solubilization, partial purification

Plant Physiol. (1992) 98, 215-2200032-0889/92/98/0215/06/$01 .00/0

Received for publication August 9, 1991Accepted September 10, 1991

Solubilization, Partial Purification, and Immunodetection ofSqualene Synthetase from Tobacco Cell

Suspension Cultures1

Kathleen Hanley and Joseph Chappell*Plant Physiology/Biochemistry/Molecular Biology Program, Department of Agronomy, University of Kentucky,

Lexington, Kentucky 40546-0091

ABSTRACT

Squalene synthetase, an integral membrane protein and thefirst committed enzyme for sterol biosynthesis, was solubilizedand partially purified from tobacco (Nicotiana tabacum) cell sus-pension cultures. Tobacco microsomes were prepared and theenzyme was solubilized from the lipid bilayer using a two-stepprocedure. Microsomes were initially treated with concentrationsof octyl-j@-d-thioglucopyranoside and glycodeoxycholate belowtheir critical micelle concentration, 4.5 and 1.1 millimolar, respec-tively, to remove loosely associated proteins. Complete solubi-lization of the squalene synthetase enzyme activity was achievedafter a second treatment at detergent concentrations above or attheir critical micelle concentration, 18 and 2.2 millimolar, respec-tively. The detergent-solubilized enzyme was further purified bya combination of ultrafiltration, gel permeation, and Fast ProteinLiquid Chromatography anion exchange. A 60-fold purificationand 20% recovery of the enzyme activity was achieved. Thepartially purified squalene synthetase protein was used to gen-erate polyclonal antibodies from mice that efficiently inhibitedsynthetase activity in an in vitro assay. The apparent molecularmass of the squalene synthetase protein as determined by im-munoblot analysis of the partially purified squalene synthetaseprotein separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis was 47 kilodaltons. The partially purified squal-ene synthetase activity was optimal at pH 6.0, exhibited a Km forfamesyl diphosphate of 9.5 micromolar, and preferred NADPH asa reductant rather than NADH.

Squalene synthetase is an integral membrane protein oftheendoplasmic reticulum that catalyses the condensation oftwoFPP2 molecules to form squalene, an isoprenoid intermediatededicated solely to sterol biosynthesis (1 1). Squalene synthe-tase also resides at a branch point in the isoprenoid biosyn-thetic pathway, representing a potentially important controlpoint for sterol biosynthesis and other branch pathways com-peting for the available FPP (5). However, examples of squal-

'Supported by the Kentucky Agricultural Experiment Station, a

cooperative grant from the U.S. Department of Agriculture, andNational Science Foundation grant DCB-8806273. This is journalpaper 91-3- 100 of the Kentucky Agricultural Experiment Station.

2 Abbreviations: FPP, farnesyl diphosphate; CMC, critical micelleconcentration; GDC, glycodeoxycholate; OTGP, octylthioglucopyr-anoside; FPLC, Fast Protein Liquid Chromatography.

215

ene synthetase regulation are limited. Faust et al. (4) andJames and Kandutsch (8) reported a suppression of squalenesynthetase enzyme activity in mammalian cells grown insterol-supplemented media, but concluded such regulationwas of secondary importance relative to the control providedby 3-hydroxy-3-methylglutaryl coenzyme A reductase. Theregulation of squalene synthetase enzyme activity in tobaccocell suspension cultures has also been described (18). Uponaddition of fungal elicitors to tobacco cell cultures, sterolaccumulation and biosynthesis cease, and the cells start syn-thesizing and secreting sesquiterpenes (3, 18). The inductionof sesquiterpene biosynthesis has been correlated with theinduction of a sesquiterpene cyclase enzyme, which is regu-lated by the transcription rate of the cyclase gene (20). Themechanism controlling the suppression ofsqualene synthetaseenzyme activity in elicitor-treated tobacco cells is currentlyunknown.To further investigate the mechanism(s) controlling the

suppression ofsqualene synthetase enzyme activity in tobaccocells, we are intent on developing antibody probes to thepurified protein to determine if the level of squalene synthe-tase protein is correlated with the respective enzyme activity.But because this enzyme is integrally associated with themembrane lipid bilayer, it was first necessary to develop asolubilization procedure that would result in continued cata-lytic activity and maintenance of stability for subsequentpurification steps. The anionic detergent deoxycholate wasused successfully to solubilize the yeast squalene synthetaseprotein, but recoverable activity was very low (1). The deter-gents octylglucopyranoside and Lubrol PX were also used tosolubilize squalene synthetase activity from yeast microsomes,but large micelles were formed, hindering further purificationsteps (9). Sasiak and Riling (13) were successful in purifyingthe yeast squalene synthetase enzyme to homogenity whenLubrol PX was removed during the latter stages of the puri-fication and octylglucopyranoside detergent was used alone,but the enzyme activity was very unstable. Similar attemptsto solubilize and purify squalene synthetase activity fromplant tissue have either failed (our laboratory) or not beenreported.

In the present investigation, we describe the solubilizationof squalene synthetase from tobacco microsomes by a two-step protocol using a combination of anionic and nonionicdetergents. Additionally, we report on the partial purification

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HANLEY AND CHAPPELL

and generation of polyclonal antibodies for the immunode-tection of a putative squalene synthetase enzyme.

MATERIALS AND METHODS

Cell Cultures

Tobacco (Nicotiana tabacum) cell suspension cultures weremaintained in Murashige-Skoog medium as previously de-scribed (3). Cells were harvested during their rapid growthphase (3 d after subculturing) and kept frozen at -80°C untilused for enzyme isolation.

Microsome Preparation

Microsomes were prepared in batches of 600 g frozentobacco cells. Cells were mixed with 800 mL of 100 mMpotassium phosphate buffer, pH 7.0, 4 mM MgCl2, 25 mMsucrose, and 5 mm f,-mercaptoethanol (buffer A), and kept atroom temperature for 15 min. Cells were then homogenizedtwice (once with 800 mL buffer A, once with 400 mL bufferA) using a Polytron (30 s, medium speed). Homogenates werecollected by filtration through two layers of nylon mesh andfiltrates were centrifuged at 10OOg for 10 min at 4°C. Thesupernatant was adjusted to 25% saturation with solid am-monium sulfate, allowed to equilibrate for 20 min at 4°C, andcentrifuged at 10,000g for 50 min at 4°C to precipitate themicrosomal fraction (14). The supernatant was discarded andthe pellet was resuspended in 30 mL of 50 mm Tris buffer,pH 7.5, 2 mm MgC92, and 2 mm ,#-mercaptoethanol (bufferB), centrifuged at 10,000g for 20 min, and the pellet againresuspended in buffer B. The microsomes were kept frozen at-20C and enzyme activity remained stable for several weeks.

Solubilization Procedure

Microsomal preparations were assayed for total protein andadjusted to a final concentration of 2.0 mg/mL with buffer Bprior to detergent solubilization. The microsomal fraction wasfirst treated with detergents at concentrations below theirCMC to remove loosely associated proteins, and then atconcentrations above their CMC to complete solubilizationof squalene synthetase. Initially, the diluted microsomes weremade to 4.5 mm OTGP (Ultrol grade, Calbiochem) and 1.1mM GDC (Calbiochem), kept on ice 15 min, and centrifugedat 100,000g for 1 hr at 4°C. The supernatant (SN 1) wasdecanted and the pellet resuspended with buffer B to theoriginal starting volumes. The resuspended pellet was adjustedto 18 mM OTGP (2 x CMC) and 2.2 mM GDC (1 x CMC)and treated as above. After ultracentrifugation, the superna-tant (SN2) contained greater than 80% of the squalene syn-thetase activity and was used for further purification steps.

Enzyme Purification Procedures

All of the purification steps were performed without inter-ruption at 4°C or on ice. The detergent-solubilized enzymeextract was concentrated to approximately one-third of theoriginal volume by ultrafiltration (YM 30 Diaflo membrane,Amicon). The concentrated enzyme preparation was loadedonto an HR S-300 Sephacryl (Pharmacia) gel filtration col-

umn (1.5 x 60 cm) equilibrated in 20 mM Tris, pH 7.5, 9 mMOTGP, 1.1 mm GDC, 2 mM MgCl2, and 5 mM f3-mercapto-ethanol (buffer C). Fractions containing squalene synthetaseenzyme activity were pooled and loaded onto a 1.0 mL FPLCMono Q anion exchange column (Pharmacia) equilibrated inbuffer C minus the GDC detergent. Bound proteins wereeluted with a 22 mL two-step buffer C-KCI salt gradient, 0 to0.3 M KC1 in 16 mL, then 0.3 to 1.0 M KC1 in the remaining6 mL.

Gel Electrophoresis and Immunoblotting

SDS-PAGE was according to Laemmli (10) in an 11.5%(w/v) acrylamide/bis gel. Proteins were detected after stainingwith Coomassie brilliant blue. Immunoblot analysis of squal-ene synthetase protein was performed as previously described(19).

Polyclonal Antibody Production and Precipitation of anEnriched Antibody Fraction

Three mice were immunized with a primary intraperitonealinjection of 50 ug partially purified squalene synthetase pro-tein in complete Freund's adjuvant. After 12 d, a secondaryinjection of 50 ,g squalene synthetase protein in incompleteFreund's adjuvant was administered. Twelve to 14 d after thesecondary injection, the mice were bled and serum collected.The antibody fraction was enriched from immune serumusing ammonium sulfate fractionation (14). A 100 uL aliquotof antiserum or preimmune serum was adjusted to 33%saturation with ammonium sulfate, kept on ice 15 min, spunat 14,000g for 10 min, and the supernatant was discarded.The pellet was resuspended in Tris-buffered saline (20 mMTris, pH 7.5, 500 mm NaCI) solution and ammonium sulfatewas removed using a centricon-10 microconcentrator (Ami-con). The final volume of the enriched antibody fraction wasadjusted to 100 ,uL with Tris-buffered saline. The isolatedantibody fraction was tested for antisqualene synthetase activ-ity in an in vitro assay and used for immunodetection on awestern blot.

Enzyme Assay and Protein Determination

The squalene synthetase enzyme assay was slightly modifiedfrom that previously reported (18). Briefly, a 1 to 2 AL (0.1-5.0 ,g protein) aliquot of sample was incubated in 500 mmpotassium phosphate buffer, pH 6.0, 40 mM MgCl2, 25 mM,B-mercaptoethanol, 5 mm NADPH, and 40 uM trans,trans-[1-3H]FPP (35 ,Ci/,umol) (18) in a final assay volume of 50ML for 20 to 30 min at 35C. The reaction was stopped by theaddition of 100 ,uL of petroleum ether containing 0.4% squal-ene as carrier, vortexed for 10 s, and then spun in a micro-centrifuge for 10 s. A 20 gL aliquot of the organic phase wasseparated on TLC silica gel plates (EM Merck) developed incyclohexane:ethyl acetate (85:15). The squalene product wasvisualized by iodine vapor and the spot scraped from the platefor counting of radioactivity. Proteins were determined afterTCA precipitation and assayed using a modified Lowryreaction (2).

216 Plant Physiol. Vol. 98, 1992

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SQUALENE SYNTHETASE FROM TOBACCO

RESULTS AND DISCUSSION

Solubilization and Purification

Squalene synthetase is a low abundant, integral membraneprotein of the endoplasmic reticulum (11). The low abun-dance of this enzyme made it initially necessary for us todevelop a procedure to isolate large quantities of microsomes.Because conventional preparations of microsomes by ultra-centrifugation results in relatively small sample sizes, an al-ternative method of precipitating membranous material witha low concentration ofammonium sulfate (14) was evaluated.By comparison, the recovery of microsomal squalene synthe-tase activity for the equivalent of 1 g frozen tobacco cells was0.5 nmol/min with a specific activity of 3.2 nmol/min-mgprotein for the ammonium sulfate precipitation method ver-

sus 0.47 nmol/min and specific activity of 3.0 nmol/min *mgprotein prepared by centrifuging the cell-free homogenate at100,000g for 1 h. In addition, preparation of microsomesresults in a 5 to 10-fold enrichment of squalene synthetasefrom the initial cell-free homogenate.Membrane proteins can be dissociated from the lipid bilayer

by various methods (17). Typically, nondenaturing detergentsare used when it is necessary to maintain catalytic activityafter solubilization. The choice of detergent(s) and concentra-tions needed must be determined empirically for each indi-vidual membrane protein (6). Initially, the detergents TritonX-100, Tween 20, Brij 96, and CHAPS were tested at 5 and50 mm for inhibition of squalene synthetase activity (data notshown). Although both OTGP and GDC did not inhibitsqualene synthetase activity, neither alone solubilized signifi-cant amounts of enzyme activity. However, in combination,

100

80

1 2 3 4

Protein for solubilzatlon (mglml)

Figure 1. Distribution of squalene synthetase enzyme activity re-covered after detergent treatment of tobacco microsomes. Micro-somes were prepared and protein was adjusted to 0.5 to 3.5 mg/mLand used in a two-step detergent-solubilization procedure as de-scribed in "Materials and Methods." The supernatents (SN1 and SN2)and remaining pellet were each assayed for squalene synthetaseactivity and plotted as a percentage of the initial microsomal squalenesynthetase activity. Each value is the mean of two separateexperiments.

Table I. Partial Purification of Squalene Synthetase Enzyme ActivitySqualene synthetase enzyme activity was solubilized from tobacco

cell microsomes using the detergents OTGP and GDC. Further puri-fication of enzyme activity was achieved by chromatography on HRS-300 Sephacryl gel filtration and FPLC Mono 0 anion exchangecolumns. All purification steps were performed at 40C withoutinterruption.

Active Total Enzyme Specific R PurificationFraction Protein Activity Activity ry Factor

mg nmol/min nmollminm. %protein

Microsomes 45.0 125 2.8 100 1.0Solubilized 17.6 104 5.9 83 2.1Concentrated 13.1 98.9 7.5 79 2.7Gel filtration 4.1 63.2 15.4 50 5.5Mono Q 0.16 26.2 164 21 59

OTGP and GDC solubilized greater than 80% of the micro-somal enzyme activity (Fig. 1) and the solubilized activityremained stable for further purification steps.

Detergent solubilization, by definition, results in the disin-tegration of the membrane lipid bilayer (17). This can beachieved by determining the correct ratio of detergent mole-cules to cell membrane lipids. Because it is generally morecumbersome to assay for total membrane lipid content thantotal membrane proteins, the latter can be quantified and thedetergent:protein ratio can be assessed for solubilization (6).The optimal ratio for detergent solubilization of tobaccosqualene synthetase was determined empirically by varyingthe total membrane protein concentration. Crude tobaccomicrosomal proteins were solubilized using a two-step solu-bilization procedure as described in "Materials and Methods."This two-step protocol initially removed the loosely associatedmembrane proteins, followed by the solubilization of integralmembrane proteins, including squalene synthetase, duringthe second detergent treatment. When crude extracts havinga total microsomal protein concentration of 3.5 mg/mL weredetergent-treated, 55, 40, and 5% of the starting microsomalsqualene synthetase activity was recovered in the superna-tants, SN2 and SN1, and pellet fractions, respectively (Fig. 1).When solubilization was attempted on microsomes havingless than 1.75 mg/mL total protein, not more than 30% ofthe original microsomal enzyme activity was recovered in anyof the fractions, with total combined activity not exceedingabout 50% (Fig. 1). Optimal recovery of squalene synthetaseactivity in the SN2 fraction was achieved at a protein concen-tration of approximately 1.75 to 2.5 mg/mL (Fig. 1). Thisstep also resulted in a twofold purification of the enzyme(Table I). Care was taken that all subsequent solubilizationsfor protein purification were done at approximately2.0 mg/mL.

Detergent solubilization of microsomal proteins results ina mixture of protein-detergent aggregates, protein-detergentmicelles, and detergent-protein-lipid mixed micelles (7, 16).Because the diverse composition of the micelles interfereswith the separation of individual proteins by conventionalbiochemical methods, the protein-detergent aggregates wereseparated from the large molecular mass micelles by gel

217

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HANLEY AND CHAPPELL

1.0

0 5 10 15column fractions

400

300

200

NC o5

1X E100

20 25

Figure 2. Separation of squalene synthetase activity from solubilizedmembrane proteins by gel filtration chromatography. Tobacco micro-somes were detergent-solubilized, concentrated by ultrafiltration, andloaded onto an HR S-300 Sephacryl column. Two milliliter fractionswere collected and assayed for enzyme activity and total protein. Themolecular mass of the detergent-enzyme complex was calculated as

1 10 kD using molecular mass standards.

MW xk(D I

228-

109-

70.3- tI. 'r...

44,1- ..

27.9-

2 3 4 540w 4*^ A"""

18.1-

Figure 3. SDS-PAGE of proteins associated with microsomal andpartially purified squalene synthetase active fractions. One hundredmicrograms of total protein from each of the different steps in thepurification protocol were separated on an SDS-polyacrylamide/bisgel and proteins stained with Coomassie brilliant blue. Fractionsseparated in each lane; 1, crude microsomes; 2, detergent-solubilizedSN2; 3, concentrated SN2; and active fractions after gel filtration (4)and Mono 0 chromatography (5).

-U nz activty200.20 - KCI

E150 51 2

0 ~~~~~~~~~~~000.iO

500

0 0.00 00 5 1 0 1 5 20 25

Column fractionsFigure 4. Separation of squalene synthetase enzyme activity usinganion exchange chromatography. Active enzyme fractions collectedfrom the gel filtration column were pooled and loaded on an FPLCMono Q column. Proteins were eluted with a two-step KCI gradientand 1 mL column fractions assayed for enzyme activity and totalprotein.

permeation. To improve resolution of the solubilized proteinmixture on a gel filtration column, it was first necessary toreduce the volume of our sample. When the SN2 fraction wasconcentrated by ultrafiltration using a membrane with amolecular mass cut-off of 30 kD, a significant purificationwith very little loss of enzyme activity was achieved (Table I).The concentrated sample was separated by gel filtration andthe large molecular mass micelles (600-200 kD) appeared ascloudy fractions just after the void volume. All of the recover-able detergent-solubilized squalene synthetase activity wasdetected in fractions 9 through 13 (Fig. 2), corresponding toa molecular mass of 1 10 kD. A broad range of different sizedproteins were recovered from the active fraction, as detectedafter separation by SDS-PAGE (Fig. 3). This possibly resultsfrom different sized proteins complexed with one or moredetergent or membrane molecules or mixed micelles, thusincreasing the apparent molecular mass anticipated for theprotein alone (16). Fractions 10 through 13 were pooled andused for further purification.

Separation ofenzyme proteins by charge or hydrophobicitymay be particularly difficult when trying to resolve detergent-solubilized proteins. Detergent moieties can interfere with thespecificity of the protein binding, or elution from the columnmaterial may destabilize the protein-detergent complex andresult in loss of catalytic activity (7). Preliminary experimentsindicated that detergent-solubilized squalene synthetasebound to DEAE anion exchange resins, but enzyme activityeluted with a very broad elution pattern (data not shown). Toincrease resolution, the pooled gel filtration fraction wasloaded onto an FPLC Mono-Q column and proteins separatedwith a two-step KC1 gradient (Fig. 4). Squalene synthetaseactivity was eluted in two fractions at approximately 550 mmKCl. Approximately 20% of the starting enzyme activity wasrecovered with a 60-fold purification (Table I).

0)E

-52a


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SQUALENE SYNTHETASE FROM TOBACCO

100

~80

60

~40

20

0.0 0.5 1.0 1.5 2.0

&d antibody fraction/gg protein

Figure 5. Inhibition of squalene synthetase enzyme activity by squal-

ene synthetase polyclonal antibodies. The enriched antibody fraction

was isolated (see "Materials and Methods") from preimmune and

immune serum from mouse-i, -2, and -3 and increasing amounts

were used to test for inhibition of the partially purified squalene

synthetase enzyme activity in an in vitro assay. Activity for preim-

mune, mouse-i, -2, and -3 represented by closed squares, open

squares, closed circles, and open circles, respectively. Activity for

Squalene synthetase activity without added serum was normalized

to 100% (20 nmol/min - mg protein).

A modest increase of specific activity from 2.8 to 15.4

nmol/min - mg protein was observed after detergent solubi-

lization and gel permeation (Table I). This specific activity

was increased further to 164 nmol/min mg protein after the

separation on a Mono Q column. Numerous unsuccessful

attempts were made to further purify the tobacco squalene

synthetase protein, including separation by chromatofocus-

ing, hydrophobic interaction, dye ligands, and hydroxyapatite.

Aliquots of proteins isolated from each step in the purification

protocol were separated by SDS-PAGE and visualized after

Coomassie blue staining. We detected a modest enrichment

of several protein bands (Fig. 3), but with no clear candidate

for the squalene synthetase protein. The enriched protein

band detected at about 00 kD is most likely not the squalene

synthetase protein. Even though squalene synthetase eluted

from the gel filtration column with an apparent molecular

mass of approximately 00 kD, this fraction represents a

protein-detergent aggregate that inflates the true molecular

mass of the protein component (16).

Immunological Studies

The partially purified squalene synthetase protein recoveredfrom the Mono Q column was used to generate polyclonalantibodies from mice. Three mice were immunized with thepartially purified squalene synthetase protein. Because serum

samples interfered with the in vitro enzyme assay (data notshown), the antibodies were enriched from both the preim-mune and immune serums using ammonium sulfate fraction-ation (14). Enriched antibodies from preimmune serum didnot inhibit the in vitro squalene synthetase enzyme assay (Fig.5). We did not detect any cross-reactivity between either thepreimmune serum or enriched antibody fraction and thepartially purified squalene synthetase protein in a western blot

(data not shown). When the enriched antibodies from mouse-

1, -2, and -3 immune serums were tested for the ability toinhibit the squalene synthetase enzyme activity, only mouse-

1 efficiently inhibited the in vitro assay (Fig. 5). Increasingamounts of mouse-1 antibodies resulted in a proportionaldecrease of enzyme activity (Fig. 5). When the polyclonalantibody serums from all three mice were used separately toprobe western blots of partially purified squalene synthetaseprotein, a prominent band located just below the 68 kDmolecular mass marker was detected in all three immunoblots(Fig. 6). This band was less intense in mouse-l as comparedwith -2 and -3. Another prominent band located at about 47kD was also detected after probing with mouse- I serum. The47 kD band was significantly less visible in the western blotprobed with mouse-2 serum, and was almost undetectablewith mouse-3 serum (Fig. 6). An enrichment of the 47 kDband in both mouse- and -2 was detected with increasingpurification of the squalene synthetase protein (Fig. 6). Theenhanced appearance of the 47 kD band from mouse- andthe decreased intensity detected using the other two serumscorrelates well with the inhibition ofenzyme activity (Fig. 5).We tentatively identify this protein as tobacco squalenesynthetase.

Biochemical Comparison

Squalene synthetase has previously been isolated and char-acterized only from yeast (1, 9, 12, 13, 15). We have observedsome similarities between the yeast and plant enzymes. Boththe tobacco and the yeast (1, 9, 12, 13) enzyme have an

absolute requirement for a divalent cation in the reactionbuffer for catalytic activity. Similar to the yeast protein (1,

-- AeM

MW

- 200

- 97

68

43

1 2 3

Pernt nhibiton of squalenesynthetat enzyme acthivy:

82 21 12

Figure 6. The immunodetection and inhibition of squalene synthetaseenzyme activity by three different mouse antiserums. Three micewere immunized with partially purified squalene synthetase proteinand their subsequent serums were tested for their ability to inhibitsqualene synthetase activity and immunodetect microsomal proteins.In each panel, 100 jg aliquots of proteins from the solubilized enzymefraction (left lane) and the gel filtration column fraction (right) wereseparated on the same SDS-polyacrylamide gel, transferred to nitro-cellulose paper, and then probed with each different mouse anti-serum. Arrow indicates location of 47 kD protein.

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HANLEY AND CHAPPELL

0.3'

0.2

0.1

#011

SM)

0.6

0.4

0.0 0.1 0.2 0.3 0.4

-0.1 0.0 0.1 0.2 03 0.4

huM FPPFigure 7. Km determination of squalene synthetase for FPP. Doublereciprocal plots of the enzyme velocity versus FPP concentration formicrosomal (A) and partially purified (through gel filtration step) (B)squalene synthetase were used to determine the apparent Km forFPP. Each value represents the mean of two replications.

13), tobacco squalene synthetase can utilize either NADH orNADPH as a reductant, but preferred NADPH (data notshown). Kinetic studies were conducted using both the crudetobacco microsomes and the partially purified gel filtrationfraction. The microsomal squalene synthetase enzyme had an

apparent Km for FPP of 26 ,uM as determined by the doublereciprocal plot of enzyme velocity versus substrate concentra-tion (Fig. 7A). After partial purification of the squalene syn-thetase enzyme, the apparent Km for FPP was 9.5 ,uM (Fig.7B). Affinity for the substrate is in close agreement withprevious kinetic studies reported in yeast (1, 13).

Squalene synthetase in yeast has been reported from twoseparate laboratories as having an apparent molecular massof 47 (13) and 55 kD (1). The correlation between stainingintensity on a western blot and inhibition of enzyme activity

also suggests that the tobacco squalene synthetase is of asimilar molecular mass. Currently, attempts are underway tofurther characterize this putative tobacco squalene synthetaseprotein.

LITERATURE CITED1. Agnew WS, Popjak G (1978) Squalene synthetase: solubilization

from yeast microsomes of a phospholipid-requiring enzyme. JBiol Chem 253: 4574-4583

2. Chappell J, Beevers H (1983) Transport of dicarboxylic acids incastor bean mitochondria. Plant Physiol 72: 434-440

3. Chappell J, Nable R, Fleming P, Anderson RA, Burton HR(1987) Accumulation of capsidiol in tobacco cell culturestreated with fungal elicitor. Phytochemistry 26: 2259-2260

4. Faust JR, Goldstein JL, Brown MS (1979) Squalene synthetaseactivity in human fibroblasts: regulation via the low densitylipoprotein receptor. Proc Natl Acad Sci USA 76: 5018-5022

5. Goldstein JL, Brown MS (1990) Regulation of the mevalonatepathway. Nature 343: 425-430

6. Helenius A, McCaslin DR, Fries E, Tanford C (1979) Propertiesof detergents. Methods Enzymol 56: 734-749

7. Hjelmeland LM, Chrambach A (1984) Solubilization of func-tional membrane proteins. Methods Enzymol 104: 305-319

8. James MJ, Kandutsch AA (1979) Inter-relationship betweendolichol and sterol synthesis in mammalian cell cultures. J BiolChem 254: 8442-8446

9. Kuswik-Rabiega G, Rilling HC (1987) Squalene synthetase: sol-ubilization and partial purification of squalene synthetase,copurification ofpresqualene pyrophosphate and squalene syn-thetase activities. J Biol Chem 262: 1505-1509

10. Laemmli UK (1970) Cleavage of the structural proteins duringthe assembly of the head of bacteriophage T4. Nature 227:680-685

11. Poulter CD, Rilling HC (1981) Conversion of famesyl pyrophos-phate to squalene. In JW Porter, SL Spurgeon, eds, Biosyn-thesis of Isoprenoid Compounds, Vol 1. John Wiley and Sons,New York, pp 413-441

12. Qureshi AA, Beytia E, Porter JW (1973) Squalene synthetase II.Purification and properties of bakers' yeast enzyme. J BiolChem 248: 1848-1855

13. Sasiak K, Rilling HC (1988) Purification to homogeneity andsome properties ofsqualene synthetase. Arch Biochem Biophys260: 622-627

14. Scopes RK (1988) Protein Purification, Principles and Practice,Ed 2. Springer-Verlag, New York, p 52

15. Shechter I, Bloch K (1971) Solubilization and purification oftrans-farnesyl pyrophosphate-squalene synthetase. J Biol Chem246: 7690-7696

16. Thomas TC, McNamee MG (1990) Purification of membraneproteins. Methods Enzymol 182: 499-520

17. van Renswoulde J, KempfC (1984) Purification of integral mem-brane proteins. Methods Enzymol 104: 329-347

18. Voegeli U, Chappell J (1988) Induction of sesquiterpene cyclaseand suppression of squalene synthetase activities in plant cellcultures treated with fungal elicitor. Plant Physiol 88:1291-1296

19. Voegeli U, Freeman JW, Chappell J (1990) Purification andcharacterization of an inducible sesquiterpene cyclase fromelicitor-treated tobacco cell suspension cultures. Plant Physiol93:182-187

20. Voegeli U, Chappell J (1990) Regulation of sesquiterpene cyclasein cellulase-treated tobacco cell suspension cultures. PlantPhysiol 94: 1860-1866


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