2 18s Biochemical Society Transactions ( 1 992) 20 Purification of ent-kaurene oxidase from Gibberelhfijikuroi and Chcurbita mima
Table 1
CAROLINE ARCHER, PHILIP J. ASHMAN,*PETER HEDDEN. JOHN R. BOWYER AND PETER M. BRAMLEY
Fraction
Department of Biochemistry, Royal Holloway and Bedford New College, University of London, Egham, Surrey, TW20 OEX and *Long Ashton Research Station, Long Ashton, Bristol, BS18 9AF The oxidation of ent-kaurene is an early step in the biosynthesis of gibberellins in higher plants and the fungus Gibberella fujikuroi [ 11. The reaction product, enr-kaurenoicacid, is formed via two intermediates, enr-kaurenol and enr-kaurenal. The enzyme catalysing these reactions, kaurene oxidase, shows properties consistent with a cyt P-450- dependent reaction [2,3] and is a membrane-bound protein [4]. Several types of plant growth retardants prevent gibberellin formation by inhibiting kaurene oxidase, eg. ancymidol [5] and paclobutrazol (PC) [6]. Previous investigations on kaurene oxidase of Gibberella have established that it can be removed from membranes with high salt concentrations and that the soluble protein shows CO- and PC- binding spectra, characteristic of cyt P-450, and is inhibited by CO [7]. This enzyme extract has been used in the present study on the purification of kaurene oxidase. Throughout the purification, kaurene oxidase was assayed by analysis of its CO-and PC-binding spectra, because catalytic activity was lost after PEG precipitation. During the isolation procedure cyt P-450 degraded to cyt P-420 (Fig. lA), but this form of the protein still exhibited ligand binding spectra (Fig IB). All buffers contained 5% glycerol and column eluates were continuously monitored at 280 and 415nm. The enzyme was precipitated with 25 % PEG 8000, prior to chromatography on a TSK DEAE 5PW anion exchange column. It was eluted with 900mM NaCI, and this fraction applied to a TSK SW (32000 gel
-
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, I
,
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,
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.
*-
,
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__ .’
I,
I
.
I
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1
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6.
1 ,
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310
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0.1 0.14 0.23 0.57 1.23
1 14 23 57 123
Metabolism of Plant Lipids (Fuller, G. and Nes, W.D., eds.), pp. 25-43, American Chemical Society, Washington DC. Murphy, P.J. and West, C.A. (1969) Arch. Biochem. Biophys. 133, 395-407 Hasson, E.P. and West, C.A. (1976) Plant Physiol. 58, 473478 Hasson, E.P. and West, C.A. (1976) Plant Physiol. 58,479484 Coolbaugh, R.C. and Hamilton, R. (1976) Plant Physiol. 57, 245-248 Hedden, P. and Graebe, J.E. (1985) J. Plant Growth Regl.
Biochim. Ashman, 111-122 Biophys. P.J., Mackenzie, Acta 1036, A. 151-157 and Bramley, P.M. (1990) 7. 4,
160
5m
YO
W J
Fig. 1. Difference spectra of kaurene oxidase. CO-binding spectra (1A) of solubilised, sodium dithionite and reduced kaurene oxidase from Gibberella fujikuroi Cucurbiru muximu (---), using sodium dithionite, and (lB), entkaurene (---) and paclobutrazol binding spectra of the DEAE-5PW fraction from Gibberella fujikuroi. The concentration of both ligands was 20 mM. (0)
(0)
140’ 29 5.1 3.1 1.4
filtration column. Several haemprotein peaks were eluted, with kaurene oxidase in a fraction of apparent M, 50-55kDa. Isoelectric focussing (pH range 3.5-6.0) of this fraction yielded several bands, of which those between PI 3-4 showed binding spectra with PC and kaurene. Individual proteins within this area are presently being tested for ligand binding spectra. Overall a 123-fold purification has been achieved up to elution from gel filtration (Table 1). In contrast to kaurene oxidase from Gibberella, that from Cucurbifa muximu was not solubilised with high salt concentrations. Instead, microsomes suspended in lOOmM NaCI, followed by sonication, yielded 90 % of the enzyme in the soluble fraction. Repeated sonication of the microsomes resulted in over 100% of the membrane-associated catalytic activity being recovered in the soluble fraction. This preparation was capable of producing, in a PC-sensitive reaction ‘C-labelled kaurenol, kaurenal and kaurenoic acid from [“Clkaurene. A COdifference spectrum of the solubilised fraction is shown in Fig 1A.
3.
..
.
&oI)
+
2.
8
Total CytP-420/ Purification c tP 420 mg protein (-fold)
’ P-450 P-420. P-450 negligible in other fractions. S,,,, soluble fraction.
A
F on2
Total Protein (mg)
Crude extract 1400 206 S,O,” PEG ppt 22 DEAE 5PW 5.4 Gel filtration 1.1
:b:+ 0.06
Purification of kaurene oxidase from Gibberella fujikuroi
Table 1
CAROLINE ARCHER, PHILIP J. ASHMAN,*PETER HEDDEN. JOHN R. BOWYER AND PETER M. BRAMLEY
Fraction
Department of Biochemistry, Royal Holloway and Bedford New College, University of London, Egham, Surrey, TW20 OEX and *Long Ashton Research Station, Long Ashton, Bristol, BS18 9AF The oxidation of ent-kaurene is an early step in the biosynthesis of gibberellins in higher plants and the fungus Gibberella fujikuroi [ 11. The reaction product, enr-kaurenoicacid, is formed via two intermediates, enr-kaurenol and enr-kaurenal. The enzyme catalysing these reactions, kaurene oxidase, shows properties consistent with a cyt P-450- dependent reaction [2,3] and is a membrane-bound protein [4]. Several types of plant growth retardants prevent gibberellin formation by inhibiting kaurene oxidase, eg. ancymidol [5] and paclobutrazol (PC) [6]. Previous investigations on kaurene oxidase of Gibberella have established that it can be removed from membranes with high salt concentrations and that the soluble protein shows CO- and PC- binding spectra, characteristic of cyt P-450, and is inhibited by CO [7]. This enzyme extract has been used in the present study on the purification of kaurene oxidase. Throughout the purification, kaurene oxidase was assayed by analysis of its CO-and PC-binding spectra, because catalytic activity was lost after PEG precipitation. During the isolation procedure cyt P-450 degraded to cyt P-420 (Fig. lA), but this form of the protein still exhibited ligand binding spectra (Fig IB). All buffers contained 5% glycerol and column eluates were continuously monitored at 280 and 415nm. The enzyme was precipitated with 25 % PEG 8000, prior to chromatography on a TSK DEAE 5PW anion exchange column. It was eluted with 900mM NaCI, and this fraction applied to a TSK SW (32000 gel
-
8 I
, ,
, I
,
$
a
,
4.
I \
.
*-
,
I
. _-.-
__ .’
I,
I
.
I
..-
5.
)
‘
1
,
> !
6.
1 ,
\_..
::II :
310
so
LlO
0.1 0.14 0.23 0.57 1.23
1 14 23 57 123
Metabolism of Plant Lipids (Fuller, G. and Nes, W.D., eds.), pp. 25-43, American Chemical Society, Washington DC. Murphy, P.J. and West, C.A. (1969) Arch. Biochem. Biophys. 133, 395-407 Hasson, E.P. and West, C.A. (1976) Plant Physiol. 58, 473478 Hasson, E.P. and West, C.A. (1976) Plant Physiol. 58,479484 Coolbaugh, R.C. and Hamilton, R. (1976) Plant Physiol. 57, 245-248 Hedden, P. and Graebe, J.E. (1985) J. Plant Growth Regl.
Biochim. Ashman, 111-122 Biophys. P.J., Mackenzie, Acta 1036, A. 151-157 and Bramley, P.M. (1990) 7. 4,
160
5m
YO
W J
Fig. 1. Difference spectra of kaurene oxidase. CO-binding spectra (1A) of solubilised, sodium dithionite and reduced kaurene oxidase from Gibberella fujikuroi Cucurbiru muximu (---), using sodium dithionite, and (lB), entkaurene (---) and paclobutrazol binding spectra of the DEAE-5PW fraction from Gibberella fujikuroi. The concentration of both ligands was 20 mM. (0)
(0)
140’ 29 5.1 3.1 1.4
filtration column. Several haemprotein peaks were eluted, with kaurene oxidase in a fraction of apparent M, 50-55kDa. Isoelectric focussing (pH range 3.5-6.0) of this fraction yielded several bands, of which those between PI 3-4 showed binding spectra with PC and kaurene. Individual proteins within this area are presently being tested for ligand binding spectra. Overall a 123-fold purification has been achieved up to elution from gel filtration (Table 1). In contrast to kaurene oxidase from Gibberella, that from Cucurbifa muximu was not solubilised with high salt concentrations. Instead, microsomes suspended in lOOmM NaCI, followed by sonication, yielded 90 % of the enzyme in the soluble fraction. Repeated sonication of the microsomes resulted in over 100% of the membrane-associated catalytic activity being recovered in the soluble fraction. This preparation was capable of producing, in a PC-sensitive reaction ‘C-labelled kaurenol, kaurenal and kaurenoic acid from [“Clkaurene. A COdifference spectrum of the solubilised fraction is shown in Fig 1A.
3.
..
.
&oI)
+
2.
8
Total CytP-420/ Purification c tP 420 mg protein (-fold)
’ P-450 P-420. P-450 negligible in other fractions. S,,,, soluble fraction.
A
F on2
Total Protein (mg)
Crude extract 1400 206 S,O,” PEG ppt 22 DEAE 5PW 5.4 Gel filtration 1.1
:b:+ 0.06
Purification of kaurene oxidase from Gibberella fujikuroi
