Isolation of cytochrome P450 from hepatopancreas microsomes of the spiny lobster, Panulirus argus, and determination of catalytic activity with NADPH cytochrome P450 reductase from vertebrate liver

ARCHIVES

OF BIOCHEMISTRY

Vol. 282, No. 1, October,

AND

pp. 8-17,

BIOPHYSICS

1990

Isolation of Cytochrome P450 from Hepatopancreas Microsomes of the Spiny Lobster, Panulirus argus, and Determination of Catalytic Activity with NADPH Cytochrome P450 Reductase from Vertebrate Liver’ Margaret

0. James

Department

of Medicinal

Received

February

15,1990,

Chemistry,

Box J-485, JHMHC,

and in revised

form

May

University

Inc.

1 This work was supported Command, Grant CA 44297.

in part

by NIH

Gainesville,

Florida

32610

29,199O

One major form of cytochrome P450 has been isolated from the hepatopancreas of untreated spiny lobsters, Panulirus argus. This form, termed here Dl, was purified to a specific content of 12.1 f 1.8 nmol/mg protein. Two minor forms, termed D2 and D3 were partially purified to 4.6 + 1.6 and 2.3 & 0.2 nmol P450/mg protein, respectively. No NADPH-cytochrome P450 reductase activity was detected in spiny lobster hepatopancreas microsomes and no purification of spiny lobster reductase was achieved in this study. Very low NADPH-cytochrome c reductase activity was found in hepatopancress microsomes and also in cytosol. Indirect evidence suggested that proteolysis of spiny lobster P450 reductase during the preparation of hepatopancreas microsomes may in part account for the lack of detectable monooxygenase activity in hepatopancreas microsomes. The catalytic activities of the Dl or D2 forms of spiny lobster P450 were measured by mixing Dl or D2 with NADPH-cytochrome P450 reductase isolated from pig or rat liver microsomes. D2 was very efficient in demethylating benzphetamine, with a turnover number of 122 per minute, and Dl was an efficient catalyst of progesterone 16 a-hydroxylation, with a turnover number of 43 per minute. Other good substrates for Dl and D2 forms were aminopyrine, testosterone, benzo(a)pyrene, and 7-ethoxycoumarin. Little activity was found with methyl-, ethyl-, pentyl-, or benzyl-phenoxazone ethers as substrates. The profile of metabolites formed by Dl or D2 with benzo(a)pyrene as substrate were more similar to those formed with uninduced rat liver microsomes than to those formed by liver microsomes from uninduced flatfish species. CC) ISSO Academic Press,

of Florida,

and the US Army

R&D

Cytochrome P450 has been found in many different animal and plant phyla, where it functions in introducing oxygen into endogenous and xenobiotic molecules present in the organism (1). Among the marine arthropods, in uiuo studies have demonstrated that several species of lobsters, crabs, and shrimp will excrete oxygenated metabolites of pollutant chemicals to which they are exposed (2). There has, however, been some difficulty in conducting in vitro studies of the functions of cytochrome P450 from decapod crustacea. The hepatopancress is the major organ of metabolism and digestion in crustaceans (3). Microsomes prepared from the hepatopancreas of lobster and crab species typically have little or no demonstrable NADPH-dependent monooxygenase activity, although relatively high concentrations of cytochrome P450 may be present (4). When added to active microsomal preparations from other species, hepatopancreas microsomes from the spiny lobster inhibit monooxygenase activity, apparently by inhibiting cytochrome P450 reductase (5, 6). Monooxygenase activity could be demonstrated in microsomes when direct oxidizing agents such as cumene hydroperoxide or sodium periodate were used (6, 7). Other organs from crab or lobster have low cytochrome P450 concentrations and low NADPH-dependent monooxygenase activity (reviewed in Ref. (4)). The objective of the present study was to isolate components of the monooxygenase system from spiny lobster hepatopancreas in order to remove the monooxygenase inhibitors and to determine if the hepatopancress P450 could function with NADPH and reductase. Another objective was to measure optimum rates of NADPH and cytochrome P450-dependent monooxygenation with several model substrates. In this way, the properties of the monooxygenase system of the spiny lobster could be compared with those of other species. A

8 All

Copyright (c> 1990 rights of reproduction

000%9861/90 $3.00 by Academic Press, Inc. in any form reserved.

CYTOCHROME

preliminary Ref. (8).

report

of these investigations

P450

was given in

METHODS

Preparation of Hepatopancreas Microsomes For each batch of microsomes, five to seven feral intermolt spiny lobsters (I’nn~lirus Argus) of mixed sex and body weight of 500-700 g were anesthetized by cooling in ice for 20-30 min, partially exsanguinated, and dissected as rapidly as possible. Each hepatopancreas was placed in a beaker containing 100 ml of ice-cold buffer H (0.15 M KCl, 0.05 M potassium phosphate, pH 7.4, 0.1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride (PMSF),” and 46,000 units/liter aprotinin). After rinsing again with chilled buffer H, each hepatopancreas was weighed and homogenized in an ice bath with 2 vol of chilled buffer H. Hepatopancreas nuclei, mitochondria, microsomes, and cytosol were isolated by fractional centrifugation as described previously (5). In some experiments, buffer H was made isotonic with spiny lobster hemolymph by including 0.3 M KC1 instead of 0.15 M KCl. It has been stated that buffers that are isotonic with hemolymph must be used to isolate microsomes from the hepatopancreas of another crustacean, the shore crab (9, 10).

Chromatographic Resolution of Microsomes and Purification of Cytochrome P450 Fractions IJnless otherwise stated, all procedures were carried out at 0-4-C. Buffer A was potassium phosphate, pH 7.4 (of various concentrations as stated), containing 20% glycerol, 0.1 mM EDTA, 0.1 mM dithiothreitol and 0.1 mM PMSF. Step I.-Concentration ofcytochrone P450 and removal of inhibitors o/ monooxygenase actiuity. As soon as they were isolated, the washed microsomes were resuspended in 0.01 M buffer A containing 0.5% (w/ v) sodium cholate (1 ml buffer per gram hepatopancreas wet weight). The solution was stirred for 90 min, then recentrifuged at 176,OOOg in a 60 Ti rotor for 90 min (Beckman L5-50 ultracentrifuge). The clear pale yellow supernatant was pipetted off and was usually discarded. The viscous, translucent dark red band near the bottom of the tube, termed the Ml fraction, was carefully pipetted out and saved. The Ml fraction has been previously described as the “red” fraction (8). The small pellet of unsolubilized microsomal membranes under the Ml fraction was usually discarded. The microsomal suspension and the Ml fraction were analyzed for cytochrome P450 and protein (see below). Step 2.-DEAE-wllulose chromatogruplz.y. The Ml fraction was dissolved in sufficient solubilizing buffer (0.1 M buffer A containing 0.5% sodium cholate and 0.2% Emulgen 911) to give a final concentration of approximat,ely 5 mg protein per milliliter. This solution was loaded at a flow rate of 0.5 ml/min onto a 2.5 X 30.cm column containing DEAE-cellulose (Whatman DE-52) which had been preequilibrated with solubilizing buffer. Fractions of 250 drops (about 5 ml) were collected. The column was eluted with 300 ml of solubilizing buffer and then with 300 ml of a linear gradient (0 to 0.5 M) of potassium chloride in soluhilizing buffer. Tubes were pooled to give three cytochrome P450-containing fract,ions, Dl, D2, D3, as shown in Fig. 1, dialyzed against solubilizing bufIer (fraction D3 only), and concentrated on calcium phosphate gel as described by Johnson (11). The fractions eluting between 0.15 and 0.5 M KC1 were assayed for NADPH cytochrome c reductase activity.

’ Abbreviations PAGE, sodium

used: PMSF, phenylmethylsulfonide fluoride; SDS dodecyl sulfateepolyacrylamide gel electrophoresis.

IN

SPINY

9

LOBSTER

Step 3.-Phenylsepharose and hydroxylapatite chromatography. The Dl and D2 fractions from step 2 were further resolved on phenylsepharose followed by hydroxylapatite. For phenylsepharose chromatography, the eluate from calcium phosphate gel (in 0.3 M buffer A) was applied directly to a 1.5 X 15.cm column containing phenylsepharose which was preequilibrated with 0.2 M buffer A containing 0.05% sodium cholate. After sample application, t,he column was washed with 50 ml of 0.2 M buffer A containing 0.05% sodium cholate and then with 100 ml of 0.005 M buffer A containing 0.05% sodium cholate. The column was eluted with 0.005 M buffer A containing 0.2% Emulgen 911. The cytochrome P450 peak was applied to a hydroxylapatite column (1 X 8 cm) which was preequilibrated with 0.005 M buffer A. After sample application, the column was washed with 50 ml of 0.005 M buffer A and then eluted stepwise with 100 ml each of 0.1,0.2, and 0.3 M buffer A containing 0.05% sodium cholate. The cytochrome P450 peak was dialyzed against 0.01 M buffer A containing 0.05% sodium cholate and stored at ~70°C. SDS-PAGE. The monomeric molecular weight and electrophoretie purity of cytochrome P450 fractions were determined by SDSPAGE on gradient gels with 4412.5% acrylamide according to the procedure recommended by Pharmacia. In some experiments, 7.5% acrylamide gels with 4% acrylamide stacking gels were prepared and used by the method of Laemmli (12). The gels were calibrated with a set of standard proteins of known molecular weight.

Purification and Assay of NADPH P4FiOReductase

Cytochrome

The enzyme was purified from hepatic microsomes of pig or phenobarbital-treated rats according to the procedure of Yasukochi and Masters (13). Each of the final preparations gave a single band on SDS-PAGE and had NADPH cytochrome c reductase activity of 40,000 units per milligram protein. Reductase activity was assayed by the method of Philips and Langdon (14) in the presence of 0.1 M Hepes buffer, pH 7.4, and 0.1 M KC1 instead of phosphate buffer. Lobster hepatopancreas microsomes, Ml fraction and fractions from DEAEcellulose chromatography of lobster hepatopancreas microsomes were similarly assayed for NADPH cytochrome c reductase activity. In one experiment hepatopancreas cytosol was desalted by passing through G25 Sephadex and loaded on to a DEAE-cellulose column as in step 2 for purification of P450.

Measurement of Monooxygenase Activity Attempts to purify NADPH-cytochrome P450 reductase from hepatopancreas were unsuccessful, so we were unable to truly reconstitut,e the spiny lobster monooxygenase system. The ability of the spiny lobster cytochrome P450 to catalyze NADPH-dependent oxidation of several substrates was determined by mixing equimolar amounts of NADPH cytochrome P450 reductase from pig or rat liver with the lobster P450 fractions as described by Miwa et al. (15). In some cases, P450 and reductase were added t,o a sonicated suspension of phosphatidylcholine for use in assays. All monooxygenase assays were conducted at 35°C with saturating concentrations of NADPH and under linear conditions of product formation. Benzo(a)pyrene monooxygenase activity was determined with [7,10-‘4C]-benzo(a)pyrene (80 PM final concentration with 0.5 PCi per assay) and metabolites were either quantitated as total water soluble-metabolites (16) or as individuals metabolites after HPLC separation (17). In these studies the incubation time was 5 min. The methyl-, ethyl-, pentyl-, and benzyl-ethers of phenoxazone were synthesized from resorufin and the appropriate alkyl iodide as described previously for ethylphenoxazone (18). After purification by TIC, the phenoxazone ethers were used as substrates at a final assay concentration of 5 PM. The amount of resorufin formed in a ii-min incubation period was determined Huorimetrically as de-

10

MARGARET

0. JAMES

scribed previously (19). Formation of umbelliferone from 7-ethoxycoumarin was measured fluorimetrically as described by Aitio (20). Production of formaldehyde from benzphetamine or aminopyrine was determined with the Nash reagent (21). Hydroxylation of testosterone and progesterone as substrates was determined as previously described (22). In some cases, substances known to modify monooxygenase activity in mammalian systems, such as a-naphthoflavone (23), lphenylimidazole (24), metyrapone (25), and ellipticine (26) were included in the assay tubes.

Other Analytical

LOW

SALT

NADPH

Reductase

Procedures

Protein was assayed by the methods of Lowry et al. (27) or the BioRad method (28). Cytochrome P450 content was determined according to the procedures described by Estabrook et al. (29). Epoxide hydrolase activity was determined by the method of Oesch et al. (30) with 8-14C-styrene oxide as substrate. Glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate was determined by the method of Habig et al. (31) as described previously for spiny lobster hepatopancreas (32). Amino acid analysis of the purified cytochrome P450 was carried out with ornithine as internal standard on a Beckmann amino acid analyzer.

RESULTS

61

Succlnate

HIGH

I3 1,

SALT

EH

6 4

Recovery and Purification of Cytochrome P450, and Absence of NADPH Cytochrome P450 Reductase Figure 1 shows the subcellular distribution of cytochrome P450, epoxide hydrolase activity, glutathione Stransferase activity with 1-chloro-2,4-dinitrobenzene, and NADPH-, succinate-, and NADH-dependent cytochrome c reductase activities in hepatopancreas. The ionic strength of the isolation buffer did not affect the subcellular distribution or activity of any of these enzymes (Fig. 1 and data not shown). The microsomal fraction was not a firm pellet, as is found with microsomes from vertebrate liver, but rather was a loosely packed layer from which the clear cytosol had to be carefully separated. Both P450 and epoxide hydrolase were concentrated as expected in the microsomal fraction, but relative NADPH cytochrome c reductase activity was similar in the microsomal and cytosolic fractions (Fig. 1). In both microsomes and cytosol, NADPH cytochrome c reductase activities were very low, 5 + 3 nmol/ min/mg protein, regardless of isolation buffer. Attempts to qualitatively measure NADPH cytochrome P450 reductase were unsuccessful in that addition of NADPH to CO-gassed microsomes gave no peak at 450 nm, but only a small peak at 420 nm (Fig. 2). For comparison, Fig. 2 also shows the spectrum obtained by adding NADPH to CO-gassed hepatic microsomes from a fish with active NADPH cytochrome P450 reductase. The fish microsomes had much less total P450 content than the spiny lobster microsomes (0.21 nmol P450/mg protein for fish, 0.9 nmol P450/mg protein for spiny lobster), as measured by the dithionite difference spectra of CO-gassed microsomes. Table I shows the concentration and recovery of cytochrome P450 in the hepatopancreas microsomal frac-

2 0

21

km 0

20

40

GSH-T

60 60

% Protein

100

Iti 0

20

40

60

80 100

% Protein

FIG. 1. Influence of the KC1 concentration of the isolation buffer on the relative specific activities or content of cytochrome P450, epoxide hydrolase (EH), and glutathione S-transferase (GSH-T), and the relative specific activities of cytochrome c reductase with NADPH, NADH, or succinate. High salt buffer had 0.3 M KC1 and low salt buffer had 0.15 M KCl. Relative specific activities were calculated by the method of de Duve (46). Nut, pellet after centrifugation of homogenate at 600g; Mit, pellet sedimenting between 600 and 13,500g; Mic, layer sedimenting between 13,500 and 176,000g; cyt, 176,OOOg supernatant.

tions and from column chromatography. Recentrifugation of solubilized microsomes to give the “Ml” fraction resulted in a 1.5- to 2-fold purification of cytochrome P450, but had no effect on epoxide hydrolase activity or NADPH cytochrome c reductase activity (8). In addition, the Ml fraction but not microsomes could be reconstituted with vertebrate reductase to give a catalytically active preparation (see below). The Ml fraction could not be isolated if a nonionic detergent such as Emulgen 911 or Lubrol were included in the solubilization buffer. After dissolution of the Ml fraction in buffer A containing cholate and Emulgen 911, the Ml fraction could be resolved into three distinct cytochrome P450 peaks (Fig. 3), with the major peak Dl eluting in the void volume. The relative proportions of peaks D2 and D3 varied somewhat between preparations. Peak Dl was always quantitatively the largest P450 fraction. As isolated

CYTOCHROME

P450

IN

SPINY

11

LOBSTER Load I

IO

Buffer 1

A

Salt gradient I

t II I: “I\;; To,

03 -

k 0.5 M KCI-

I 400

410

420

430

440

450

460

470

400

490

500

h k

FIG. 2. Difference spectrum of CO-gassed spiny lobster hepatopancress microsomes, or sheepshead fish hepatic microsomes, in the presence of NADPH. Each microsomal suspension contained 1 mg protein/ml. Fish hepatic microsomes contained 0.21 nmol P450/mg protein, as determined from the dithionite difference spectrum of COgassed microsomes, whereas spiny lobster microsomes contained 0.9 nmol/mg protein.

from the DEAE column, all three P450 fractions contained some epoxide hydrolase activity. Chromatography on phenylsepharose partially dissociated P450 from epoxide hydrolase, but even the most purified Dl fraction had measureable epoxide hydrolase activity with styrene oxide as substrate (l-5 nmol/min/mg protein). Figure 4 shows the relative mobility on SDS-PAGE of the Dl and D2 P450 peaks, after further chromatography on phenylsepharose and hydroxylapatite, and of the Ml fraction. The apparent molecular weight of the major form, Dl, by SDS-PAGE was 52,500, D2 had molecular weight 53,000, and D3 51,500 (data not shown). Duplicate samples of the most highly purified Dl preparation (14.6 nmol P450/mg protein) were subjected to acid hydrolysis and amino acid analysis. The results are presented in Table II. The molecular weight by

TABLE Purification

of Cytochrome Hepatopancreas

Note. Data show number

are expressed of experiments

a\

150

Fraction

200

Number

FIG. 3. Elution of cytochrome P450 and 418-nm absorbing material in fractions following application of solubilized hepatopancreas microsomes to DEAE-cellulose. For details of the elution, see the Methods section.

amino acid composition was 55,000. Absolute spectra of the oxidized forms of Dl and the CO-gassed, dithionitereduced form of Dl are presented in Fig. 5. The peak at 417 nm in the absolute spectrum of the native cytochrome indicates that Dl was isolated in the low spin form. The peak of the reduced-CO-gassed cytochrome

I

P450 from Spiny Lobster Microsomes nmol

Microsomes Ml fraction Dl, DEAE eluate Dl, final preparation D2, DEAE eluate D3, DEAE eluate

9

P450/mg

protein

0.93 * 0.49 (5) 1.57 4.5

t 0.36 AZ 2.6

12.1 k 1.8 4.6 2.3

+ 1.6 f 0.2

as means + SD. Numbers performed.

(6) (4)

(4) (4) (3)

in parentheses

FIG. 4. SDS-PAGE of fractions Dl, D2, and Ml relative to molecular weight standards. Lane A shows purified Dl; lane B shows D2; lane C shows molecular weight standards at 94,000, 67,000, 45,000, 30,000 and 20,000; lane D shows the Ml fraction.

12

MARGARET TABLE Amino

Acid

Amino

Composition Cytochrome

acid

II of Spiny P450 Dl Number

Lobster

of residues

Ala Asx cys Phe Gly His Ile LYS Leu Met Pro As Ser Thr Val Trp Tyr GlX Total residues Calculated molecular

0. JAMES

per nanomole

39 42 ND 34 46 17 31 39 69 3 25 31 38 42 31 ND 20 9 516 55,000

weight

0 i

350

nm.

Similar

absolute

spectra

were

500

FIG. 5. Absolute spectra of purified Dl fraction (12 nmol P450/mg protein). The solid line shows the absolute spectrum of the native oxidized form of Dl and the dotted line shows the spectrum obtained after forming the CO complex of the reduced (dithionite) cytochrome.

activity. NADPH-dependent reduction of cytochrome c was catalyzed by fractions eluting between 0.18 and 0.2 M KCl, but the total activity recovered was very low (0.1 pmol cytochrome c reduced per minute). Measurement

at 450.5

450 Nanometers

Note. Duplicate 2.5.nmol samples of Dl were completely hydrolyzed in hydrochloric acid. Ornithine was used as an internal standard. ND, not determined.

was

400

of Monooxygenase

Activity

The NADPH-dependent monooxygenase activity of each spiny lobster P450 fraction was determined in the

obtained

for D2 and D3. No the

cytochrome

KC1

gradient

c reductase eluate

from

activity

the DEAE

was

detected

in

column.

Solubilization of NADPH Cytochrome c Reductase Activity from Endoplasmic Reticulum The data in Fig. 1 show that reductase activity was partially solubilized during the preparation of microsomes. Since hepatopancreas microsomes had very low or undetectable monooxygenase activity (4, 5), experiments were conducted to determine if proteolytic enzymes in hepatopancreas were cleaving the hydrophobic ‘tail’ from NADPH cytochrome P450 reductase (33,34) and solubilizing the protein. In one experiment, hepatopancreas cytosol was isolated in the absence of PMSF or aprotinin and samples of this cytosol were incubated for 15 min with purified pig liver reductase or an equal volume of water, then subjected to SDS-PAGE, along with untreated reductase. The gel is shown in Fig. 6. Incubation of pig reductase with cytosol resulted in the appearance of a new band at molecular weight 66,000, presumably by proteolytic cleavage of the reductase protein. In other experiments, desalted hepatopancreas cytosol was chromatographed on DEAE-cellulose and the eluate that was 0.2-0.5 M in KC1 was assayed for reductase

FIG. 6. Effect of hepatopancreas cytosol on P450 reductase isolated from pig liver. Lanes A and B show the protein band present in a preincubated (15 min at 4°C) mixture of hepatopancreas cytosol and pure reductase: lane A contains 82 pg cytosolic protein and 8 rg reductase protein and lane B contains 27 pg cytosolic protein and 3 pg reductase protein. Lane C shows molecular weight standards at 94,000, 67,000, 45,000,30,000, 20,000, and 14,500. Lanes D and E respectively show 8 and 3 pg of pure pig reductase. Lanes F and G show 82 and 27 pg cytosolic protein, respectively.

CYTOCHROME

P450

IN

TABLE Monooxygenase

Activity

of Spiny

Lobster

Cytochrome P450

P&O Reductase

SPINY III

Fractions in the from Rat Liver Nanomoles

Benzphetamine Aminopyrine 7.Ethoxycoumarin Methylphenoxazone Ethylphenoxazone Pentylphenoxazone Benzylphenoxazone Benzo(a)pyrene Testosterone 16n-

W Progesterone

product

Ml

Substrate

16~

W 21.

26.3 IL 5.3 19.8 0.325 k 0.139 0.0189 0.062 f 0.051 0.002 0.010 1.43 2 0.41 1.3 0.6 4.96 + 0.78 1.18 + 0.41 0.67 i- 0.42

13

LOBSTER

Presence

of NADPH

formed/min/nmol

and

(4) (5)

(5)

(8) (8) (8)

502 0.140 0.004 0.007 0.011 0.004 1.97 8.65 7.31 43.4 0.9 0.47

Note. Values shown are means f SD (n) or individual values. Data for the xenobiotics Data for the steroids has been partially published in James and Shiverick (1984).

presence of P450 reductase which had been isolated from pig or rat liver by affinity chromatography. The abilities of the Ml fraction, the Dl fraction, and the D2 fraction to catalyze monooxygenation of several model substrates for P450 is shown in Table III. Similar activities were found in the presence of pig and rat reductase. Insufficient D3 was isolated to characterize its catalytic activity with all substrates. The highest turnover numbers were found with benzphetamine and aminopyrine, especially with the partially purified D2 fraction. Benzphetamine was used to examine the dependence of monooxygenase activity on reductase, and as expected (15), the highest activities were found when reductase and P450 were present in a 1:l molar ratio (data not shown). The lowest monooxygenase activities were observed with the phenoxazone ethers, none of which was a good substrate for the spiny lobster forms of P450, including D3 (results not shown). Considerable variability was found in the ethoxycoumarin 0-deethylase activity of Ml fractions. Neither Dl nor D2 had a higher mean ethoxycoumarin 0-deethylase activity than the Ml fraction. As shown in part previously (22), progesterone and testosterone were rapidly hydroxylated by the Ml fraction and by the Dl and D2 fractions, especially by the Dl fraction at the 16 CYposition. Progesterone 6P-hydroxylase was much higher with D2 than with Dl. The model polycyclic aromatic hydrocarbon procarcinogen benzo(a)pyrene was a good substrate for the Ml fraction and the purified Dl and D2 fractions, but there was no apparent purification of the benzo(a)pyrene hydroxylase activity, as similar activities were found with each fraction (Table III). Benzo(a)pyrene was used as a substrate

15 40 * 0.023 + 0.001 i- 0.002 +- 0.001 f 0.001 + 0.83 + 5.81 + 6.16 * 9.1 * 0.3 i- 0.02

Cytochrome

P450

Dl (8)

NADPH

D2 (4)

122 k 62 76 0.183 0.005 + 0.001 0.005 * 0.002 0.013 + 0.001 0.003 * 0.001 I .54 f 0.39 4.1 3.4 21.1 6.2 0.41

(3) (3) (4) (3) (3) (5) (3) (3) (3) (3) (3)

has been partially

presented

in a review

(4)

(3) (3) (3) (3) (4)

(James,

1989).

to determine if catalytic activity was affected by the phospholipid environment of P450 and reductase. Total activity was lowest when freshly mixed P450 and reductase were used as the enzyme source. If the P450 and reductase mixture were frozen for l-24 h before assay a IO-15% increase in activity similar to that found if P450 and reductase were added to a sonicated suspension of phosphatidylcholine (0.3% in 0.01 M buffer A) for use in assay was found. The effect of chemicals known to modify vertebrate P450-dependent monooxygenase activity was investigated, and the results for benzphetamine N-demethylase and benzo(a)pyrene hydroxylase are presented in Table IV. In the absence of reductase the only activity observed was for the Ml fraction with benzphetamine as substrate, and this was very low (Table IV). Omission of NADPH from the reaction prevented product formation. Bubbling CO through incubation vials for 2 min prior to assay inhibited benzo(a)pyrene hydroxylase activity with the Dl fraction. Ellipticine was a potent inhibitor of both benzo(a)pyrene hydroxylase and benzphetamine N-demethylase activity (Table IV). 1-Phenylimidazole did not inhibit benzo(a)pyrene hydroxylase activity but was a good inhibitor of benzphetamine N-demethylase activity (Table IV). Addition of 0.1 mM a-naphthoflavone or metyrapone to incubations either had no effect, or stimulated benzphetamine N-demethylase and benzo(a)pyrene hydroxylase activities (Table IV). Further studies were done to determine the positional metabolism of benzo(a)pyrene by spiny lobster P450 Dl and D2 fractions, and the effects of inhibitors on this

14

MARGARET

0. JAMES

TABLE

Factors Affecting

Monooxygenase

IV

Activity

of Spiny Lobster

Cytochrome

P450

Nanomoles product formed/min/nmolP450 Benzphetamine Incubation

conditions

Complete system No reductase No NADPH CO (2 min bubbling) Ellipticine, 10e5 M Ellipticine, 10-a M n-Naphthoflavone, 10m4 M l-Phenylimidazole, 10m4 M l-Phenylimidazole, 10e5 M Metyrapone, 10m4 M

Benzo(a)pyrene

Ml

Dl

Ml

Dl

26.6 0.3

43.0

0.96

2.06

1.73

ND ND

ND ND

ND ND

ND ND

ND

D2

0.63 0.08 9.0

27.2 7.6 13.6 24.4

8.7 29.0

1.35 1.23

2.13 2.31

Note. The complete appropriate substrate P450 for benzphetamine not investigated.

system contained cytochrome P450 and reductase in a 1:l molar ratio, in the presence of 0.03% phosphatidylcholine, and buffers (see text), and NADPH. ND indicates no activity was detected. Limits of detection were 0.1 nmol/min/nmol N-demethylase and 0.05 nmol/min/nmol P450 for benzo(a)pyrene hydroxylase. ~ Indicates that this condition

metabolism.

results

The

from

one experiment

are pre-

Isolation of Cytochrome Microsomes

of Benzo(a)pyrene

Addition Dl fraction None a-NF TCPO l-PI D2 fraction None n-NF l-PI

9,10-

117 212

4,5179

136

227 34

108 393

292 389

181

320 127

135 363

57 73

93 391

288 244 243

46 75 63

180 203 200

176 270 218

Note. n-NF, cu-naphthoflavone; 10m4 M. Total metabolism includes II includes 3-hydroxybenzo(a)pyrene.

TCPO, trichloropropeneoxide; all radioactivity eluting before

231

l-PI, l-phenylimidazole. benzo(a)pyrene. Phenols

P450

Phenols

6,12

370 423

Cytochromes P450

3,6-

164

481

formed/min/nmol

1,6-

179

2

product

Spiny Lobster

7,8-

247 43 207

the micro(6). This Ml

V

Diones

Dihydrodiols

Hepatopancreas

activity, was effected by partially solubilizing somes with cholate to give the Ml fraction

by Purified

Picomoles

P450 from

Although some P450 was solubilized into the cytosolic fraction during the preparation of microsomes (Fig. 1) most of the measurable P450 was associated with the microsomal fraction. A good purification of the microsomal P450, and separation from inhibitors of monooxygenase

TABLE

Metabolism

was

DISCUSSION

sented in Table V. Similar results were obtained with other Dl and D2 preparations. a-Naphthoflavone and lphenylimidazole caused increases in activity, but there was no substantial alteration in the pattern of metabolites produced. Because the P450 preparations were contaminated with small amounts of epoxide hydrolase, some dihydrodiol metabolites were observed. As expected, trichloropropene oxide markedly inhibited the formation of dihydrodiols, especially BaP-9,10-dihydrodiol, and increased phenol production.

Positional

the

63

Total metabolism

I

II

218 463 543

288

1500

589

541 400

3580 2300 2160

201 301 172

1708 2261

296 97

156 114

1980

All modifiers were added at a final concentration of I includes 7- and 9-hydroxybenzo(a)pyrene. Phenols

CYTOCHROME

P450

fraction has not been described in other species, and appears to be an aggregate of cytochrome P450 and other endoplasmic reticulum proteins (8) and lipids. The aggregate was dispersed by treatment with nonionic detergents, and anion exchange chromatography of the solubilized P450 gave three reproducible P450 peaks, one major (Dl) and two minor (D2 and D3). The molecular weight of the major (Dl) form was 52,500 by SDSPAGE (Fig. 3) and 55,000 by amino acid analysis (Table II). Similar discrepancies in molecular weight by these two methods have been reported previously for cytochromes P450 (35). The amino acid composition showed that glutamine and glutamic acid residues were very low in the spiny lobster P450 Dl compared with P450 from rat, rabbit, or mouse liver (35), while threonine and glytine were present in spiny lobster P450 Dl in higher concentrations than have been found for rat, rabbit, or mouse liver forms (35). Further studies are needed to provide more structural information for the spiny lobster P450. Lack of Recovery of NADPH Cytochrome P450 or Cytochrome c Reductase from Hepatopancreas NADPH cytochrome c reductase activity was present in hepatopancreas microsomes and cytosol at similar, low levels (Fig. 1). The low activity was not due to the presence of phosphate buffer, which has been shown to inhibit cytochrome c reductase activity of mussel digestive gland microsomes (36), as we measured activity in the presence of Hepes buffer. No cytochrome P450 reductase activity was detected in hepatopancreas microsomal fractions. When CO-gassed microsomes were reduced with NADPH no peak was observed at 450 nm, but a peak of unknown origin was observed at 420 nm (Fig. 2). Efforts to recover the cytochrome c reductase activity from DEAE-cellulose chromatography of the solubilized Ml fraction were unsuccessful. Small amounts of NADPH cytochrome c reductase activity were recovered in eluate from DEAE-cellulose chromatography of desalted cytosol, but the amount was too low to study further. Evidence that indicated that proteolysis of NADPH-cytochrome P450 reductase could occur in subcellular fractions of whole hepatopancreas was obtained. Figure 6 shows clearly that a band at 66,000 Da, which was not present in the original pig liver reductase or in hepatopancreas cytosol, was formed when cytosol and reductase were incubated for 15 min at 4°C prior to treatment with SDS and gel electrophoresis. The difference in molecular weight between the pig liver reductase and the proteolytic product was about 10,000, and the cleaved peptide could include the hydrophobic portion of NADPH cytochrome P450 reductase that is required for proper interaction of reductase with P450 and the microsomal membrane (33, 34). Although we

IN

SPINY

LOBSTER

15

have no direct evidence that NADPH cytochrome P450 reductase is present in spiny lobster hepatopancreas, we have found small amounts of cytochrome c reductase activity, which may have derived from proteolysis of cytochrome P450 reductase during preparation of microsomes. It is likely that spiny lobster hepatopancreas does contain cytochrome P450 reductase, since in vivo studies have shown that spiny lobster can monooxygenate benzo(a)pyrene to phenolic and dihydrodiol metabolites (2, 37). Even so, it appears that spiny lobster hepatopancress contains a much lower reductase:P450 ratio than is found in most vertebrate species, including fish (38). Further experiments, in which different hepatopancreas cell types with specialized functions (39) are isolated prior to preparation of microsomes, may allow us to isolate and characterize the lobster P450 reductase. Catalytic Activity of the Spiny Lobster Hepatopancreas P450 It was not possible to reconstitute the spiny lobster monooxygenase system, since the reductase component was not purified. The catalytic profile of two of the forms of P450 present in hepatopancreas of untreated spiny lobsters was determined by combining the spiny lobster P450 with reductase purified from pig or rat liver. As expected for purified cytochrome P450 proteins, the addition of both P450 reductase and NADPH were absolute requirements for catalytic activity (Table IV). The Dl and D2 forms of P450 were able to catalyze monooxygenation of several substrates, including the xenobiotics benzo(a)pyrene, ethoxycoumarin, benzphetamine, and aminopyrine and the steroids progesterone and testosterone (Table III). Although the molecular weights and spectral characteristics of the two forms were similar, there were important differences in catalytic activity between Dl and D2 which, together with the differences in retention on DEAE-cellulose columns, indicate that these are two separate forms of P450. The D2 form had two-fold higher monooxygenase activity than the Dl form with benzphetamine and aminopyrine as substrates, and sevenfold higher progesterone 6P-hydroxylase activity (Table III). Rates of hydroxylation of both testosterone and progesterone at the 16 (Y position were twofold higher for the Dl form compared with the D2 form (Table III). None of the isolated P450 fractions (Dl, D2, and D3) had good activity with any of the phenoxazone ethers that were tested. These substrates have been widely used in rats to monitor induction by phenobarbital or methylcholanthrene-type inducing agents, as they are preferentially metabolized by specific isozymes of P450 (40). There is as yet no evidence that the spiny lobster monooxygenase system can be induced by treatment with phenobarbital or methylcholanthrene (4,41). Traditional inhibitors of the different mammalian forms of P450 had variable effects on catalytic activity

16

MARGARET

(Table IV and Ref. (6)). Neither metyrapone nor (Ynaphthoflavone inhibited monooxygenation of benzo(a)pyrene, benzphetamine, or 7-ethoxycoumarin ((6); Table IV), suggesting that the active site environments of Dl and D2 are not similar to those of the phenobarbital-induced or 3-methylcholanthrene-induced forms of mammalian P45Os. The effects of 1-phenylimidazole on monooxygenase activity were substrate-dependent. Benzo(a)pyrene hydroxylase was unaffected by l-phenylimidazole, whereas benzphetamine and 7-ethoxycoumarin were inhibited by concentrations in the micromolar range ((6); Table IV). Detailed studies of the mechanism of inhibition have not been carried out. The pattern of metabolism of benzo(a)pyrene by the spiny lobster P450 preparations was similar for both forms, and indicated no ring preference for oxygen incorporation (Table V). The metabolites formed were more similar to those produced by microsomes from uninduced rat than those produced by microsomes from several fish species, including the carcinogen-sensitive flatfish species which form primarily benzo ring phenol and dihydrodiol metabolites of benzo(a)pyrene (42,43). The position of attack of benzo(a)pyrene may have important implications in determining the carcinogenicity of this environmental contaminant to a given species. Those animals which preferentially form benzo(a)pyrene-7,8-dihydrodiols, which can be further metabolized by cytochrome P450 to benzo(a)pyrene-7,8-dihydrodiol9,10-oxides, ultimate carcinogenic metabolites of benzo(a)pyrene, are more likely to suffer DNA damage by adduct formation than those species which form less of the ultimate carcinogenic metabolites. It is of interest that crustacean species are generally resistant to chemical carcinogens of the polycyclic aromatic hydrocarbon type (44). The formation of dihydrodiol metabolites of benzo(a)pyrene by spiny lobster P450 fractions and the inhibition by TCPO gave further evidence that epoxide hydrolase was present even in the most purified preparations. The SDS gels of the purified Dl preparations showed no evidence of any bands at 49,000 Da, the molecular weight of rat microsomal epoxide hydrolase (45) although a prominent band at this molecular weight (see Fig. 3) was present in the Ml fraction and D2 preparations, which had high styrene oxide hydrolase activity. Previous studies have shown that the spiny lobster microsomal epoxide hydrolase has good activity with benzo(a)pyrene-4,5-oxide even at low substrate concentrations ((32) and unpublished observations): if this extends to other benzo(a)pyrene oxides, then this could explain the high percentage of dihydrodiols formed even though very little epoxide hydrolase activity was found in some Dl preparations. In summary, this study has shown that at least three forms of P450, one major and two minor, are present in hepatopancreas microsomes from untreated spiny lob-

0. JAMES

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