of a cytochrome p-450-dependent camphor hydroxylase tissue … · pseudomonas putida upon which...

Plant Physiol. (1993) 101: 1231-1237

lnduction and Characterization of a Cytochrome P-450-Dependent Camphor Hydroxylase in Tissue

Cultures of Common Sage (Salvia officinalis)’

Christoph Funk and Rodney Croteau*

lnstitute of Biological Chemistry and Department of Biochemistry and Biophysics, Washington State University, Pullman, Washington 991 64-6340

(+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), i s catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor i s oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O,-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Severa1 well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCI2, camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor- 5-exo-hydroxylase from Pseudomonas putida and cytochrome P- 450 limonene-6s-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn’+-induced sage cells. These results suggest that camphor-6- exo-hydroxylase from sage i s a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases.

The biosynthetic capacity of plant cell cultures to produce various monoterpenoid, sesquiterpenoid, and diterpenoid substances has been demonstrated; however, the accumulation of monoterpenes in cultured cell systems is only rarely observed (Banthorpe et al., 1986; Charlwood et al., 1989). Studies with undifferentiated suspension cultures of common sage (Salvia officinalis) showed that the enzymes directly responsible for the conversion of the ubiquitous isoprenoid precursor, geranyl pyrophosphate (Fig. 1, structure l)’, to (+)- camphor (Z), a major monoterpene of the intact plant, were readily detected during the late logarithmic phase of growth. Yet, no significant accumulation of camphor (C0.3 ng/g fresh

This investigation was supported in part by U.S. Department of Energy Grant DE-FG06-91ER13869, by Project 0268 from the Wash- ington State University Agricultura1 Research Center, and by a Swiss National Science Foundation Postdoctoral Fellowship (to C.F.).

* In the text, boldface arabic numerals refer to the structures shown in Figure 1.

* Corresponding author; fax 1-509-335-7643. . *

weight) was observed in these undifferentiated cells, even when provisions wer; made for trapping this volatile, hydro- phobic product (Falk et al., 1990). This lack of accumulation was attributed to the high rate of camphor catabolic activity of suspension cultures (Falk et al., 1990), a process that may mimic the metabolic turnover of camphor produced in the oil glands of the intact plant (Croteau et al., 1984, 1987). Altematively, many monoterpenes are toxic to plant suspension cultures (Brown et al., 1987) and the metabolism of camphor by sage cells might thus represent such a detoxification system (Benveniste et al., 1982; Hendry, 1986). Similar detoxification systems are known in animal tissues in which inducible, microsomal Cyt P-450-dependent monooxygenases of broad specificity are involved in xenobiotic metabolism (Walker-Griffin et al., 1979), including the metabolism of dietary monoterpenes (Waller, 1969; Karp and Croteau, 1988).

Feeding experiments with cultured sage cells have demonstrated that camphor is transformed, in sequence, to 6-exo- hydroxycamphor (3), 6-oxocamphor (4), a-campholonic acid (5), and 2-hydroxycampholonic acid (6 ) (Fig. 1) (Funk et al., 1992). The hydroxylation of camphor is, seemingly, the first step in the metabolism of this monoterpene ketone by micro- organisms and animals as well (Waller, 1969). Indeed, based on the substrate and the nature of the reaction catalyzed, the presumptive sage hydroxylase would appear to resemble Cyt P - ~ ~ O C A M , the camphor-5-exo-hydroxylase isolated from Pseudomonas putida upon which much of our current knowl- edge of Cyt P-450 monooxygenases is based (Sligar and Murray, 1986).

In this paper, we describe the properties of camphor-6- em-hydroxylase from sage. The results indicate that this enzyme is an inducible, microsomal Cyt P-450 monooxygenase that shares many characteristics with other monoterpene hydroxylases, and that it may have at least one epitope in common with the bacterial Cyt P - 4 5 0 ~ ~ ~ and the Cyt P-450 limonene-6-hydroxylase from spearmint.

MATERIALS A N D METHODS

Suspension Cultures and Reagents

Suspension cultures of common sage (Salvia officinalis), initiated from leaves, were maintained in Murashige and

Abbreviations: FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide.

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1232 Funk an id Croteau Plant Physiol. Vol. 101, 1993

ppo2 1 ---O+ 2 3

HOO 1 1 H O O C

4 5 6

Figure 1. Pathway for (+)-camphor metabolism in sage. The structures are geranyl pyrophosphate ( I ) , (+)-camphor (2), 6-exo-hydroxycamphor (3), 6-oxocamphor (4), a-campholonic acid (S), and 2-hydroxycampholonic acid (6) . PPO is the pyrophosphate moiety.

Skoog medium (Murashige and Skoog, 1962), supplemented with 3% Suc as well as 2,4-D and kinetin (1 mg/L each) as described earlier (Funk et al., 1992). For induction experiments, aliquots of suspension cultures in early stationary or late linear growth were taken by means of a sterile pipette and treated with the different inducers. Stock solutions of inducers, in water or 30% aqueous ethanol, were sterilized by filtration (0.2 Mm), and control cultures were treated with the corresponding amounts of these sterilized solvents.

(+)-[3-3H2]Camphor (7.4 Ci/mol) was prepared as described previously and 6-exo-hydroxycamphor was isolated from a camphor-treated sage suspension culture (Funk et al., 1992). Acrylamide and bisacrylamide were obtained from Hoefer Scientific (San Francisco, CA), polyvinylidene diflu- onde (Immobilon-P) membranes were from the Millipore Corp. (Bedford, MA), and, unless otherwise noted, a11 other reagents for SDS-PAGE, western blotting, and immunostaining were obtained from Bio-Rad Laboratories or Sigma Chem- ical Co. Other reagents and biochemicals were from Sigma or Aldrich Chemical Co.

lsolation and Assay of Camphor-6-exo-Hydroxylase

For the isolation of (+)-camphor-6-exo-hydroxylase, tissue cultures were harvested in late logarithmic growth phase (7- 9 d after subcultivation). During a11 isolation steps, the tem- perature was maintained at O to 4OC. Cells (50 g) were extracted in phosphate buffer (200 mL, 100 mM NaP04, pH 7.0, 1 mM EDTA, 1 mM DTT) by means of a glass Ten-Broeck homogenizer. After addition of polyvinylpolypyrrolidone (2%, w/v) and stimng on ice for 10 min, large particulate matter was removed by centrifugation (3000g, 15 min). A further centrifugation step (lO,OOOg, 15 min) yielded the crude enzyme preparation from which the light membranes were obtained by ultracentrifugation (195,00Og, 90 min). The pellet containing the microsomes was either resuspended in phosphate assay buffer (see below) or immediately frozen under argon and stored at -7OOC.

The assay for (+)-camphor-6-hydroxylase activity in crude extracts (10,OOOg supematants) and microsomal preparations

'

(195,OOOg pellets) was performed in 10-mL screw-cap culture tubes containing a total volume of 1 mL of 50 m~ sodium phosphate buffer (pH 7.0) with 1 mM EDTA, 1 mM DTT, 1 mM NADPH, and 10% (v/v) glycerol. The reaction was initiated by addition of (+)-[3Hz]camphor (70 nmol, 7.4 Ci/ mol) and incubated at 32OC for 90 min. The reaction mixture was then chilled on ice, 5 pg of 6-exo-hydroxycamphor was added as carrier, and the products were extracted with diethyl ether (2 X 1 mL). This extract was dried by passage through a short column of anhydrous MgS04, the eluate was concen- trated under NZ, and the products contained therein were separated by TLC (0.2-mm silica gel sheets, Kodak) using ether:pentane (1:1, v/v) as developing solvent. After visuali- zation by exposure to I2 vapor, the zone containing 6-hydroxycamphor (RF = 0.2) was excised, and the radioactivity contained therein was detennined by liquid scintillation spec- trometry (in 10 mL of cocktail consisting of 0.4% [w/v] Omnifluor [New England Nuclear] dissolved in 30% ethanol in toluene) (3H efficiency = 40%).

When inhibitors of the camphor hydroxylase were tested, they were added to the reaction mixture 5 min prior to the addition of substrate and subsequent incubation. For testing inhibition with CO, the reaction mixtures were saturated (by bubbling for 5 min) with gas mixtures containing 10,50, and 90% CO with 10% O2 and the remainder N2 before addition of substrate and incubation in the dark. Light reversal of CO inhibition was evaluated by incubation of treated samples and controls under light of 450 nm maximal output (Karp et al., 1987).

CO-difference spectra were recorded as described by Es- tabrook and Wemngloer (1978) and the amount of Cyt P- 450 was calculated by assuming an extinction coefficient of 91 m ~ - ' (Omura and Sato, 1964). I'rotein concentration was estimated by the method of Bradford (1976), utilizing the dye-binding reagent and bovine 7-globulin standard from Bio-Rad Laboratories.

Determination of Cinnamate-4-Hydroxylase Activity and Total Phenolics

For the estimation of the cinnamate-4-hydroxylase activity, a spectrophotometric assay described by Lamb and Rubery (1975) was used. Total phenolic concentration was estimated using Folin-Ciocalteau reagent as described by Koumba- Koumba and Macheix (1982).

SDS-PAGE and Western lmmunoblotting

SDS-PAGE was performed in a Hoefer-Scientific gel electrophoresis apparatus (SE 600), utilizing 1.5-mm-thick discontinuous gels containing 10% total acrylamide (2.7% bisacrylamide) and 0.1% SDS (Laemmli, 1970). Proteins (20- 100 Pg/lane) were diluted in sample loading buffer, heated on a steam bath for severa1 minutes, and then subjected to electrophoresis at 45 V for 18 h. Separated polypeptides were electroblotted onto polyvinylidene difluoride membranes with a Hoefer TE-52 Transphor apparatus using Tris-Gly buffer containing 15% methanol (Towbin et al., 1979). Blot- ted proteins were visualized either by staining with Coo- massie .brilliant blue or by immunostaining. Molecular mass

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Cyt P-450 Camphor Hydroxylase 1233

markers (Diversified Biotech) were used to estimate the molecular mass of peptides in western blot experiments.

Protein blots were blocked with 3% nonfat dry milk and incubated with primary antibody (1 : lOOO dilution) or preimmune controls. Polyclonal antiserum, raised in rabbits against Pseudomonas putida camphor 5-exo-hydroxylase, was obtained from Oxygene (Dallas, TX) (as anti-Cyt P-450 CIAl immunoglobulin G fraction). Polyclonal antibodies were raised in rabbits against the solubilized, anion-exchange chro- matography- and SDS-PAGE-purified limonene-6-hydroxylase from spearmint (Mentha spicata) (Karp et al., 1990) using the subcutaneous implanted-ball technique (Ried et al., 1992). Alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (1:3000 dilution, Pierce) was used as the secondary antibody with 5-bromo-4-chloro-3-indolyl phosphate as substrate (Garfin and Bers, 1989).

RESULTS AND DISCUSSION

Demonstration and Localization of Camphor-6-exo-Hydroxylase Activity

The first step in the catabolism of (+)-camphor in undifferentiated sage cells is the transformation of this monoterpene ketone to 6-exo-hydroxycamphor (Funk et al., 1992). The reaction resembles that catalyzed by the well-known Cyt P-45OCAM from Pseudomonas (Sligar and Murray, 1986), and the responsible enzyme is one of the growing number of plant Cyt P-450-dependent monooxygenases (Donaldson and Luster, 1991). Whereas camphor-6-hydroxylase func- tions in the catabolism of a natural product, other monoterpene hydroxylases isolated from higher plants are involved in biosynthetic pathways (Karp et al., 1987, 1990; Karp and Croteau, 1988, 1993) that are expressed in highly differen- tiated glandular tissues (Gershenzon et al., 1987, 1989, 1991).

From in vivo feeding experiments with unlabeled substrate, the capacity for (+)-camphor catabolism in sage cell-suspension cultures was estimated to be 10 nmol h-' g-' fresh weight in early stationaryllate linear growth. However, optimum camphor-6-hydroxylase activity measured in crude cell-free extracts (10,OOOg supernatants with [+]-[3-3H2]camphor as substrate) was only 10 to 20% of the in vivo flux (i.e. 1-2 nmol h-' g-' fresh weight). The poor extractability and stability of higher plant P-450 Cyts are well known (Donald- son and Luster, 1991; Mihaliak et al., 1993), and in the present instance the loss of activity was most likely due to inefficient isolation of this membranous enzyme system as well as to degradation; species absorbing at 420 nm in the CO-difference spectra were observed in the soluble protein fraction as well as in the growth medium itself (Yu and Gunsalus, 1974; McMurry and Groves, 1986).

Differential centrifugation experiments indicated that roughly 90% of the camphor hydroxylase activity was localized in the light membrane fraction (195,0008 pellet). Only a small portion (approximately 10%) of the total hydroxylase activity was found in the heavy membrane fraction (lO,OOOg), most likely due to aggregation of light membranes with other organelles, and no detectable hydroxylase activity was found in the soluble protein fraction (195,OOOg supernatant). These results indicate that camphor-6-hydroxylase is a microsomal

enzyme system, as are other plant and animal Cyt I'-450 systems (West, 1980; Mihaliak et al., 1993).

During subcellular fractionation, a considerable part of the camphor hydroxylase activity observed in the crude extracts was lost and could not be accounted for by reassembly of fractions. This loss of activity could result from the afore- mentioned degradation of the Cyt or by separation of the various components required for hydroxylase activity (i.e. Cyt P-450 reductase, prosthetic groups, etc.). The addition of a small amount of the soluble enzyme fraction (up to 20% of total assay volume) or flavins (FAD and FMN to 2.5 ~ L M ) to the resuspended microsomes did enhance the camphor-6- hydroxylase activity of this fraction by roughly 3-fold (to approximately 4 pkat/mg protein). Such stimulation of Cyt P-450 catalysis has been reported previously for severa1 systems (West, 1980; Karp et al., 1987, 1990).

Product ldentification and Reaction Parameters

Incubation of microsomal preparations of sage cell-suspension cultures with (+)-[3-3H2]camphor and appropriate cofac- tors gave rise to only one radioactive product, coincident with authentic 6-hydroxycamphor, by radio-GLC analysis. This biosynthetic product was confirmed as the 6-exo epimer (>96%) by capillary GLC-MS. These analytical methods have been described previously in the context of the in vivo metabolism of (+)-camphor (Funk et al., 1992).

The microsomal camphor-6-exo-hydroxylase system required molecular oxygen and a reduced pyridine nucleotide (Table I). Incubations without a pyridine nucleotide revealed no detectable hydroxylation, whereas simply flushing the head space of the reaction mixture with argon reduced the hydroxylation rate by over 75%. NADPH could be replaced with NADH, but the latter supported the reaction at a lower rate (21%), probably due to less efficient redox coupling via

Table I. Reaction conditions and inhibition of camphor hydroxylase The (+)-camphor-6-exo-hydroxylase from suspension cultures of

common sage (S. officinalis) was assayed at a microsomal protein concentration of 1.5 mg/mL and at a (+)-camphor concentration of 70 p ~ . A relative rate of 100% = 2.5 pkat/mL, and the SD was in all cases within 16% of the mean of the triplicate determinations.

Conditions Relative Rate

%

1 mM NADPH + 0, 1 O0 1 mM NADPH + Ar 23 0, O 1 mM NADH + 0 2 21 1 mM NADPH + O2 + 2.5 p~ each FAD + FMN 240

Complete system (1 mM NADPH + 02) plus 10% co + 10% 0 2

50% CO + 1 O% O2

90% co + 10% o, 50% CO + 10% O2 + 450 nm of light

66 44 27

, 89 49

20 p~ Clotrimazole 29 50 p~ Clotrimazole 17

90% C O + 10% O2 + 450 nm of light 50 pM Cytc 5

1 r n M ,LI-Mercaptoethanol 73

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1234 Funk and Croteau Plant Physiol. Vol. 101, 1993

0.008Ldo

0.006

0.004

0.002

0.000400 450

WAVELENGTH (nm)

500

Figure 2. CO-difference spectrum of microsomal proteins from asage cell-suspension culture (600 Mg protein/mL) that were reducedwith Na2S2O4. The maximum absorption of the CO adduct at 450nm (minus the average 470- to 500-nm background) was used toestimate the Cyt P-450 content at 28 pmol/mg of protein.

NADH:Cyt b5 rather than NADPH:Cyt P-450 reductase. Theflavin moiety of the reductase component of plant Cyt P-450systems is known to be especially labile during the isolationof these membranous proteins (Madyastha and Coscia, 1978).As indicated previously, inclusion of both FAD and FMN inthe incubation mixture enhanced hydroxylation activity sev-eralfold (Table I).

The pH optimum for camphor hydroxylation was deter-mined in several buffers to be about 7.0, with activity reducedto about 60% at a half pH unit above or below the maximum.Under optimum conditions, linear with respect to proteinconcentration and time, an apparent Km value for (+)-cam-phor was estimated at 34 JIM based on double reciprocalplots of initial hydroxylation velocity versus substrateconcentration.

Inhibition of Camphor-6-Hydroxylase

Several classical inhibitors of hydroxylation reactions areoften used to examine the involvement of a Cyt P-450

Table II. Effect of potential inducers on camphor hydroxylationCamphor-6-exo-hydroxylase activity in 7-d-old suspension cul-

tures of common sage (5. officinalis) exposed to different inducersfor 18 h was measured after gel filtration of the enzyme extracts toremove residual inducer. For description of the assay system, seeTable I.

Inducer Concentration Relative Activity

None(+)-Camphor(+)-Fenchone(— )-MenthoneMgCI2MnCI2HgCI2FeCI3MnCI2 + FeCI32,4-DEthanolDMSO

1 mM1 ITIM1 mM

30 mM30 mM10 MM50 MM25 mM + 10 MM20 MM

0.2%1%

100363457

11369421312544639463

- 1 . 5 «

Q0.0

20 30 40 50

MnCI2 CONCENTRATION (mM)

Figure 3. Dose dependence of the induction of camphor-6-hy-droxylase (C6H) activity in MnCI2-treated sage cell-suspension cul-tures. Cultured cells (7 d after transfer) were treated with increasingconcentrations of MnCI2 and incubated for an additional 18 h, andthe cell fresh weight (T) and microsomal camphor hydroxylaseactivity (•) were determined. The microsomal protein content wasunchanged over the course of the 18-h experiment.

Pre- anti- anti-immune (P-450CAM) (P-450LH)

C M C S M

Coomassieblue

Mw

Figure 4. Western blot analysis of cultured sage cell proteins.Cultured cells were harvested in late logarithmic growth and ex-tracted, and the 10,000g supernatant, 195,000g supernatant, and195,000g pellet were prepared by differential centrifugation. Fiftymicrograms of the crude protein fraction (C), the soluble proteins(S), and the microsomal proteins (M) were resolved on a 10%discontinuous SDS-polyacrylamide gel, transferred to a polyvinyli-dene difluoride membrane, and probed with the preimmune con-trol, polyclonal antibodies raised against camphor-5-exo-hydroxyl-ase from P. put/da [anti-(P-450CAM)L and polyclonal antibodiesraised against limonene-6-hydroxylase from M. sp/cata [anti-(P-450LH)]. Proteins were stained with Coomassie brilliant blue, andmolecular mass markers (Mw) are indicated in kD.

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o>E

0.3 -

XCOo

CLin0.0

0 10 20 30 40 50 60 70

INCUBATION TIME (h)

0.0

1 ct- in2 -^< I

Q_

0 3.5 7 12 17 24 36 72

INCUBATION TIME (h)

Figure 5. Time course of induction of (+)-camphor-6-hydroxylase(C6H) following treatment with 30 mM MnCI2. (+)-Camphor-6-hydroxylase activity (•, O) and total phenolics (A, A) in induced(closed symbols) and noninduced (open symbols) cells are plottedin A. Dot blots of total microsomal protein probed with polyclonalantibodies raised against limonene-6-hydroxylase from M. spicata[anti-(P-450LH)] are provided in B. Total microsomal protein contentdid not change significantly over the 72-h time course. The slightoffset in hydroxylase activity and blot intensity may be due to thehigh background and apparent accumulation of inactive protein.

hydroxylase activity was almost completely inhibited (TableI).

Spectral Evidence

It was not possible to demonstrate the presence of Cyt P-450 in crude extracts by CO-difference spectra because ofinterfering pigments. However, microsomal preparations, re-duced with sodium dithionite, yielded useful CO-differencespectra (Fig. 2) from which a total Cyt P-450 content of 28pmol/mg protein (15 pmol/g fresh weight) was estimated.

Induction of Camphor-6-exo-Hydroxylase

Many Cyt P-450 monooxygenases involved in catabolictransformations in animals, microbes, and plants are inducedby treatment with the corresponding oxygenase substrate(Parke, 1957; Benveniste et al., 1982; Sligar and Murray,1986). Cyt P-450 monooxygenase systems in plants have alsobeen induced by treatment with growth hormones, heavymetals, and other xenobiotics (Reichhart et al., 1980; Adeleet al., 1981).

The influence of several potential inducers on camphorhydroxylase activity in sage suspension cultures following 18h of exposure is summarized in Table II. Camphor and othermonoterpene ketones did not enhance the rate of camphorhydroxylation in extracts of treated cells. However, if cellswere treated with metal ions the activity was induced, re-sulting in an up to 7-fold increase in camphor-6-hydroxylaseactivity after Mn2"1" (MnCl2) treatment. Very similar resultshave been reported for the induction of cinnamate-4-hydrox-ylase activity in Jerusalem artichoke slices exposed to 25 mM

monooxygenase, among which the photoreversible inhibitionby CO is the most definitive (West, 1980; Mihaliak et al.,1993). Increasing levels of CO (from 10-90% with constantO2 at 10%) revealed increasing inhibition of camphor hy-droxylation (Table I). These inhibition rates, ranging from 34to 73% inhibition relative to the corresponding control, aretypical of those observed with other plant-derived monoter-pene hydroxylases (Karp et al., 1987,1990; Karp and Croteau,1993). Partial light-reversal of CO inhibition (Table I) wasdemonstrated by incubation of reaction mixtures under bluelight (450 nm maximum output).

N-Substituted imidazoles have been used to inhibit Cyt P-450 terpene hydroxylases (Karp et al., 1990), and, in thepresent instance, clotrimazole was shown to be very effectivein inhibiting camphor hydroxylase activity by nearly 90% ata concentration of 50 pM. Inhibition of camphor hydroxyl-ation by |8-mercaptoethanol (Table I) has also been shownfor the bacterial P-450CAM system (Yu and Gunsalus, 1974).

NADPH-Cyt P-450 reductase, a key component of themicrosomal monooxygenase system, conducts the transfer oftwo electrons from NADPH to Cyt P-450. This flavoproteinis also able to transfer electrons to Cyt c and may be assayedby this means (Donaldson and Luster, 1991). Because Cyt cserves as an alternate electron acceptor, this Cyt can be usedto inhibit Cyt P-450-dependent monooxygenases (West,1980) and, in the presence of 50 /UM Cyt c, camphor-6-

anti-(P-450LH)

CM C+ S+ M+ Mw

9 6

5 5

362 9

Figure 6. Western blot analysis of the crude (C) and microsomal(M) proteins from untreated sage cells and of the crude (C+), soluble(S+), and microsomal (M+) proteins from suspension cells treatedwith 30 mM MnCI2 for 18 h. The blots were probed with polyclonalantibodies raised against limonene-6-hydroxylase from M. spicata[anti-(P-450LH)L and the molecular mass markers (Mw) are indicatedinkD.

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1236 Funk and Croteau Plant Physiol. Vol. 101, 1993

Mn2+ (Reichhart et al., 1980). The optimal concentration for hydroxylase induction in sage cell suspensions (18 h of exposure) was determined to be about 30 mM MnCh (Fig. 3); at this concentration, there was no observable lysis or adverse effect on cell fresh weight or protein content.

Antigenic Cross-Reactivity of Sage Proteins with Heterologous Polyclonal Antibodies

Very little information is available on the immunological relatedness of bacterial, animal, and plant Cyt P-450 enzymes that catalyze similar reactions. Antibodies raised against bac- teria1 Cyt P-450 species have been shown to cross-react with severa1 microsomal, wound-inducible proteins from pea epi- cotyls (Stewart and Schuler, 1989) and an anti-(Cyt P-450) monoclonal antibody raised against rat Cyt P-450 isoform c recognizes a bean microsomal protein tentatively identified as cinnamate hydroxylase (Bolwell and Dixon, 1986); rabbit anti-rat Cyt P-450 also inhibits the P-450-dependent hydroxylation of the monoterpene (+)-sabinene in S. officinalis microsomes (Karp and Croteau, 1987).

To evaluate the possible immunological similarity between the sage camphor hydroxylase and other Cyt P-450 monoterpene hydroxylase proteins, different cell fractions were subjected to SDS-PAGE and the separated peptides were transferred onto polyvinylidene difluoride membranes. These blots were then probed with two different -heterologous polyclonal antibodies, one raised in rabbits against the camphor-5-hydroxylase from P. putida (anti-[P-450~~~]), the other raised in rabbits against the limonene-6-hydroxylase from spearmint (M. spicata) (anti-[P-45OLH]), No polypeptides in crude extracts of sage cells (containing both soluble and membranous proteins) cross-reacted with preimmune controls (Fig. 4). However, two polypeptides in these extracts (one at 56 kD, the other at 58 kD) showed strong crossreactivity with both polyclonal antibodies. The analysis of microsomal and soluble proteins separately showed that the 58-kD polypeptide was localized in the microsomes, as was the Cyt P-450 camphor hydroxylase activity, whereas the 56- kD polypeptide was localized in the soluble fraction, which contained negligible hydroxylase activity. The soluble fraction showed a strong 420-nm absorbance in the CO-difference spectrum, suggestive of degraded Cyt P-450 (Yu and Gunsalus, 1974). Other plant Cyt P-450 proteins show similar molecular masses, such as cinnamate hydroxylase from Je- rusalem artichoke at 56 kD (Gabriac et al., 1985), a Cyt P- 450 purified from avocado at 47 kD (O’Keefe and Leto, 1989), and a Cyt P-450 purified from tulip bulbs at 52.5 kD (Higashi et al., 1985).

The induction of camphor-6-exo-hydroxylase activity in Mn2+-treated sage cells offered the possibility of examining the time-course coordination of catalysis and the level of the 58-kD microsomal polypeptide presumed to represent the Cyt P-450 hydroxylase. The specific activity of the camphor- 6-exo-hydroxylase of Mn2+-treated sage suspension cells reached maximum (7-fold) about 12 h after exposure (Fig. 5A) and the level of the microsomal protein recognized by anti-(Cyt I‘-450 limonene hydroxylase) followed closely (maximum at about 17 h) in dot-blot experiments (Fig. 5B). Because the anti-(limonene hydroxylase) polyclonal antibody

cross-reacted essentially with only the 58-kD polypeptide in microsomal preparations from both Mn2+-treated and untreated cells (Fig. 6), the results are consistent with the assumption that the 58-kD protein does represent the camphor-6-hydroxylase and that the induction of this activity is accompanied by a coordinate increase in enzyme protein. However, the possibility cannot be eliminated that other Mn2+-inducible Cyt I’-450 species at 58 kD are recognized by the antibodies and thus contribute to the observed time- course of protein accumulation and may obscure the identity of the camphor hydroxylase.

In general properties, the camphor-6-hydroxylase resembles other monoterpene hydroxylases (Karp et al., 1987,1990; Karp and Croteau, 1993; Mihaliak et al., 1993) and fulfills the established criteria for a Cyt P-450 monooxygenase (West, 1980). The induction by Mn2+ is reminiscent of other plant P-450 species involved in defense responses (Reichhart et al., 1980) and detoxification reactions (Adele et al., 1981; Mougin et al., 1990). Cinnamate-4-hydroxylase is induced by Mn2+ in some species (Reichhart et al., 1980), and, al- though cinnamate hydroxylase was readily observed in sage cell microsomal preparations (approximately 3 pkat/mg protein), this activity was unaltered by treatment of the suspension cultures with Mn2+; total phenolic content was also unaltered by Mn2+ treatment (Fig. 5A). The connection between Mn2+ induction of camphor-6-hydroxylase and the apparent role of this enzyme in monoterpene catabolism in sage is at present unclear

ACKNOWLEDGMENTS

We thank Charles Mihaliak for preparing the anti-(limonene hydroxylase) polyclonal antibodies and for assistance with the westem blots, A1 Koepp for preparing the substrate, and Joyce Tamura-Brown for typing the manuscript.

Received November 16, 1992; accepted January 11, 1993. Copyright Clearance Center: 0032-0889/93/101/1231/07.

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