Nov 5, 2015 - The reaction is catalyzed by a membrane-bound mon- ooxygenase system, isolated as the microsomal fraction of cell-free homogenates, and is ...
Vol. 261, No.31, Issue of November 5,pp. 14651-14657,1986 Printed in 11.S.A .
T H E JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Societyof Biological Chemists, Inc.
Microsomal Enzymesof Cholesterol Biosynthesis PURIFICATIONOF LANOSTEROL 14~~-METHYL DEMETHYLASE CYTOCHROME P-450 FROM HEPATIC MICROSOMES* (Received for publication, July 10, 1986)
James Trzaskos, Sumio Kawata, and JamesL. Gaylor From the Central Research and DevebDment DeDartment, E. I. du Pont de Nemours & Company, Experimental Station, Wilmington, Delaware 19898
Employing reconstitution assays and measurement of cytochrome P-450 content, lanosterol 14a-demethylase and cholesterol 7a-hydroxylase have been studied in solubilized preparations of rat hepatic microsomes. Both activities have been resolved from other cytochrome P-450 isozymes and each other by chromatography on DEAE-Sephacel andadsorption on hydroxylapatite. The demethylase has been further purified to homogeneity by cation exchange chromatography on Mono-S resin. The purified cytochrome displays a specific content of 15.8 nmol of heme/mgof protein and a single band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis with an apparent M, of 51,000. A Soret maximum for the reduced/CO binding complex at 448nm is observed. Reconstitution of the purified cytochrome with NADPH-cytochromec reductase, dilaurylphosphatidylcholine,NADPH, and 0,supports the demethylationprocess which is inhibited by CO. Reconstitution also affords accumulation of oxygenated, metabolic intermediateswithsingle catalytic turnover of the cytochrome, thus supporting the hypothesis that a single isozyme of cytochrome P450 isresponsible for all three oxidations and the activity involved in the lanosterolC-32demethylation sequence. Low oxidase activity toward several xenobiotic substrates and selectivity toward endogenous sterolsubstrates is observed forthepurified cytochrome. These results indicate a high degree of substrate specificity for the cytochrome, which would be expected for a constitutiveP-450 involved in anabolic biochemical processes.
Microsomal monooxygenase systems consisting of cytochrome P-450, cytochrome P-450 reductase, and phospholipid are responsible for the oxidative metabolism of numerous xenobiotics, carcinogens, and endogenous substrates (1). Study of these monooxygenase systems has been facilitated by the use of various inducing substancesand induction protocols which permit manipulations that increase the level of the desired cytochrome (2). Thus, purification and detailed study of the heme-protein with respect to protein structure, substrate specificity, and mechanism of induction have been obtained (2-4). Recently, considerable interest and effort has been given to the purification and characterization of constitutive cytochrome P-450 species ( 5 , 6). Constitutive cyto* This is Publication 3594 from the Central Research and Development Department, E. I. du Pont de Nemours & Co. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact.
chromes, unlike their inducible counterparts, are present in microsomes in much smaller amounts and are inherentlyless easily purified, characterized, and studied. The constitutive cytochrome P-450 species presumably function in the metabolism and biochemical transformation of endogenous metabolites, a role consistent with the biochemistry and intermediary metabolism of their respective substrates. As one would expect for any enzyme involved in intermediary metabolism, a great degree of substrate specificity is seen for constitutive cytochrome P-450-dependent oxidations (5). Thus, employment of enzymic activity measurements as a means to purify constitutive cytochrome P-450 species should prove useful. This is indeed the case for the lanosterol demethylating cytochrome P-450 of mammalian microsomes which is described in this report. The initial reaction in the biosynthesis of cholesterol from lanosterol is the oxidative demethylation of C-32 oflanosterol (7-9). The reaction is catalyzed by a membrane-bound monooxygenase system, isolated as the microsomal fraction of cell-free homogenates, and is dependent upon 02,NADPH, NADPH-cytochrome-c reductase, and cytochrome P-450 (7, lyase 10-13). We have described conditions for a sensitive and easy assay of the lanosterol’ C-32 demethylation reaction employing radiolabeled dihydrolanosterol as substrate and HPLC analysis of metabolic products (13). In this same report, we demonstrated that oxidative demethylation could beobserved in solubilized and partially purified preparations. Wenow report the purification of the lanosterol demethylating cytochrome P-450 from hepatic microsomes. In addition, we show by monitoring in a reconstituted assay that lanosterol demethylating cytochrome P-450 is resolved from cholesterol 7ahydroxylase, a second constitutive sterol metabolizing cytochrome P-450-dependent enzyme. Characterization of the purified demethylase activity with respect to substrate specificity, electron transport component-dependence, and spectral properties is also presented. EXPERIMENTALPROCEDURES
Materials-24,25-Dihydrolanosterol and [24,25-3H2]-24,25-dihydrolanosterol were isolated as described previously (13). Cholesterol (Eastman Kodak) and [4-“C]cholesterol (New England Nuclear) were purified by HPLC in the chromatographic system described previously (13). Triton WR-1339 was obtained from Ruger Chemical CO. (Irvington, NJf. Triton N-101was from Rohm and Haas (Philadelphia, PA). Dilaurylphosphatidylcholine was from Avanti Polar Lipids (Birmingham, AL). DL-Isocitric acid, trisodium salt, Type I; isocitrate dehydrogenase, pig heart,Type V; reduced glutathione;
* The abbreviations and trivial names used are: lanosterol, 4 , 4 , 1 4 ~ trimethyl-5a-cholesta-8,24-dien-3P-ol; dihydrolanosterol, 4,4,14a-trirnethyl-5a-cholest-8-en-3~-01; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.
14651
14652
Lanosterol Demethylase P-450
cytochrome c, horse heart, Type VI; @-NAD,grade 111; p-NADH, terol or [4-"C]cholesterol was suspended with the aid of the detergent, disodium salt, grade 111; NADPH,tetrasodiumsalt,Type I; and Triton WR-1339, in the ratio of 75:l(w/w) detergent:sterol. The sodium cholate were purchased from Sigma. DEAE-Sephacel and stock substrate suspension was made to a final concentration of 1000 Mono-S cation exchange columns and agarose-octylamine were pur- nmol/ml, 10,000 dpm/nmol. Incubation flasks contained in a final chased from Pharmacia P-L Biochemicals. Hydroxylapatite (Bio-Gel volume of2.0 ml: 100 mM PEDG, buffer, pH 7.4, 0.02% Triton NHT) was obtained from Bio-Rad. All other chemicals were from 101, 100 pg of dilaurylphosphatidylcholine,0.1 mM NADH, 1.0 mM commercial sources and of the best grade available also as described NADPH, 10 mM isocitrate, 0.5 units of isocitrate dehydrogenase, 0.4 mM MgCI,, 50 p M sterol substrate, 1 unit of NADPH-cytochrome-c (13). Purification of Cytochrome P-450,,.,-,~-Microsomeswere pre- reductase, cytochrome P-450 fraction (0.1-0.5 nmol). Incubations pared from male, Sprague-Dawley rats maintained on adiet contain- were started by the addition of sterol substrate and continued at ing cholestyramine (13) and washed with a 10%solution of WR-1339 37 "C for 20 min. Reactions were terminated by the addition of 2 ml (14). Microsomes (2-3 g of protein) were suspended in 100 mM of 15% potassium hydroxide (w/v) in 95% methanol (also containing potassium phosphate, 0.1 mM EDTA, 0.1 mM dithiothreitol, 20% 100 pg/ml butylated hydroxytoluene) and sterols were extracted and glycerol (100 mM PEDGbuffer), pH 7.4, containing 0.6% (w/v) analyzed by HPLC asdescribed previously (13). Enzymic activity was sodium cholate to a final protein concentration of 4 mg/ml. After calculated from the relative amounts of radiolabeled substrate and stirring for 30 min a t 4 "C, soluble material was isolated by centrif- detected radiolabeled products in incubated samples compared to ugation at 105,000 X g for 1 h. Polyethylene glycol precipitation (0- nonenzyme controls. 16% w/v) of the soluble protein fraction was performed at 4 "C by the dropwise addition of a 50% (w/v) stock solution of polyethylene RESULTS glycol 6000 with stirring overnight. The precipitated material isolated It was our intent to resolve chromatographically the isoby centrifugation was solubilized in a minimal volume (-80 ml) of 100 mM PEDG buffer, pH 7.25, containing 0.6% sodium cholate and zymes of cytochrome P-450 which catalyze lanosterol C-32 applied to an agarose-octylamine column (2.6 X 30 cm) equilibrated demethylation and cholesterol 7a-hydroxylation by monitorin the same buffer. The column was washed with equilibration buffer ing both enzymic activities in reconstituted assay systems. until the A4,,.,of the column effluent returned to base-line (-200 Solubilization of hepatic microsomes and enrichment of cyml). Bound protein fraction was eluted with 1000 mlof 100 mM PEDG buffer, pH 7.25, containing 0.2% (w/v) Triton N-101 tochrome P-450 isozymes by polyethylene glycol precipitation (PEDGT). The octylamine fractions from three preparations were and octylamine column chromatography was followed byseppooled and concentrated against an Amicon PM-30 membrane to a aration of the twoenzymic activities by DEAE-Sephacel final volume of 125 ml. The concentrated sample was dialyzed over- column chromatography (Fig. 1). Lanosterol demethylase is night against 4 liters of 5 mM PEDGT buffer, pH 7.8, diluted to 260 very weakly bound to the anion exchange resin under the ml with a solution of 20% (w/v) glycerol, 0.1 mM EDTA, 0.1 mM conditions employed and two peaks of activity are routinely dithiothreitol, and 0.2% Triton N-101, and applied to a DEAESephacel column (2.6 X 25 cm) equilibrated with 5 mM PEDGT observed with the reconstituted assay system. The two activbuffer, pH 7.8. The column was washed with 100 ml of equilibration ities demonstrated identical metabolite profiles as detected buffer followed by 200 ml of 10 mM PEDGT buffer, pH 7.8. A 780- by radio-HPLC analysis and therefore were combined for ml linear gradient composed of equal volumes of 10 mM PEDGT further purification. buffer, pH 7.8, and 100 mM PEDGT buffer, pH 7.8, was used to elute Cholesterol 7a-hydroxylase was eluted from the DEAE bound protein fractions. Fractions were pooled as shown in Fig. 1and column with a linear gradient of potassium phosphate which those containing active lanosterol demethylase or cholesterol 7ahydroxylase were adjusted to pH 7.25 by the dropwise addition of 1 resulted in hydroxylase activity in the fractions designated C M potassium monobasic phosphate. The fractions were then applied and D. These data suggest that theP-450 isozyme for cholesbroad elution profile to an hydroxylapatite column (1.6 X 6 cm) equilibrated with 10 mM terol 7a-hydroxylation hasarather PEDGT buffer, pH 7.25. The column was eluted with a step gradient under the gradient conditions employed, but dramatically of 60 ml each of 50,90, and 200 mM PEDGT buffer, pH 7.25. Active illustrates the distinction between demethylase and hydroxdemethylase eluted with the 100 mM PEDGT buffer. Cholesterol 7a- ylase chromatographic behavior. For further purification of hydroxylase eluted with the 200 mM PEDGT wash. Pooled fractions were dialyzed overnight against 3 litersof 10 mM PEDGT buffer, pH cholesterol 7a-hydroxylase, only fraction D wasemployed 6.8, and applied to a Mono-S cation exchange column equilibrated since the catalytic efficiency of this fraction was 2-fold higher/ with the same buffer. Fractions containingactive enzyme were eluted nmol of cytochrome P-450 when compared to fraction C. with a 0-0.15 M KC1 gradient in 10 mM PEDGT buffer, pH 6.8. A further demonstration that the two isozymes are indeed Active fractions were pooled and represented purified lanosterol distinct is provided by chromatography on hydroxylapatite demethylase cytochrome P-450. (Fig. 2). Under identical column conditions, the lanosterol Other Enzyme Purifications and Actiuity Determinations-Deterdemethylase activity in the pooled DEAE fractions A plus B gent-solubilized NADPH-cytochrome-c reductase and cytochrome b6 were purified from rabbit liver microsomes as described by Yasukochi is eluted with 90 mM phosphate buffer (Fig. 2 A ) . Cholesterol 7a-hydroxylase activity which is contained in DEAE fraction and Masters (15) and Spatz and Strittmatter ( X ) , respectively. Cytochrome b, and cytochrome P-450 were determined as described D is only minimally affected by 90 mM phosphate. The bulk by Estabrook and Werringloer (17). Protein was determined by the of the hydroxylase activity requires 200 mM phosphate buffer method of Lowry et al, (18) using bovine serum albumin as standard. to be eluted from the hydroxylapatite column (Fig. 2B). It Activities of p-nitroanisol0-demethylase and aminopyrine N-demethylase were measured by formaldehyde release according to the should be noted, that in both the DEAE fractions andhydroxmethod of Nash (29) and described by Kawata et al. (30). Aniline ylapatite fractionsdisplaying lanosterol demethylase activity, hydroxylase was assayed by p-aminophenol using phenol reagent (30, no cholesterol 7a-hydroxylase activity could bedetected. Sim31). All assays were done in a reaction mixture containing 1.0 mg of ilarly, no demethylase activity was detected in any pooled aminopyrine (10 mM), or fractions displaying cholesterol 7a-hydroxylase activity. microsomal protein; p-nitroanisole (1 aniline (10 mM) as substratein a final volume of 1 ml of 0.1 M PEDG These results clearly demonstrate a separation of enzymic buffer, pH 7.4. Samples were preincubated at 37 "C for 3 min and the reaction was started by addition of 0.1 mM NADPH. The reaction activities for these distinct reactions in sterol biosynthesis was terminated after 10 min by adding 0.25 ml of 20% trichloroacetic and metabolism, respectively, and prove that unique isozymes of cytochrome P-450 are required for each reaction. acid. Xenobiotic assays in a reconstitutedsystem were done as described Purification of lanosterol demethylase and cholesterol 7aabove, but utilizing purified components in the following proportions: hydroxylase was continued for both activities by chromatog0.3nmol of cytochrome P-450, 1 unit of NADPH-cytochrome-c raphy on Mono-S cation exchange resin (Fig. 3). This step reductase, 25 pg of dilaurylphosphatidylcholine,0.02%Triton N-101, 0.1 M PEDG buffer, pH 7.4, in a total volume of 1 ml. Reactions were proved to be successful for the demethylase activity, but did initiated with 0.1 mM NADPH at 37 'C and terminated after 10 min. not result in an active preparation of hydroxylase enzyme. To Reconstitution Assays-Substrate [24,25-3H2]-24,25-dihydrolanos-date, an active preparation of cholesterol 7a-hydroxylase be-
14653
Lanosterol DemethylaseP-450
FRACTION NUMBER
FIG. 1. Elution profile of lanosterol demethylase and cholesterol 7a-hydroxylase from DEAE-Sephacel. Cytochrome P-450 (1500 nmol) isolated by octylamine column chromatography was applied to a column of DEAE-Sephacel (2.6 X 20 cm) equilibrated with 5 mM PEDGT buffer pH 7.8. The column was washed with 100 ml of equilibration buffer and then eluted with 200 ml of 10 mM PEDGT buffer, pH 7.8, followed by a 600-ml linear gradient composed of equal volumes of 10 mM and 100 mM PEDGT buffer, pH 7.8. Fractions were pooled based upon 417 nm absorbance, as indicated, and then assayed for enzymic activity. Column flow rate was 0.5 ml/ min with 10-ml fractions. El, dihydrolanosterol demethylase activity (24.6 nmol/min total activity); 0,cholesterol 7a-hydroxylase (48.0 nmol/min total activity).
I A I
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8 1216
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11100
I
k-R4
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400- 1500
.20
20 24 28 32 36 40
FRACTIONNUMBER FRACTION NUMBER
T
-* t
60 2 6 a -I
4oF
ec z W
20
8w
P
' 0
4
8
12162024
0
28 32 36 40
FRACTIONNUMBER
FIG. 2. Hydroxylapatite column elution profile for lanosterol demethylase and cholesterol 7a-hydroxylase activities. DEAE fractions A plus B or fraction D (Fig. 1) were applied to an hydroxylapatite column (1.6 X 7.0 cm) equilibrated in 10 mM PEDGT buffer, pH 7.25. The column was washed with 20 ml of equilibration buffer and eluted with 60 ml each of 50, 90, and 200 mM PEDGT buffer, pH 7.25. Fractions were pooledbased upon 417 nmabsorbance and assayed for lanosterol demethylase or cholesterol 7a-hydroxylase. A , chromatogram of DEAE A plus B pool. B , chromatogram of DEAE D pool. Symbols are as described in the legend to Fig. 1.
FIG. 3. Chromatography of dihydrolanosterol demethylase activity on Mono-S fast protein liquid chromatography. 82 nmol of pooled hydroxylapatite fraction was loaded on a Mono-S column (1 X 10 cm) equilibrated in 10 mM PEDGT buffer, pH 6.8. The column was washed with 5 ml of equilibration buffer and eluted with a linear salt gradient inthe same buffer.
yond the hydroxylapatite step has not been obtained. A 14-fold purification of demethylaseactivity over the hydroxylapatite fraction isobserved with the Mono-Scolumn step when compared on a per mg protein basis. This reflects a 4.5-fold purification when compared on cytochrome P-450 content. At this stage, the lanosterol demethylase cytochrome P-450 is homogeneous. Table I summarizestheseresults which reflect both enzymic activity and cytochrome P-450 content a t various steps in the purification. The overall yield from a partially enriched cytochrome P-450 preparation isolated by octylamine column chromatography was 1%when based upon cytochrome P-450 recovery and 28% when based upon demethylase activity. The low recovery of cytochrome P-450 in the active demethylase fraction does not appear to be due to inherent lability of the cytochrome, but reflects the small amount of this constitutive cytochrome present inhepatic microsomes. The finalpreparation of purifiedcyto-
Lanosterol Demethylase P-450
14654
TABLE I Purification of lanosterol demethylase cytochrome P-450
drolanosterol emethylase" contentSpecific P-450 Protein Procedure
Recovery mg
Octylamine 3.3 DEAE-Sephacel 285 Hydroxylapatite 5.23 355 Mono-S 15.8
643 120 38 1.5
%
nmol pmol/rninlmg pmol/min/nmol nmollmg
2,104 99.0 495 200 24.3
914 4.1
53 223
175
(1,300)b
100 7.1 (28.3)b
(20,540)
Microsomal values average 1 nmol of P-450/mg; 421 > 67 pmol/min/mg; respectively. Values in parentheses were obtained in incubationswith 100 pg of dilaurylphosphatidylcholine.
m-6
-a
Mr = 51,000
.......................
.7
\"/H""
400
3 4
700
FIG.5. Absolute absorbance spectra for purified cytochrome P-45014a.DM. Spectra were recorded in 0.1 M potassium phosphate buffer (also containing 0.1 mM EDTA, 0.1 m M dithiothreitol, 20% glycerol, and 0.02% Triton N-101), p H 7.8, using 0.055 nmol of purifiedcytochrome. Oxidized, (-); reduced, (- - -); reduced/CO binding, (. . . ). A UFS, absorbance units a t full scale.
%
1 2
500 600 WAVELENGTH (nm)
5 6
FIG.4. SDS-PAGE of purified cytochrome P-45014a-DM. bound cholesterol. Employing the standard reconstitution assay which was used for the purification of enzymic activity (Fig. 6), it was possible to show enrichment of the demethylase to1.3 nmol/ min/mg of proteininthe presence of phospholipid. The modest increase in catalyticefficiency over crude microsomes (0.42 nmol/min/mg) suggested that further optimization of reconstituted activity could be obtained. In this regard, demethylase activity wasshown to haveadependenceupon phospholipid and NADPH-cytochrome-c reductase as would chrome displayed a specific content of 15.8 nmol of heme/mg be expected for a P-450-dependent reaction (Fig. 7). Surprisof protein. A single band on SDS-PAGE(Fig. 4) shows a n M , ingly, saturation of demethylase activity with NADPH-cytoof 51,000 and suggests that only one isozyme of cytochrome chrome-c reductase required unusually high amounts of the P-450 is responsible for the entire lanosterol (3-32 demethyl- reductase enzyme. This may be do, in part, to thepresence of ase process. This is in agreement with previous results found detergents, Triton N-101 (0.02%) and WR-1339 (0.16%), in for the yeast enzyme (10, 11).The final preparation is stable thereconstitutedassaysystem.For purposes of studying upon storage a t 4 "C on ice for several months without any demethylase activity with the purified cytochrome, 3 units of detection of conversion to cytochrome P-420 or loss of en- reductase enzyme and 50 pg of dilaurylphosphatidylcholine were employed. zymic activity. Results of effectors upon the purified enzyme are shown in Absolute spectra of the purified cytochrome are shown in Fig. 5. Absorbance maxima at 570, 530, and 417 nm are seen Table 11. An absoluterequirement for molecular oxygen, purifiedcytochrome, and NADPH-cytochrome-creductase for the oxidized spectrum indicative of low spin character. The dithionite reduced cytochrome has maxima at 545 and was observed, thus, defining the obligatory molecular com418 nm and thereduced/CO binding complex displays aSoret ponents necessary for catalysis. CO inhibition of the reconpeak a t 448 nm with an absorbance maximum at 550 nm. stituted enzyme is also seen, which is consistent withprevious Upon occasion during thecourse of this study, some high spin results for the microsomalenzyme and indicative of cytocharacter hasbeen observed forthe purified cytochrome. This chrome P-450 involvement. The inclusion of other micromay reflectthe association of some endogenoussubstrate with somal electron transport components such as cytochrome bs the cytochrome which may vary among preparations. High or NADH-cytochrome-c reductase, either alone or in combispin character has been observed for other sterol metabolizing nation, were without significanteffect. This latterobservation P-450 species such as cytochrome P-450,, from adrenal mi- was also maintained when the NADPH concentration of the tochondria (19) which is routinely isolated with substratereconstituted systemwas lowered to 100 p~ (data notshown).
SDS-PAGE was performed as described by Laemmli (20) employing a 10% acrylamide separation gel. Proteins bandswere visualized with Coomassie Blue staining. Lane I , 0.2 pg of p-45014,.DM; Lane 2,0.4 pg Lane 3, highmolecularweightmarkers:myosin, of P-45Ol4,.~~; 200,000; &galactosidase, 116,000; phosphorylase, 92,500; bovine serumalbumin, 67,000; ovalbumin, 43,000; Lane 4, low molecular weight markers: phosphorylase; bovine serum albumin; ovalbumin; carbonic anhydrase, 31,000; soybean trypsin inhibitor, 21,000; lysozyme, 14,300; Lane 5,P-45014n.DM,0.6 pg; Lane 6, P-45014,.0~,1.2 pg.
14655
Lanosterol Demethylase P-450
P - 4 5 0 REDUCTASE (UNITS)
TIME (mid I
3
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1
B
P-450 CONTENT (nmol)
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100
200
300
400
500
DLPC ( ugm)
FIG. 7. Optimization of reconstituted dihydrolanosterol deFIG. 6. Assay linearity of reconstituted dihydrolanosterol demethylating activity. Reconstitution of dihydrolanosterol de- methylase activity with purified components. Activity was opmethylating activitywas performedas described under “Experimental timized employing 0.05 nmol of purified cytochrome P-450 in a 20Procedures” employinga cytochrome P-450 fraction purified throughmin incubation as described under “Experimental Procedures.” A, in presence hydroxylapatite chromatography.A ; time was varied employing 0.2 the amount of cytochrome P-450 reductase was variedthe B , The amount of nmol of cytochrome P-450. B, amount of added cytochrome P-450 of 100 pg of dilaurylphosphatidylcholine(DLPC). dilaurylphosphatidylcholine was varied in the presence of 3 units of was varied in a 20-min assay. cytochrome P-450 reductase. TABLEI1 Results of effectors upon purified demethyhe cytochrome P-450 activity lanost-8-ene-3@,32-diol, 3P-hydroxylanost-8-en-32-aldehyde, Reconstitution assays were performed as described under “Experand 4,4-dimethyl-5a-cholesta-8,14-dien-3@-ol. These products imental Procedures”employing 0.05 nmolof cytochrome P-450fracare identical to the metabolites observed under interruption tion and 3 units of NADPH-cytochrome-c reductase activity. Results conditions with intactmicrosomes (13,20).It should be noted are the average of duplicate assays which did not varyby more than 2%. that even with this extremely sensitive radio-HPLC assay Demethylase with purified components, it has not been possible to detect Conditions ‘ activity a third oxygenated species necessary for demethylation. Alnmol/min/nmol though failure to trap or detect this intermediate does not Complete 4.1 disprove the involvement of an isolable metabolite in the P-450 0 demethylase reaction, it does suggest that alternative mech- P-450 reductase 0 anisms which preclude the isolation of such an intermediate + CO/air (80/20) 0.1 or trap a penultimate + N, (air) (80/20) may exist. The inabilitytodetect 2.6 intermediate is alsoobserved in the aromatase reaction which + 1 mM CN” 3.8 3.6 is cytochrome P-450-dependent and catalyzing a similar de- + 1 nmol of cytochrome b5 + 1 nmol of cytochrome b5 plus 1unit 3.5 methylation reaction (21). of b, reductase Finally, substrate specificity of the purified lanosterol de3.5 + 1 unit of b, reductase methylating cytochrome P-450 was determined towardseveral xenobiotics substrates. As can be seen in Table111, the purified cytochrome appears to be specific in its oxidation toward ated with broad substrate specificity for cytochrome P-450 14a-methyl sterols withvery poor activity toward any of the isozymes. xenobiotic compounds tested. This low activity with purified DISCUSSION cytochrome reflects the rather poor activity toward these same substrates seen for isolated microsomes from cholestyramineLanosterol demethylation and cholesterol-7a hydroxylation fed animals. These data are consistent with a high degree of are two cytochrome P-450-dependent reactions of sterol mesubstrate specificity for constitutive cytochrome P-450 iso- tabolism which are catalyzed by hepatic microsomes. These zymes necessary for catalyzing biosynthetic reactions as op- activities are similar since they are both enhanced by cholesposed to detoxification processes which are normally associ- tyramine treatment (13,22),catalyze reactions on the a-face The observed metabolic products generated by the reconstituted demethylase system are shown in Fig. 8 and include:
14656
Lanosterol Demethylase P-450
1
FIG. 8. Radio-HPLC chromatogram of dihydrolanosterol demethylase reaction mixtures employing purified components. Reconstitution assays were performed as described in the legend to Table 11. Panel A is minus cytochrome P-450 fraction.Panel B is a
complete reaction mixture. Sterols identified by retention time are: 13.4 min, lanost-8-en-30,32-diol; 14.4 min, 38hydroxy-lanost-8-en-32-aldehyde; 21.0 min, 4.4-dimethyl-5a-cholesta-8,14dien-30-01;and 27.8 min, dihydrolanosterol.
”-
”
2t
0
8
16
24
32
-
0
8
16
24
0
+
32
TIME (min)
TABLE I11 Xenobiotic metabolism with hepatic microsomesand purified lanosterol demethylase cytochrome P-450 from cholestyramine-fed
448 nm for the reduced CO binding complex and is isolated primarily in the low spin state. The reconstituted enzymic activity shows anabsoluterequirement for NADPH-cytorats chrome-creductase, molecularoxygen, and purified P-450 Xenobiotic metabolism was conducted with isolated microsomal fraction (Table 11) in keeping with previous results for the fraction or with a reconstituted system as described under “Experi- microsomal, membrane-bound enzyme (13). Optimal demethmental Procedures.” Resultsare the average of duplicate assays. ylase activity was seen in the presence of phospholipid, an Lanosterol essential component of most reconstituted cytochrome P-450 Substrate demethylase P-450 _ _Microsomes systems (2). nmoljminjnmol Purified preparations of the isolated cytochrome do not 1.2 4.17 Aminopyrine display substantialactivitytoward xenobiotic substrates
