their final length. Geranylgeraniol was a gift from Dr. T. Takigawa of Kurare Co. Ltd. (Kurashiki, Japan). Copalic acid (Nakano and Djerassi, 1961) and natural resin "Brazil copal" (which contains copalic acid) were gifts from Prof. T. Nakano of the Venezuela National Institute of Science (Caracas, Venezuela) and Prof. Y. Ichinohe and Dr. H. Sakamaki of Nihon University (Tokyo, Japan), respectively. r3H]Sodium borohydride (296 GBq mmol-') was purchased from Amersham. [l-3H]Geranylgeranio1 (74 GBq mmol-') and [15-3Hlcopalol (74 GBq mmol-') were prepared by reduction of geranylgeranial and copalal with [3Hlsodium borohydride (Bensen and Zeevaart, 1990). Tri- tium-labeled GGPP and CPP were prepared through chlo- rination and subsequent pyrophosphorylation (Davisson et

Abbreviations: CBB, Coomassie brilliant blue R-250; CPP, copalyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; KSA, ent-kaurene synthase A; KSB, ent-kaurene synthase 8; KSAB activity, overall ent-kaurene synthase A and B activity; KPi buffer, potassium phosphate buffer.

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1240 Saito et al. Plant Physiol. Vol. 109, '1995

KSA activity KSB activity

OPP 4 @= '%e

Geranylgeranyl Copalyl pyrophosphate ent-Kaurene pyrophosphate

Figure 1. The reactions catalyzed by KSA and KSB. KSA catalyzes the cyclization of GCPP to CPP, and KSB catalyzes the cyclization of CPP to enf-kaurene.

al., 1986). The pyrophosphates were purified by reverse- phase chromatography on a Bond-Elut C,, cartridge (1 mL [Varian, Harbor City, CAI) with stepwise elution using 20, 30, 50, and 70% (v/v) aqueous methanol. Optically pure uniconazole (Izumi et al., 1985) was obtained from Sumi- tomo Chemical Co. (Tokyo, Japan).

Enzyme Assay and Protein Assay

KSB activity was measured in the incubation mixtures consisting of enzyme solution in KPi buffer (50 mM, pH 8.0, 100 pL) containing glycerol (10%, v/v), DTT (2 mM), MgC1, (5 mM), uniconazole (20 p ~ ; Izumi et al., 1985), and [15-3HlCPP (1.0 kBq). When KSAB activity (conversion from GGPP to ent-kaurene) was measured, [1-3H]GGPP (1.0 kBq) was used as a substrate. The mixture was incubated at 30°C for 30 min, and the enzyme reaction was terminated by the addition of acetone (200 pL) and water (100 pL). The mixture was extracted with n-hexane (400 pL), and 300-pL aliquots of the extract were concentrated in vacuo and applied to a silica gel plate. After developing with n-hexane, the radioactivity of the silica gel of the ent-kaurene region (RF 0.6-1.0) was determined. One unit of kaurene synthase activity was defined as the production of 1 pmol of ent-kaurene per min. The protein concentration was determined using Bio-Rad microassay (Bradford, 1976) using BSA as the standard.

Purification of KSB

A11 procedures were performed at O to 4°C. The follow- ing buffers were used: buffer A, 50 mM KPi buffer (pH 8.0) containing 2 mM DTT; buffer B, buffer A containing 10% glycerol; buffer C, 20 mM Tris-HC1 buffer (pH 7.4) containing 10% glycerol and 2 mM DTT; buffer D, 1 mM KPi buffer (pH 6.0) containing 20% glycerol, 2 mM DTT, and 5 mM MgC1,.

Crude enzyme extract (1 L) was prepared by the method of Graebe et al. (1974). Butyl-Toyopearl 650s gel (Toso, Tokyo, Japan) was packed into a glass column (AP-1, 10 mm X 100 mm [Waters, Millipore Corp.]), and the column was equilibrated with buffer A containing 1.7 M (NH,),SO,. (NH,),SO, was added to the crude enzyme preparation (77 mL) to 1.7 M final concentration and stirred for 10 min. The enzyme solution was centrifuged at 20008 for 10 min, and the supernatant was loaded onto the column at a flow rate of 3.0 mL min-'. The column was eluted with a 30-min linear gradient of 1.7 to O M (NH,),SO, and then with buffer B for 45 min. The fraction size was 6 mL,

and a 5-pL aliquot of each fraction was assayed. The major active fractions (32-37) were concentrated by ultrafiltration (YM-10 filter, Amicon, Beverly, MA), and the active fractions from severa1 runs were pooled (53 mL). The butyl- purified enzyme preparation equilibrated with buffer C (8.4 mL) was loaded onto a DEAE-8HR column (10 m m X 100 mm [Waters]). The column was eluted with a 50-min linear gradient of O to 500 mM NaCl in buffer C. The flow rate was 1.0 mL min-' and the fractionation size was 2.0 mL. The active fractions (33-38) were pooled and concentrated by ultrafiltration (YM-10). (NH,),SO, was added to the DEAE-purified enzyme preparation (7.9 mL) to 1.7 M

final concentration. The enzyme solution was loaded onto a TSK phenyl-5PW column (7.5 mm X 75 mm [Toso]), which was equilibrated with buffer B containing 1.7 M

(NH,),SO,. The column was eluted with a 60-min linear gradient of 1.7 to O M (NH,),SO, and then with buffer B for 30 min. The flow rate was 1.0 mL min-', and 2.0-mL fractions were collected. The major KSB activity was found in fractions 37 to 41. For the second DEAE ion-exchange chromatography, the phenyl-purified enzyme preparation (2.0 mL) was charged onto the DEAE column and eluted as described above. The fractions containing most of the KSB activity (36-38) were concentrated to 300 pL by ultrafiltration (Centricon 30, Amicon). This preparation was charged onto a hydroxyapatite column (TONEN TAPS-050805 HG, 8.0 mm X 50 mm [Tonen, Tokyo, Japan]) equilibrated with buffer D. The column was eluted with a 60-min linear gradient of 1, to 500 mM phosphate. The flow rate wds 1.0 mL min-', and the fraction size was 1 mL. The major KSB active fractions (23-25) were concentrated by ultrafiltration (Centricon 30) and stored at -80°C (70 pg, 673 units).

Cel-Permeation Chromatography

The phenyl-purified enzyme preparation was charged onto the gel-permeation column (TSK G3000SWx1, 7.13 mm X 300 mm [Toso]) that was pre-equilibrated with buffer B containing 200 mM NaC1. The column was eluted with the same buffer at a flow rate of 0.4 mL min-' and fractions were collected every 30 s.

SDS-PAGE

Active enzyme fractions of the sequential purification steps were analyzed by SDS-PAGE using a 7.5% (w/v) gel (Laemmli, 1970). Approximately 3 pg of total proteiii was applied to each lane. After electrophoresis, proteins were visualized by CBB R250 staining. Ovalbumin (45.0 kD), BSA (66.2 kD), phosphorylase b (97.4 kD), p-galactosidase (116 kD), and myosin (200 kD) were used as standard proteins (Bio-Rad).

Properties of KSB

When the properties of KSB were examined, the phenyl- purified enzyme preparation (0.47 pg of total protein) was used as the enzyme solution. The amount of [1-3H]CPP was 2.2 kBq and the incubation period was 15 min. The other conditions were the same as those for the enzyme assay. To determine the optimal pH, KPi buffer (50 mM) adjusted to

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Purification of ent-Kaurene Synthase B from Pumpkin 1241

--

a particular pH (pH 4.5-9.0) was used. To determine the K , value, the phenyl-purified enzyme preparation (1.2 pg of total protein) was used, and the concentrations of [l-3H]CPP were in the range of 7 to 480 nM. The production of ent-kaurene was calculated and the K , value was determined from a Lineweaver-Burk plot.

N-Terminal Amino-Acid and interna1 Peptide Sequence Analysis

The final purified enzyme preparation (14.5 pg) was purified on a 7.5% (w/v) polyacrylamide gel. Proteins were electroblotted to a polyvinylidene difluoride membrane and visualized (Matsudaira, 1987). The 81-kD band on the membrane was cut out and the amino acid sequence of the protein was analyzed by a protein sequencer (477A protein sequencer [Applied Biosystemsl). For interna1 peptide sequence analysis, the 81-kD protein was digested in situ (Kawasaki et al., 1990) with the Acromobacter protease I (a gift from Dr. T. Masaki of Ibaraki University, Ibaraki, Japan). The generated peptide fragments were extracted from the gel and separated by reverse-phase HPLC using a Supersphere RP-Select B column. Aqueous 0.09% (v/v) TFA and 80% (v/v) acetonitrile containing 0.075% (v/v) TFA were used as eluants A and B, respectively. The flow rate was 0.2 mL min-’, and a 32-min linear gradient of O to 80% of eluant B was used (1090M HPLC system, Hewlett- Packard). A,,,, AZ7,, and A,,, were monitored. Amino acid sequences of separated peptides were analyzed with the protein sequencer.

- 1.0

E, O

- M hl

-0.5 ’ c)

9 s - 4

> 3

0.0

Polyclonal Antibodies for the 81 -kD Protein

A major peptide fragment of an 81-kD protein, ASQIITHPDESVLENINSWT, was custom synthesized by Quality Controlled Biochemicals, Inc. (Hopkinton, MA). The synthetic peptide (3.1 mg, 1.4 nmol) was combined to keyhole limpet hemocyanin by using an Imjet immunogen EDC conjugation Kit (Pierce) according to the manufactur- er’s instructions. The combined protein (1 mg) was injected into a rabbit three times with intervals of 2 weeks, and 4 weeks after the last injection antiserum was collected by centrifugation of the blood. IgG was purified from the antiserum using a HiTrap Protein G column (1 mL [Pharmacia]).

About 10 p L of each fraction of the second DEAE chromatography step was resolved on two 7.5% SDS-PAGE gels. One of the gels was stained with CBB solution, and proteins on the other gel were electroblotted onto a nitrocellulose membrane. The nitrocellulose membrane was treated with the IgG at room temperature for 2 h and then with alkaline-phosphatase-conjugated anti-rabbit IgG at the same temperature for another 2 h. The membrane was treated with nitroblue tetrazolium chloride and 5-bromo- 4-chloro-3’-indolyl-phosphate (Pierce) to visualize proteins.

RESULTS

Purification of KSB

The crude enzyme prepared from immature seeds was first purified by a hydrophobic interaction chromatogra-

phy to remove metal ions and GAs and to concentrate the enzyme. The majority of the KSB activity was eluted at an (NH,),SO, concentration of 0.6 to 0.1 M, whereas the majority of the KSAB activity was eluted after the (NH,),SO, gradient was complete (Fig. 2). This suggested that KSA and KSB were separable by hydrophobic interaction chromatography. Fractions corresponding to an (NH,),SO, concentration of 0.6 to 0.1 M were collected as the butyl- purified KSB preparation, although this preparation contained some KSA activity.

The butyl-purified KSB preparation was purified further by DEAE ion-exchange chromatography with a NaCl gradient. Both KSA and KSB co-eluted at fractions corresponding to 240 to 360 mM NaCI. Separation of KSA and KSB was not achieved by DEAE chromatography, but the specific activity of KSB was increased (Table I). These fractions were collected and were further purified by high-perfor- mance hydrophobic interaction chromatography using a Phenyl-5PW column. The majority of KSB activity eluted at (NH,),SO, concentrations of 0.5 to 0.2 M, and KSAB activity eluted later.

About 40% of the phenyl-purified enzyme preparation was further purified on the same DEAE column. The main peak of KSB activity was detected in fractions of 250 to 280 mM NaCl (Fig. 3), which were collected as the second DEAE-purified enzyme preparation. This enzyme preparation contained four major proteins by SDS-PAGE (Fig. 4) and was further purified on a hydroxyapatite column. The KSB activity was eluted at phosphate concentrations of 90 to 140 mM, and the peak shape of KSB activity corre- sponded with that of UV A,,, (Fig. 5). The purest KSB preparation did not show KSAB activity, suggesting that KSA was not present in the preparation. KSB was purified 291-fold from endosperm of pumpkin (Table I), and 70 pg of purified enzyme was obtained.

- KSB activity

- - - Protein concentration - UV absorbance at 280 nm I..... (NH4)2SO4 concentration

KSAB activity

20 40 60 80 I00 120

I

Retention time (min)

Figure 2. A typical elution profile of hydrophobic interaction chromatography of the crude enzyme preparation on the butyl-Toyopearl 6505 column. The sample was loaded onto the column equilibrated with buffer containing 1.7 M (NH,),SO, and eluted with a linear 1.7 to O M gradient of (NH,),SO,. Each fraction was assayed for KSAB and KSB activity. Protein concentration and A,,, are indicated with dotted and solid lines, respectively.



1242 Saito et al. Plant Physiol. Vol. 109, 1995

Table 1. Purification o f KSB from immature seeds o f pumpkin

Enzyme Preparation Total Protein Total Activity Yield Specific Activity Purification

mg uni1s” % unitsa mg- ’ - fold

Crude enzyme 530 17,500 1 O0 33.0 1

First DEAE-purified 13.9 5,170 29.6 3 72 11.3

Second DEAE-purifiedb 0.407 2,820 1 6.2b 6,930 21 o

Butyl-purified 168 11,000 63.8 65.4 1.98

Phenyl-purified 4.43 3,340 19.2 754 22.8

HA-purified 0.070 673 3.87 9,610 291

a One unit of KSB activity was defined as the production of 1 pmol of ent-kaurene min-’ at 30°C when 29 pmol (2.2 kBq) of [’HICPP was used as a substrate. Forty percent of the phenyl-purified enzyme preparation was used for the second DEAE chromatography, so the yield of this step is corrected by a factor 0.4.

SDS-PACE Analysis

Figure 4 shows the SDS-PAGE of active enzyme fractions of the sequential purification steps. The hydroxyapatite- purified enzyme preparation showed a major band at apparent molecular mass of 81 kD. The amount of protein corresponding to the 81-kD band estimated by SDS-PAGE in each fraction of the second DEAE chromatography correlated with KSB activity (Fig. 6).

Molecular Mass Estimation

To determine the molecular mass of KSB by means of gel-permeation HPLC, we used a TSK G3000 SWxl column. On this column the elution profile of KSB was broad but centered on a retention time of 23.5 min, which corre- sponds to an apparent molecular mass of approximately 90 kD.

Properties of KSB

The properties of KSB were examined with the phenyl- purified enzyme preparation, since the amount of final purified enzyme was limited. The optimal pH for KSB was 6.8 to 7.5 in 50 mM KPi buffer. The K , value for [15-3H]CPP was 0.35 PM. In the presence of 5 mM Mg2+, KSB could convert CPP to ent-kaurene, but was not active without exogenous cations. Since EDTA (1-100 mM) did not change KSB activity, the phenyl-purified enzyme preparation did not contain endogenous cations that affected KSB activity. Some divalent cations (0.1-50 mM, as chlorides) were added to the incubation mixture instead of 5 mM Mg2+, and KSB activity was measured. As shown in Figure 7, Mg2+, Co2*, and Mn2+ promoted KSB activity, and their optimal concentrations were around 10 mM. Ni2+ and Fez+ weakly promoted KSB activity, whereas Cu2+, Ca2+, and Ba2+ showed no effects. When Cu2+, Ca”, and Ba2+ were added to the incubation mixture in combination with 5 mM Mg2+, all three inhibited KSB activity promoted by Mg”. In particular, Cu2+ completely inhibited the conversion promoted by 5 mM Mg2+ at concentrations above 0.5 mM (data not shown).

Peptide Sequence Analysis

The N-terminal peptide sequence of the 81-kD protein from SDS-PAGE was analyzed, and the N terminus of the

protein was found to be blocked. The 81-kD protein band was cut out from the polyacrylamide gel and treatecl with a lysyl endopeptidase, Acromobacter protease I (Masaki et al., 1981). The resulting peptides were separated by reverse-phase HPLC and the sequences of the major peptides were analyzed (Kawasaki et al., 1990). Sequence.5 and approximate amounts of the peptides recovered are: 1, ASQIITHPDESVLENINSWT (74 pmol); 2, EAEWSTNK (45 pmol); 3, RAMESYSGDIVRISK (37 pmol); 4, HGLSSIISVW (24 pmol); 5, LQDWSMVMQYQRK (17 pmol).

Western Blot Analysis and lmmunoprecipitation

Western blot analysis (Fig. 6) showed that the polyclonal antibody raised against the synthetic peptide ASQIITHPDESVLENINSWT, combined with keyhole limpet hemocyanin, selectively recognized the 81-kD protein in each fraction from the second DEAE chromatography, and the pattern showed a good correlation with the pat- terns of KSB activity and CBB staining. In an immuiiopre- cipitation experiment using the phenyl-purified enzyme and the polyclonal antibody, the 81-kD protein was detected in the supernatant fraction and not in the precipitate regardless of the amount of antibody used (data not shown).

- KSB activity - UV absorbance at 280 nm ...... NaCl concentrahon

m; 1 1

x 20 h II 10.6

Retention time (min)

Figure 3. lon-exchange chromatography of the phenyl-puriiied enzyme preparation on the second Protein-Pak DEAE-8HR column. The phenyl-purified enzyme preparation was charged to the column and eluted with a linear O to 500 mM gradient of NaCI. Each fraction was assayed for KSB activity. A,,, is also indicated.



Purification of enf-Kaurene Synthase B from Pumpkin 1243

kD

-* 45.0

i

Figure 4. SDS-PAGE of purified enzyme preparations. Active en-zyme fractions from the sequential purification steps were analyzedon a 7.5% SDS-PAGE gel. About 3 ^g of total protein was applied toeach lane. Proteins were visualized by CBB staining. Lane 1, Crudeenzyme; lanes 2 to 6, butyl-, first DEAE-, phenyl-, second DEAE-, andhydroxyapatite-purified enzymes.

in butyl chromatography, in which the earlier fractionscontain a higher concentration of (NH4)2SO4. This isconsistent with the results obtained from wild cucumber.KSA and KSB from M. macrocarpus could be resolvedusing anion-exchange chromatography on a QAE Seph-adex column. We also tried a QAE column as the fourthstep of purification. Preliminary experiments showedthat KSB appeared to bind tightly to the QAE column,whereas recovery of KSB activity from the column wasonly about 10%. We abandoned the QAE column andused a DEAE column twice. The elution profile of KSBfrom gel-permeation chromatography was broad, andthe molecular mass was estimated to be approximately

- SlkDa

DISCUSSION

We report the purification of KSB from pumpkin en-dosperm. The purification includes five chromato-graphic steps using hydrophobic interaction twice,DEAE ion exchange twice, and hydroxyapatite columnchromatography. Duncan and West (1981) used(NH4)2SO4 precipitation as the first step of their purifi-cation of enf-kaurene synthase of wild cucumber, Marahmacrocarpus. They found most of the KSA activity in the0 to 45% (NH4)2SO4 fraction and most of the KSB activityin the 45 to 80% fraction. We used hydrophobic interac-tion chromatography as the first step in our purificationbecause a reverse gradient of (NH4)2SO4 concentration isused in hydrophobic interaction chromatography. Asexpected, the KSB activity eluted before the KSA activity

o"* 20 -\

£> 10 •'

paC/3

— KSB activity— UV absorbance at 280 i...... j^pj concentration

0.6

0.4

10 20 30 40 50Retention time (min)

60 70

-4

-3

-2

-0

Figure 5. Hydroxyapatite chromatography of the second DEAE-pu-rified enzyme preparation. The second DEAE-purified enzyme prep-aration was charged to the hydroxyapatite column equilibrated with1 mM potassium phosphate buffer. The column was eluted with alinear 1 to 500 mM gradient. Each fraction was assayed for KSBactivity. A2BO is also indicated.

M 34 35 36 37 38 39 40 41

X

aex

J'>03

IN

W

- 81kDa

34 35 36 37 38 39 40 41

3435363738394041Fraction

Figure 6. Western blot analysis of each fraction from the secondDEAE chromatography. Each fraction (10 /LI!) of the second DEAE-chromatography was resolved on two 7.5% SDS-PAGE gels. Theupper part shows the CBB-stained gel, and the middle part showswestern blotting using the polyclonal antibody raised against thecombined protein as a primary antibody. The lower part shows KSBactivity of each fraction (5 ju.L). www.plantphysiol.orgon December 4, 2018 - Published by Downloaded from

Copyright © 1995 American Society of Plant Biologists. All rights reserved.


1244 Saito et al. Plant Physiol. Vol. 109, 1995

400

2

Ba 0.01 0.1 1 10 1 O0

Cation concentration (mM)

Figure 7. Effect of cations on KSB. The phenyl-purified enzyme preparation (0.47 Wg) and [l -'H]CPP (2.2 kBq) in a potassium phosphate buffer (50 mM, pH 8.0, 1 O0 wL) were incubated in the presence of each cation. Each point represents the mean KSB activity 2 SE of four or six incubations as a percentage of the control.

90 kD. The final purified enzyme preparation showed a single band with an apparent molecular mass of 81 kD on SDS-PAGE (Fig. 4). The molecular masses of KSA and KSB from M. macrocarpus were estimated to be approximately 82 kD by gel-filtration and sedimentation velocity determinations (Duncan and West, 1981). Recombinant KSA from Arabidopsis, produced in Esckerichia coli, gave a protein with an apparent molecular m a s of 86 kD that was processed to a smaller protein by pea chloroplasts (Sun and Kamiya, 1994). Another diterpene cyclase, abietadiene synthase, was partially purified from grand fir (Abies grandis Lindl.) and identified as an 80-kD protein (LaFever et al., 1994). These results are consistent with the apparent molecular mass of the pumpkin KSB. Cas- tor bean KSB was partially purified, but unfortunately its apparent molecular mass was not reported (Spickett et al., 1994)

The optimal pH and the K , value of KSB are in good agreement with those reported by Frost and West (1977), who found an optimal pH of 6.9 and K , value of 0.49 p~ in M. macrocarpus. As for the effect of metal ions on KSB, there are some differences between pumpkin and wild cucumber. For the pumpkin enzyme, Mn2+ enhanced KSB activity most effectively, followed by Co2+ and Mg2+. For the M. macrocarpus enzyme, Mg2+ also enhanced KSB activity, whereas Mn2+ and Co2+ gave lower rates at an optimal concentration of 0.1 miv and inhibited enzyme activity at higher concentrations.

Since we found that the N terminus of the 81-kD protein was blocked, it was partially hydrolyzed by lysyl endopeptidase, which selectively hydrolyzes at the C-terminal end of Lys residues. The longest peptide fragment, with an amino acid sequence of ASQIITHPDESVLENINSWT, was synthesized and combined with'the keyhole limpet hemocyanin. Polyclonal antibodies raised against this combined protein selectively recognized the 81-kD protein on SDS- PAGE. In fractions from the second DEAE chromatography, recognition of the 81-kD protein by the antibodies

showed a good correlation with both KSB activity and CBB staining. This I-esult indicated that the 81-kD protein was probably KSB. However, the antibodies could not recognize any proteins on a native PAGE gel. The antibodies did not reduce KSB activity in vitro, and immunoprecipitation of KSB was also unsuccessful. These results suggest that the synthetic peptide is not located on the surface of the protein molecule but is hidden inside and that the antilbody could neither effectively bind to nor recognize the 81-kD protein in the native form.

Using synthetic oligonucleotides deduced from peptides sequenced as primers for reverse-transcription PCR, we have recently cloned the KSB cDNA. All the peptide sequences were part of the deduced amino acid sequence encoded by the cDNA. Lysogenized E. coli expressing the cDNA as a fusion protein contained KSB activity. This confirms that the major protein in the purest enzyme iprep- aration is the KSB. The cloning of the KSB gene will be reported elsewhere.

ACKNOWLEDCMENTS

We thank Dr. T. Takigawa of Kurare Co. Ltd., Prof. T. Nakano of Venezuela National Institute of Science, Prof. Y. Ichinohe and Dr. H. Sakamaki of Nihon University, and Dr. T. Masaki of Ibaraki University for generous gifts of geranylgeraniol, copalic acid, natural resin "Brazil copal," and Acromobacter protease I, respectively. We also thank Dr. N. Dohmae and Dr. A. Ozaki of R1KE:N for analysis of amino acid sequence and preparation of polyclonal antibodies, respectively. The authors thank Dr. S. Swain for his critica1 comments on the manuscript.

Received May 15, 1995; accepted September 4, 1995. Copyright Clearance Center: 0032-0889/95/109/1239/07.

LITERATURE ClTED

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Bensen RJ, Johal GS, Crane VC, Tossberg JT, Schnabel PS, Meeley RB, Briggs SP (1995) Cloning and characterization of the maize Anl Gene. Plant Cell 7: 75-84

Bensen RJ, Zeevaart JAD (1990) Comparison of ent-kaurene synthetase A and B activities in cell-free extracts from young tomato fruits of wild-type and gib-2, gib-2, and gib-3 tomato plants. J Plant Growth Regul 9: 237-242

Bradford MM (1976) A rapid and sensitive method for the quan- titation of microgram quantities of protein utilizing the priinciple of protein-dye binding. Ana1 Biochem 72: 248-254

Chung CH, Coolbaugh RC (1986) ent-Kaurene biosynthesis in cell-free extracts of excised parts of tal1 and dwarf pea seedlings. Plant Physiol 80: 544-548

Coolbaugh RC (1983) Early stages of gibberellin biosynthesis. In A Crozier, ed, The Biochemistry and Physiology of Gibberellins, Vol 1. Praeger Publishers, New York, pp 53-98

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Duncan JD, West CA (1981) Properties of kaurene synthetase from Marah macrocaypus endosperm. Evidence for the participation of separate but interacting enzymes. Plant Physiol 6 8 1128-1134



Purification of ent-Kaurene Synthase B from Pumpkin 1245

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Purification and Properties of ent-Kaurene - Plant Physiology

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