ARCHIVES
OF BIOCHEMISTRY
AND
Vol. 298, No. 2, November
BIOPHYSICS
1, pp. 395-402,1992
Comparative Study of Monomeric Reconstituted and Membrane Microsomal Monooxygenase Systems of the Rabbit Liver I. Properties of NADPH-Cytochrome P450 LM, (2B4) Monomers
P450 Reductase
and Cytochrome
Irina P. Kanaeva, Ilya R. Dedinskii, Elena D. Skotselyas, Arkadii G. Krainev, Irina Irina F. Sevryukova, Yakov M. Koen, Galina P. Kuznetsova, Galina I. Bachmanova,’ and Alexander I. Archakov Institute
Received
of
Biological
December
and Medical
6,1991,
Chemistry,
and in revised
form
Russian Academy of Medical
May
Academic
Press,
Inc.
0003~9861/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Russia
8, 1992
Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LMz (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LMz oligomers were 14.8 f 1.7 and 19.2 + 1.4 nm, respectively. Twentyfour-hour incubation with Emulgen 913 at 4°C at a molar ratio of 1:lOO led to the monomerization of NADPHcytochrome P450 reductase and cytochrome P450 LMz oligomers, the particle sizes diminishing to 6.15 1.3 and 5.2 f 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LMz monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LMz oligomers and the microsomal hemoprotein, cytochrome P450 LM, monomers formed complexes with type I and II substrates, but with & values higher than those of microsomes and cytochrome P450 LMz oligomers. Kinetic parameters (V,,,, and K,) of HzOz- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LMz oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LMz oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein. 0 1992
Sciences, Moscow,
V. Guleva,
Cytochrome P450-containing membrane monooxygenase systems can be reconstituted in solution in the presence of phospholipids (l-8), as well as ionic, zwitterionic, and nonionic detergents (9-13). The effect of phospholipids and detergents on the activity of such systems is associated with their deaggregating action on the oligomers of isolated proteins. Maximum rates of NADPHdependent cytochrome P450 reduction and substrate oxidation were observed in reconstituted systems at the equimolar reductase-to-cytochrome P450 ratio. However, Dean and Gray (9) and Wagner et al. (12), using the non-
ionic detergent octylglucoside and the zwitterionic detergent 3-([3-cholamidopropyl]dimethylammonio)-l-propanesulfonate ( CHAPS),2 demonstrated that a reconstituted system containing purified rabbit liver microsomal NADPH-cytochrome P450 reductase and cytochrome P450 LM2 exhibited maximum activity at a reductase-to-cytochrome P450 ratio of 1:5 without the formation of stable complexes between the proteins. A considerable body of evidence indicates that the two pro-
teins interact
in reconstituted
systems by way of random
collisions (7, 14-17). Muller-Enoch et al. (18) showed that it is possible to reconstitute monooxygenase activities in a system containing high concentrations of the reductase 1 To whom correspondence should be addressed at the Institute of Biological and Medical Chemistry Russian AMS, Pogodinskaya St., 10, Moscow 119832, Russia. ’ Abbreviations used: CHAPS, 3-[(3-cholamidopropylhlimethylammonio]-l-propanesulfonate; reductase, NADPH-cytochrome P450 rem ductase isolated from phenobarbital-induced rabbit liver microsomes; LM2, cytochrome P450 LM2 (2B4) isolated from phenobarbital-induced rabbit liver microsomes; CHP, cumene hydroperoxide; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 395
Inc. reserved.
396
KANAEVA TABLE
ET
AL.
I
Determination of Particle Sizes of Reductase and LM2 in the Absence and Presence of Emulgen 913 System 5 pM 5 @M 5 /tM 5 pM
reductase reductase + 0.25g/liter Emulgen913 LMz
LMp + 0.25g/liter Emulgen913
Diameter b-4 14.8 f 1.7 6.1 + 1.3 19.2 f 1.4
5.2 f 0.4
Note. Proteinmonomerization in the presence of detergentanddeterminationof particlesizeswerecarriedout asdescribedunderExperimentalProcedures. Meansof four or five measurements arepresented+ SE.
401 0
3
9
TIME (8MlN) of oligomers (solid line) and monomers (dashed 1. Inactivation of oxidized LMz in the absence (0, A) and presence (0, A) of 1 mM benzphetamine at 30°C (0, 0) and 37’C (A, A). The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5, and 0.5 @M cytochrome P450. In the case of monomers the buffer contained 0.25 g/ liter Emulgen 913. The content of LMz was determined in 2-min intervals from the difference spectrum of its dithionite-reduced CO complex, with 100% corresponding to q&chrome P450 content at zero incubation time. FIG. line)
and cytochrome P450 LMz at a ratio of 2:l without phospholipids and detergents after prolonged incubation. Dean and Gray (9) and Wagner et al. (12), using high concentrations of octylglucoside and CHAPS, obtained the monomers of NADPH-cytochrome P450 reductase and cytochrome P450 which were unable to catalyze cyclohexane oxidation, but did catalyze CHP-dependent toluene hydroxylation at a high rate. The lack of hydroxylase activity at high concentrations of the detergent was attributed by the authors to its inhibitory effect on cytochrome-reductase interaction. Earlier in our laboratory a monooxygenase system containing NADPH-cytochrome P450 reductase (reductase) and cytochrome P450 LMz (LM,) was reconstituted in the presence of the nonionic detergent Emulgen 913. Prolonged incubation of the reductase and LMz with Emulgen 913 at a protein-to-detergent ratio of 1:lOO led to the dissociation of protein oligomers to monomers. No formation of stable binary complexes upon mixing reductase and LM2 monomers was observed. Such a monomeric reconstituted monooxygenase system catalyzed NADPH-dependent LMz reduction and benzphetamine demethylation (19-24). The present study deals with the influence of the aggregation state of LM2 and the reductase on their thermal stability as well as on LMz substrate-binding ability and peroxidase activity. It was demonstrated that the oligomers and monomers of isolated LM2 were less thermostable than the hemoprotein of the microsomal membrane. Monomerization decreased the thermal stability of LMz, but not of the reductase. LMz oligomers and monomers possessed high peroxidase activities in the presence of hydrogen peroxide and CHP. The replacing of protein-protein interactions in LM2 oligomers by protein-detergent interactions in LM2 monomers affected values for the cosubstrates and substrates only without influence on values. K,,,
V,,
EXPERIMENTAL PROCEDURES Chemicals. EDTA and cytochrome c were purchased (Germany). Sodium dithionite was obtained from Merck
from Serva (Germany),
cumene hydroperoxide from Fluka (Switzerland), and NADPH from Beanal (Hungary). Catalase from beef liver was a product of BoehringerMannheim (Germany). EmuIgen 913 was from Kao-Atlas (Japan). Other chemicals were purchased from Reakhim (Russia). Phenobarbital-induced rabbit liver microsomes, LM, and reductase preparation. The microsomal fraction was isolated from the liver of male New Zealand rabbits treated with 0.1% (w/v) sodium phenobarbital in drinking water for 1 week (25). The reductase (specific activity 4013-13.5 43 pmol cytochrome c min-’ mg-’ at 3O”C, specific content
\ ‘0.
0
-.
..
9
3
TIME ;MIN) FIG. 2. Inactivation of oligomers (solid line) and monomers (dashed line) of reduced LMz in the absence (0, A, and presence (0, A, +) of 1 mM benzphetamine at 25°C +), 30°C (0, l ), and 37°C (A, A). The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5, and 0.5 pM cytochrome P450. In the case of monomers the buffer contained 0.25 g/liter Emulgen 913. The difference spectra of cytochrome P450 were recorded in P-min intervals after CO bubbling and sodium dithionite addition, with 100% corresponding to cytochrome P450 content at zero incubation time. 0)
(0,
RECONSTITUTION
OF
MONOMERIC
MICROSOMAL
MONOOXYGENASE
SYSTEM,
I
397
0.01
A
0
/ 8 2
\
-0.01
5 P 4 ---
xl.02 -
MICROSOMRS OLIGOMERS
---
400
450 WAVELENGTH
OLIGOHERS ..
500
550
.
).JONOmRS
I
-0.03 350
MICROSOMES
350
400
450 WAVELENGTH
(rim)
500
550
(nm)
0.01
C
I \
!
-
j,
MICROSOMES ---
1,’
...‘....’
OLIGOMERS MONOMERS
-0.02 350
400
450 WAVELENGTH
500
550
(nm)
FIG. 3. Binding spectra of 2 mM benzphetamine (A), 5 mM dimethylaniline (B), and 20 mM aniline (C) with cytochrome P450 from microsomal fraction, LM2 oligomers, and LM1 monomers. The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5, and 1 pM LM2 in the oligomeric or monomeric state or the same concentration of cytochrome P450 from rabbit liver phenobarbital-induced microsomes. In the case of LMp monomers the buffer contained 0.25 g/liter Emulgen 913. T = 3O’C.
nmol reductase mg protein-‘) and LM2 (specific were purified as described we’, &W/&T = 0.5-l) proteins showed a single band on SDS-PAGE.
content 17-18 earlier (25,26).
nmol Both
Monomerization of the proteins. Monomerization of the oligomers of isolated reductase and LMP was carried out as follows: to 5 nmol(2030 al) of either protein 13 ~1 of 2% (w/v) Emulgen 913 solution was added. After incubation for 5-10 min at room temperature, the concentrations of the proteins and Emulgen 913 were brought to 5 PM and 0.25 g/liter, respectively, with 100 mM K-phosphate buffer, pH 7.5, and the mixtures were incubated at 4°C for another 24 h. The determination of the amount of Emulgen 913 tightly bound by the protein monomers was carried out after gel chromatographic removal of free detergent
by the modified method of Horigone and Sugano (27) applied to Emulgen 913. Determination of the sizes of protein particles. Translation diffusion constants (D) of the reductase and LM, in the absence and presence of 0.25 g/liter Emulgen 913 in 100 mM K-phosphate buffer were determined by laser correlation spectroscopy using a Coulter N4 instrument (Coultronix, France) operated at 21°C. The reductase and LM, concentrations were 5 pM; the sample volume was 1 ml. The Coulter N4 uses a heliumneon laser (X = 632.8 nm) as a light source. Instrument settings were a scattering angle of 90”, an acquisition time of 200 s, particle size ranges of l-1000 and l-10,000 nm. Correlation time was varied from 10 to 50 p.s. Steady solutions of the reverse problem in the range l-300 nm were
398
KANAEVA
ET
TABLE Parameters
of Cytochrome
P450
Difference
Microsomes LM2 oligomers LMP monomers
&in (nm)
Lx bm)
421 423
385 383 385
421
Note. The incubation mixture contained 0.25 g/liter Emulgen
wnax (X103) 43 f 41 t 41 f
0.5 0.4 0.3
II
Binding
Benzphetamine
System
AL.
Spectra
with
Type
I and
II Substrates
Dimethylaniline &in Kd
0.03 0.3 0.6
bM)
f
0.003 + 0.05
+ 0.1
(nm)
425 418 423
Lx
Aniline
uwu (X103)
bm)
389 379 383
&
28 f 0.3 21 f 0.1 15 2 0.2
6-d
Substrate binding to cytochrome P450. The binding spectra of cytochrome P450 with type I and II substrates were recorded by the differential scheme using a Hewlett-Packard 8451A diode array spectrophotometer. The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5, 1 fiM LM2 in the oligomeric or monomeric state or the same concentration of cytochrome P450 in phenobarbital-induced rabbit liver microsomes and various concentrations of benxphetamine, dimethylaniline, or aniline. Spectral binding constants (&) and maximum absorbance change (AA,) were calculated by the Eadie-Hofstee equation using a computer program based on the linear regression procedure.
Hemoprotein peroridase activity measurements. The rates of HzOzand CHP-dependent benzphetamine N-demethylation and aniline phydroxylation were estimated by measuring the amounts of formaldehyde and p-aminophenol formed, respectively. The incubation mixture (0.5 ml) contained 100 mM K-phosphate buffer, pH 7.5, and various concentrations of benzphetamine or aniline. The concentration of LM2 or microsomal cytochrome P450 was 1.0 pM. The reactions were started by the addition of various amounts of HzOz or CHP and carried out at 30°C for 1 min. In the case of H,O,-supported reactions, unreacted HzOz was removed by addition of 16,000-48,000 U/ml catalase to the incubation mixture. The reactions were stopped by the addition of trichloroacetic acid to a final concentration of 7.5%. For formaldehyde determination 0.5 ml of Nash reagent consisting of 4 M ammonium acetate, 0.1 M glacial acetic acid, and 0.04 M acetylacetone was added to 0.5 ml of the supernatant and incubated at 37°C for 30 min. The extinction coefficient of the colored product at 412 nm was 4 mM-’ cm-‘. For paminophenol determination, 0.25 ml of 20% Na,CO, and 0.75 ml of 2% phenol were added to 0.5 ml of the supernatant and incubated at 37’C for 30 min. The extinction coefficient of the colored product at 630 nm was 10 mM-’ cm-i. K,,, and V,, values for the substrates and cosubstrates were calculated by the nonlinear regression technique using a modified KINFIT program (28). Other assays. The concentration of cytochrome P450 was determined by the method of Omura and Sato (29) in a Hewlett-Packard 8451A spectrophotometer (USA) from the CO-difference spectrum of dithionitereduced microsomes or LMp based on an extinction coefficient of 91 mM-’ cm-i. The concentration of the purified reductase was determined from its absolute absorption spectrum based on an extinction coefficient of 21.4 mM-’ cm-’ (30). NADPH-cytochrome c reductase activity was determined in an Ultraspec spectrophotometer (Sweden) from the cytochrome c reduction rate at 3O”C, based on the extinction coefficient of reduced cytochrome c, 21.1 mM-’ cm-’ for 550 nm (31). Inactivation of LM, oligomers and monomers was evaluated by recording difference absorption spectra of the reduced CO-LM2 complex. Heat resistance of
Lax (nm)
unax (XlOs)
439 441 450
34 + 0.4 25 + 0.3 16 I!Z 0.4
0.05 0.6
+ 0.01 + 0.03
399 407
0.9
+ 0.1
413
contained 100 mM K-phosphate buffer, pH 7.5, and 1 pM cytochrome 913. 7’ = 30°C. Means of five or six measurements are presented *SE.
taken into consideration. Data presented are means from four or five measurements f SE. The Coulter N4 software uses the Stokes-Einstein equation for spherical particles to derive particle diameter as an estimation of particle size.
Lain (nm)
P450.
&
0.8 f 0.1 1.3 k 0.1 3.8 f 0.3
In the case of monomers
the reductase oligomers and monomers NADPH-cytochrome c reductase activity.
was estimated
bM)
the buffer
by measuring
RESULTS AND DISCUSSION Particle Sizes of the Purified Reductase and LA!, It was shown earlier in this laboratory that the incubation of purified 5 PM reductase and 5 PM LM2 with 0.25 g/liter Emulgen 913 in 100 mM K-phosphate buffer led to the dissociation of protein oligomers. The molecular weights of the reductase and LM2 monomers estimated by gel filtration were 135 and 65 kDa, respectively (19, 20,22). Such great values may be due to the large amounts of detergent tightly associated with the proteins. Measurement of amount of detergent tightly bound (27) to the proteins showed that lo-20 and 50-60 detergent molecules are associated with 1 molecule of LMP and reductase, respectively (19, 20, 22). Taking into account the molecular weight of Emulgen 913, which is equal 792 Da,
1.25
I
I,
I.,
,
I. l
.
1.05
f
.
0.85 l
.5 -0 y
:/~
0.65
0.45
Ii Ii
0.25
" 0.00
" 0.01
a 0.02
IEmulgenl,
1 0.03
" 0.04
0.05
%
FIG. 4. Dependence of spectral binding constants Kd for benxphetamine on Emulgen 913 concentration. The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5,1 pM LM,, and various concentrations of the detergent. T = 3OOC. The spectral binding constants of LMP with benzphetamine were determined at the detergent concentrations indicated under Experimental Procedures.
RECONSTITUTION
OF
MONOMERIC
MICROSOMAL TABLE
Kinetic Parameters of H,O,-Dependent
MONOOXYGENASE
For HzOz
III
Microsomes LM2 oligomers LMx monomers
110 + 13 420 f 22 340 ?I 61
VJK,,,
For
399
I
Benzphetamine (BP) N-Demethylation
Km (mM) System
SYSTEM,
BP
V,..
0.23 f 0.04 0.54 + 0.08 0.61 + 0.08
(mM
min-‘)
0.16 + 0.03 0.10 f 0.02 0.090 f 0.001
(X
10x mini)
For H202
For BP
0.15 0.02 0.03
70 19 15
Note. The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5, and 1 PM cytochrome P450. In the case of monomers the buffer contained 0.25 g/liter Emulgen 913. The reaction was initiated by the addition of various concentrations of H202. When evaluating Km for H202 and BP the concentrations of BP and HzOz were 2 mM and 1 M, respectively. The incubation was carried out for 1 min at 30°C. Means of three or four measurements are presented *SE.
the aforementioned molecular weights of the protein monomers seem reasonable. Thus, Emulgen 913 induced monomer formation under experimental conditions. To prove that monomerization actually takes place under the above conditions the sizes of the reductase and LMB particles were measured in the absence and presence of Emulgen 913 using laser correlation spectroscopy. From Table I it is seen that incubation of oligomers with 0.25 g/liter Emulgen 913 leads to the dissociation of the oligomers, with particle sizes decreasing to 6.1 + 1.3 and 5.2 + 0.4 nm, respectively, which agrees well with the values of 5.6 and 4.7 nm we estimated from earlier reported molecular weights for the monomeric reductase and LM2. Apparent monomeric values derived from SDS-PAGE analysis for NADPH-cytochrome P450 reductase and LM, purified from rabbit liver microsomes were reported by Muller-Enoch et al. (18) and corresponded to 74,000 and 49,000 respectively. These values were used for estimation of the diameters of the proteins, assuming a globular shape of these molecules and a density of proteins of 1.35 X lo3 kg rnp3 (32). Thus, the results obtained by two independent methods provide convincing evidence that under these conditions the dissociation reveals protein particles corresponding to monomeric ones.
TABLE Kinetic
Parmeters
Thermal Stability of the Reductase and LM, Oligomers and Monomers To study how the change from protein-protein interactions in oligomers to protein-detergent interactions in monomers influences the protein thermal stability, LMz content, and cytochrome c reductase activity of the reductase were estimated before and after the incubation of oligomers and monomers at 25, 30, and 37°C for 10 min. The oligomers and monomers of ferric LM2 were not inactivated at 25°C. At 3O”C, insignificant inactivation (by as much as 10%) of only LMz monomers was observed (Fig. 1). At 37”C, LM, oligomers were inactivated by 10% and monomers by 40% (Fig. 1). The addition of benzphetamine to ferric LM2 oligomers and monomers had no effect on their thermal stability (Fig. 1). The inactivation of dithionite-reduced LMz oligomers occurred at 37°C only (by 40%) (Fig. 2). The reduced form of LM2 monomers was inactivated by 30% at 25°C and by about 45-50% at 30 and 37°C (Fig. 2). In the case of ferrous LM2, inactivation was slowed down by benzphetamine so that oligomeric LM2 was inactivated at 37°C by only 10% (Fig. 2). With monomers, the greatest protective effect was observed at 25 and 30°C. Unlike LM2, the reductase showed considerably higher thermal sta-
IV
of CHP-Dependent Benzphetamine (BP) N-Demethylation KmbM)
V,,/K,
System
For CHP
For BP
Microsomes LMx oligomers LM2 monomers
0.12 f 0.01 0.11 + 0.03 0.28 + 0.04
0.50 + 0.08 1.8 -c 0.2 0.90 f 0.15
V,,,
(mM
mini)
0.013 + 0.004 0.010 + 0.001 0.010 f 0.002
(min-*)
For CHP
For BP
0.11 0.09 0.04
0.026 0.006 0.011
Note. The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5, and 1 PM cytochrome P450. In the case of monomers the buffer contained 0.25 g/liter Emulgen 913. The reaction was initiated by addition of various concentrations of CHP. When evaluating K,,, for CHP and BP the concentrations of BP and CHP, respectively, were 2 mM. The incubation was carried out for 1 min at 30°C. Means of three or four measurements are presented +SE.
400
KANAEVA TABLE Kinetic
Parameters
of HeOe-Dependent
ET
AL.
V Aniline
(AN)
p-Hydroxylation V&K,,,
Km (mM) System
For H202
For AN
Microsomes LM2 oligomers LM2 monomers
66k a 80+ 10 800 + 105
0.32 + 0.06 0.40 f 0.05 3.4 f 0.6
V,,
(mM
0.038 0.024 0.020
mini)
+ 0.005 f 0.003 f 0.004
(X
10’
mini)
For HzOz
For AN
0.06 0.03 0.003
12 5.6 0.6
Note. The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5, and 1 pM cytochrome P-450. In the case of monomers the buffer contained 0.25 g/liter Emulgen 913. The reaction was initiated by the addition of various concentrations of HzOz. When evaluating Km for HzO, and AN, the concentrations of H202 and AN were 1 M and 20 mM, respectively. The incubation was carried out for 1 min at 30°C. Means of three or four measurements are presented -+SE.
bility, and the NADPH-cytochrome c reductase activity of reductase oligomers and monomers did not change after incubation for 10 min at 25, 30, and 37°C (data not shown). Therefore, in all cases LM2 oligomers were more thermostable than monomers. In other words, the substitution of protein-protein interactions in oligomers with proteindetergent interactions in monomers reduces hemoprotein stability. In rabbit liver phenobarbital-induced microsomes the oxidized and reduced forms of cytochrome P450 were not inactivated at the above temperatures (33). Substrate Binding to LM, Oligomers, LM, Monomers, and Cytochrome P450 of Phenobarbital-Induced Rabbit Liver Microsomes The binding spectra of microsomal cytochrome P450 and isolated oligomeric and monomeric LM2 with benzphetamine, dimethylaniline, and aniline are presented in Fig. 3 and their parameters in Table II. It is seen that similar to microsomal hemoprotein, LM2 oligomers and monomers are capable of binding both type I and II substrates. With benzphetamine and dimethylaniline the positions of the absorption maxima (379-385 nm) and minima (418-427 nm) of the microsomal hemoprotein, oligomers, and monomers of LM2 were typical of type I substrates. The binding of the type II substrate aniline to monomers produced a maximum at 450 nm, whereas with oligomers and microsomes the maxima were at 441 and 439 nm, respectively. The magnitudes of maximum absorbance changes of LMz-benzphetamine complexes were the same for LM2 oligomers, LM2 monomers, and the hemoprotein of the microsomal fraction. With dimethylaniline and aniline these values for microsomal cytochrome P450 were 1.32 times those for LM2 oligomers and monomers. As seen from Table II, spectral binding constants (&) decreased in the order monomers > oligomers > microsomes. With monomers, Kd values for benzphetamine and dimethylaniline were 1.5-2 times those with oligomers and an order of magnitude greater than those with microsomes. With
aniline, the Kd values for monomers was 3 times that for oligomers and 5 times that for microsomal cytochrome P450. The different spectral binding constants but similar maximum absorbance changes may be due to the presence of the detergent, either in the tightly protein-bound form in the case of LM2 oligomers or in solution (0.25 g/liter Emulgen 913) of LM2 monomers. This assumption was confirmed by experiments on the influence of various concentrations of Emulgen 913 on the Kd for benzphetamine. The Kd increased with detergent concentration (Fig. 4), indicating the possibility of competition between the detergent and the substrate for binding sites on the hemoprotein. Peroxidase Activities of LM2 Oligomers, LM2 Monomers, and Cytochrome of Phenobarbital-Induced Rabbit Liver Microsomes To elucidate the influence of protein-protein and protein-detergent interactions on the catalytic activity of LM2 the oxidation rates of type I and II substrates were measured in the presence of the active oxygen donors HzOz and CHP which differ substantially in hydrophobicity. These studies were of particular interest since some authors believe that the quarternary structure of LM2 plays an important role in its catalytic activity (34). As seen from Table III, in the case of benzphetamine (type I substrate), K,,, values for the substrate and for hydrogen peroxide, as well as V,,, values, were close to those for the oligomers and monomers. CHP-dependent benzphetamine oxidation (Table IV) was characterized by the same V,, values for oligomers and monomers but different K,,, values for the substrate and cosubstrate. With monomers, the K, for CHP was 2.5 times higher and the K,,, for benzphetamine 2 times lower than that for oligomers. As a consequence, the V,,,/K, ratio for CHP was greater in the case of oligomers and that for benzphetamine was greater in the case of monomers. Thus, the substitution of protein-protein with proteindetergent interactions has no effect on the ability of LM2 to catalyze type I substrate oxidation as evidenced by vir-
RECONSTITUTION
OF
MONOMERIC
MICROSOMAL TABLE
Kinetic
Parameters
of CHP-Dependent
MONOOXYGENASE
SYSTEM,
VI
Aniline
(AN) p-Hydroxylation
Km bM) System Microsomes LM2 oligomers LMP monomers
401
I
(min-‘)
V,,/K,
For CHP
For AN
0.15 f 0.02 0.28 f 0.04 1.2 + 0.3
1.6 f 0.2 4.4 +0.6 6.8 f 0.8
V,,
(mM
0.021 0.016 0.015
min-‘)
+ 0.003 f 0.002 f 0.003
For CHP
For AN
0.14 0.06 0.013
0.013 0.004 0.002
Note. The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5,l pM, hemoprotein. In the case of monomers the buffer contained 0.25 g/liter Emulgen 913. The reaction was initiated by the addition of various concentrations of CHP. When evaluating K, for CHP and AN, the concentrations of CHP and AN were 2 and 20 mM, respectively. The incubation was carried out for 1 min at 30°C. Means of three or four measurements are presented +SE.
tually identical V,,, values in both systems. It may be concluded therefore that the quarternary (oligomeric) structure of LM2 does not affect its peroxidase activity. On the other hand the activity of the hemoprotein in the microsomal membrane was 1.5 times higher than in solution, which confirms our previous conclusion that the “liquid” membrane fixes LM2 in the active state (17, 35, 36). With the type II substrate aniline no differences were found between LM2 oligomers and monomers with respect to V,,, (Tables V and VI). However, the K,,, values for HzOz, CHP, and aniline did differ. In the H202-dependent system, the K,,, values for LM2 monomers were one order of magnitude higher than those for oligomers, which resulted in different V,,,/K, ratios. With the CHP-dependent system the K,,, values for the substrate and cosubstrate were also higher in the case of monomers. The differences can be due to competition for the binding site(s) in the active center of LM2 monomers between the substrate or cofactor, on the one hand, and the nonionic detergent Emulgen 913, on the other hand. The HzOzand CHP-dependent aniline hydroxylase activities of cytochrome P450 in microsomes were higher than in solution. It may be concluded therefore that the peroxidase activity of LM2 is independent of its aggregation state. LM2 monomers, like the oligomers and microsomes, can utilize hydrogen peroxide and CHP as active oxygen donors. The substitution of protein-protein interactions in LM2 oligomers by protein-detergent interactions in monomers affects only the Km values for the cosubstrates and for type II substrate aniline, but not for benzphetamine. The microsomal hemoprotein has higher thermal stability and catalytic activity as compared to LM2 oligomers and monomers, which can be accounted for by the stabilizing action of the membrane on the hemoprotein and the ability of the phospholipid bilayer to maintain the enzyme in a conformational state close to the transitional one (17, 35, 36).
ACKNOWLEDGMENT The authors of the English
thank Dr. A. A. Zhukov for useful version of the manuscript.
discussion
and revision
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K. P., and Coon,
M. J. (1979)
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3. Sato, R., Imai, Y., and Taniguchi, H. (1979) in Induction of Drug Metabolism (Estabrook, R. W., and Lindenlaub, E., Eds.), pp. 213224, Schattauer Verlag, Stuttgart/New York. 4. Vatsis, K. P., Oprian, D. D., and Coon, M. J. (1979) Acta Biol. Med. Germ.38,456-479. 5. French, J. S., Guengerich, F. P., and Coon, M. J. (1980) J. Biol. Chem. 265,4112-4119. 6. Lu, A. Y., Miwa, G. T., and West, S. B. (1980) in Microsomes and Drug Oxidations, Chemical Carcinogenesis (Coon, M. J., Conney, A. H., Estabrook, R. W., Gelboin, H. V., Gillette, J. R., and Brien, P. J., Eds.), pp. 59-66, Academic Press, New York. 7. Kitada, M., Sakamoto, R., Rikihisa, Biochem. Pharmacol. 33,3971-3976.
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8. Tamburini, P. P., Jansson, J., Favreau, (1986) Biochem. Biophys. Res. Commun. 9. Dean, W. L., and Gray, 14685.
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10. Martsev, S. P., Chashchin, V. L., and Akhrem, A. A. (1983) Vestnik AN BSSR 1,77-84. 11. Kominami, S., Hara, H., Ogishima, T., and Takemori, S. (1984) J. Biol. Chem. 259,2991-2999. 12. Wagner, S. L., Dean, 259,2390-2395. 13. Kaminsky, Biochemistry
W. L., and Gray,
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14. Taniguchi, H., Imai, Y., Iyanagi, Biophys. Acta 530,341-356.
R. D. (1984)
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F. P., and Lee, J. J. (1987)
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16. Taniguchi, chemistry & Francis,
H., and Pyerin, and Biophysics London.
17. Archakov, and Active
A. I., and Bachmanova, G. I. (1990) Oxygen, Taylor & Francis, London.
R. (1979) Chem.
Biochim.
257,
4783-
W. (1989) in Cytochrome P-450: Bio(Schuster, I., Ed.), pp. 312-315, Taylor Cytochrome
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18. Muller-Enoch, D., Churchill, P., Fleischer, S., and Guengerich, F. P. (1984) J. Biol. Ckem. 259,8174-8182. 19. Bachmanova, G. I., Skotselyas, E. D., Kanaeva, I. P., Kuznetsova, G. P., Gordeev, S. A., Korneva, E. N., Karyakin, A. V., and Archakov, A. I. (1986) Biochem. Biophys. Res. Commun. 139, 883-888. 20. Bachmanova, G. I., Skotselyas, E. D., Kanaeva, I. P., Petrachenko, E. V., Davydov, D. R., Gordeev, S. A., Karyakin, A. V., Kuznetsova, G. P., Korneva, E. N., and Archakov, A. I. (1987) in Drug Metabolism-From Molecules to Man (Benford, D. J., Bridges, J. W., and Gibson, G. G., Eds.), pp. 419-422, Taylor & Francis, London. 21. Kanaeva, I. P., Skotselyas, E. D., Turkina, I. F., Petrachenko, E. V., Davydov, D. R., Kondrashin, S. K., Dzhuzenova, Ch. S., Bachmanova, G. I., and Archakov, A. I. (1987) Biochem. Biopkys. Res. Commun. 147,1295-1299. 22. Skotselyas, E. D., Kanaeva, I. P., Dzhuzenova, Ch. S., Gordeev, S. A., Karyakin, A. V., Bachmanova, G. I., and Archakov, A. I. (1987) Dokl. AN USSR 293,748751. 23. Bachmanova, G. I., Kanaeva, I. P., and Skotselyas, E. D. (1988) Vectnik AMN USSR 1, 43-52. 24. Bachmanova, G. I., Kanaeva, I. P., Kondrashin, S. K., Skotselyas, E. D., Kuznetsova, G. P., and Archakov, A. I. (1989) in Cytochrome P-450: Biochemistry and Biophysics (Schuster, I., Ed.) pp. 300-304, Taylor & Francis, Vienna. 25. Karuzina, I. I., Bachmanova, G. I., Mengazetdinov, D. E., Myasoedova, K. N., Zhikhareva, V. O., Kuznetsova, G. P., and Archakov, A. I. (1979) Biokhimia 44, 1049-1057.
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OF BIOCHEMISTRY
AND
Vol. 298, No. 2, November
BIOPHYSICS
1, pp. 395-402,1992
Comparative Study of Monomeric Reconstituted and Membrane Microsomal Monooxygenase Systems of the Rabbit Liver I. Properties of NADPH-Cytochrome P450 LM, (2B4) Monomers
P450 Reductase
and Cytochrome
Irina P. Kanaeva, Ilya R. Dedinskii, Elena D. Skotselyas, Arkadii G. Krainev, Irina Irina F. Sevryukova, Yakov M. Koen, Galina P. Kuznetsova, Galina I. Bachmanova,’ and Alexander I. Archakov Institute
Received
of
Biological
December
and Medical
6,1991,
Chemistry,
and in revised
form
Russian Academy of Medical
May
Academic
Press,
Inc.
0003~9861/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
Russia
8, 1992
Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LMz (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LMz oligomers were 14.8 f 1.7 and 19.2 + 1.4 nm, respectively. Twentyfour-hour incubation with Emulgen 913 at 4°C at a molar ratio of 1:lOO led to the monomerization of NADPHcytochrome P450 reductase and cytochrome P450 LMz oligomers, the particle sizes diminishing to 6.15 1.3 and 5.2 f 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LMz monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LMz oligomers and the microsomal hemoprotein, cytochrome P450 LM, monomers formed complexes with type I and II substrates, but with & values higher than those of microsomes and cytochrome P450 LMz oligomers. Kinetic parameters (V,,,, and K,) of HzOz- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LMz oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LMz oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein. 0 1992
Sciences, Moscow,
V. Guleva,
Cytochrome P450-containing membrane monooxygenase systems can be reconstituted in solution in the presence of phospholipids (l-8), as well as ionic, zwitterionic, and nonionic detergents (9-13). The effect of phospholipids and detergents on the activity of such systems is associated with their deaggregating action on the oligomers of isolated proteins. Maximum rates of NADPHdependent cytochrome P450 reduction and substrate oxidation were observed in reconstituted systems at the equimolar reductase-to-cytochrome P450 ratio. However, Dean and Gray (9) and Wagner et al. (12), using the non-
ionic detergent octylglucoside and the zwitterionic detergent 3-([3-cholamidopropyl]dimethylammonio)-l-propanesulfonate ( CHAPS),2 demonstrated that a reconstituted system containing purified rabbit liver microsomal NADPH-cytochrome P450 reductase and cytochrome P450 LM2 exhibited maximum activity at a reductase-to-cytochrome P450 ratio of 1:5 without the formation of stable complexes between the proteins. A considerable body of evidence indicates that the two pro-
teins interact
in reconstituted
systems by way of random
collisions (7, 14-17). Muller-Enoch et al. (18) showed that it is possible to reconstitute monooxygenase activities in a system containing high concentrations of the reductase 1 To whom correspondence should be addressed at the Institute of Biological and Medical Chemistry Russian AMS, Pogodinskaya St., 10, Moscow 119832, Russia. ’ Abbreviations used: CHAPS, 3-[(3-cholamidopropylhlimethylammonio]-l-propanesulfonate; reductase, NADPH-cytochrome P450 rem ductase isolated from phenobarbital-induced rabbit liver microsomes; LM2, cytochrome P450 LM2 (2B4) isolated from phenobarbital-induced rabbit liver microsomes; CHP, cumene hydroperoxide; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 395
Inc. reserved.
396
KANAEVA TABLE
ET
AL.
I
Determination of Particle Sizes of Reductase and LM2 in the Absence and Presence of Emulgen 913 System 5 pM 5 @M 5 /tM 5 pM
reductase reductase + 0.25g/liter Emulgen913 LMz
LMp + 0.25g/liter Emulgen913
Diameter b-4 14.8 f 1.7 6.1 + 1.3 19.2 f 1.4
5.2 f 0.4
Note. Proteinmonomerization in the presence of detergentanddeterminationof particlesizeswerecarriedout asdescribedunderExperimentalProcedures. Meansof four or five measurements arepresented+ SE.
401 0
3
9
TIME (8MlN) of oligomers (solid line) and monomers (dashed 1. Inactivation of oxidized LMz in the absence (0, A) and presence (0, A) of 1 mM benzphetamine at 30°C (0, 0) and 37’C (A, A). The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5, and 0.5 @M cytochrome P450. In the case of monomers the buffer contained 0.25 g/ liter Emulgen 913. The content of LMz was determined in 2-min intervals from the difference spectrum of its dithionite-reduced CO complex, with 100% corresponding to q&chrome P450 content at zero incubation time. FIG. line)
and cytochrome P450 LMz at a ratio of 2:l without phospholipids and detergents after prolonged incubation. Dean and Gray (9) and Wagner et al. (12), using high concentrations of octylglucoside and CHAPS, obtained the monomers of NADPH-cytochrome P450 reductase and cytochrome P450 which were unable to catalyze cyclohexane oxidation, but did catalyze CHP-dependent toluene hydroxylation at a high rate. The lack of hydroxylase activity at high concentrations of the detergent was attributed by the authors to its inhibitory effect on cytochrome-reductase interaction. Earlier in our laboratory a monooxygenase system containing NADPH-cytochrome P450 reductase (reductase) and cytochrome P450 LMz (LM,) was reconstituted in the presence of the nonionic detergent Emulgen 913. Prolonged incubation of the reductase and LMz with Emulgen 913 at a protein-to-detergent ratio of 1:lOO led to the dissociation of protein oligomers to monomers. No formation of stable binary complexes upon mixing reductase and LM2 monomers was observed. Such a monomeric reconstituted monooxygenase system catalyzed NADPH-dependent LMz reduction and benzphetamine demethylation (19-24). The present study deals with the influence of the aggregation state of LM2 and the reductase on their thermal stability as well as on LMz substrate-binding ability and peroxidase activity. It was demonstrated that the oligomers and monomers of isolated LM2 were less thermostable than the hemoprotein of the microsomal membrane. Monomerization decreased the thermal stability of LMz, but not of the reductase. LMz oligomers and monomers possessed high peroxidase activities in the presence of hydrogen peroxide and CHP. The replacing of protein-protein interactions in LM2 oligomers by protein-detergent interactions in LM2 monomers affected values for the cosubstrates and substrates only without influence on values. K,,,
V,,
EXPERIMENTAL PROCEDURES Chemicals. EDTA and cytochrome c were purchased (Germany). Sodium dithionite was obtained from Merck
from Serva (Germany),
cumene hydroperoxide from Fluka (Switzerland), and NADPH from Beanal (Hungary). Catalase from beef liver was a product of BoehringerMannheim (Germany). EmuIgen 913 was from Kao-Atlas (Japan). Other chemicals were purchased from Reakhim (Russia). Phenobarbital-induced rabbit liver microsomes, LM, and reductase preparation. The microsomal fraction was isolated from the liver of male New Zealand rabbits treated with 0.1% (w/v) sodium phenobarbital in drinking water for 1 week (25). The reductase (specific activity 4013-13.5 43 pmol cytochrome c min-’ mg-’ at 3O”C, specific content
\ ‘0.
0
-.
..
9
3
TIME ;MIN) FIG. 2. Inactivation of oligomers (solid line) and monomers (dashed line) of reduced LMz in the absence (0, A, and presence (0, A, +) of 1 mM benzphetamine at 25°C +), 30°C (0, l ), and 37°C (A, A). The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5, and 0.5 pM cytochrome P450. In the case of monomers the buffer contained 0.25 g/liter Emulgen 913. The difference spectra of cytochrome P450 were recorded in P-min intervals after CO bubbling and sodium dithionite addition, with 100% corresponding to cytochrome P450 content at zero incubation time. 0)
(0,
RECONSTITUTION
OF
MONOMERIC
MICROSOMAL
MONOOXYGENASE
SYSTEM,
I
397
0.01
A
0
/ 8 2
\
-0.01
5 P 4 ---
xl.02 -
MICROSOMRS OLIGOMERS
---
400
450 WAVELENGTH
OLIGOHERS ..
500
550
.
).JONOmRS
I
-0.03 350
MICROSOMES
350
400
450 WAVELENGTH
(rim)
500
550
(nm)
0.01
C
I \
!
-
j,
MICROSOMES ---
1,’
...‘....’
OLIGOMERS MONOMERS
-0.02 350
400
450 WAVELENGTH
500
550
(nm)
FIG. 3. Binding spectra of 2 mM benzphetamine (A), 5 mM dimethylaniline (B), and 20 mM aniline (C) with cytochrome P450 from microsomal fraction, LM2 oligomers, and LM1 monomers. The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5, and 1 pM LM2 in the oligomeric or monomeric state or the same concentration of cytochrome P450 from rabbit liver phenobarbital-induced microsomes. In the case of LMp monomers the buffer contained 0.25 g/liter Emulgen 913. T = 3O’C.
nmol reductase mg protein-‘) and LM2 (specific were purified as described we’, &W/&T = 0.5-l) proteins showed a single band on SDS-PAGE.
content 17-18 earlier (25,26).
nmol Both
Monomerization of the proteins. Monomerization of the oligomers of isolated reductase and LMP was carried out as follows: to 5 nmol(2030 al) of either protein 13 ~1 of 2% (w/v) Emulgen 913 solution was added. After incubation for 5-10 min at room temperature, the concentrations of the proteins and Emulgen 913 were brought to 5 PM and 0.25 g/liter, respectively, with 100 mM K-phosphate buffer, pH 7.5, and the mixtures were incubated at 4°C for another 24 h. The determination of the amount of Emulgen 913 tightly bound by the protein monomers was carried out after gel chromatographic removal of free detergent
by the modified method of Horigone and Sugano (27) applied to Emulgen 913. Determination of the sizes of protein particles. Translation diffusion constants (D) of the reductase and LM, in the absence and presence of 0.25 g/liter Emulgen 913 in 100 mM K-phosphate buffer were determined by laser correlation spectroscopy using a Coulter N4 instrument (Coultronix, France) operated at 21°C. The reductase and LM, concentrations were 5 pM; the sample volume was 1 ml. The Coulter N4 uses a heliumneon laser (X = 632.8 nm) as a light source. Instrument settings were a scattering angle of 90”, an acquisition time of 200 s, particle size ranges of l-1000 and l-10,000 nm. Correlation time was varied from 10 to 50 p.s. Steady solutions of the reverse problem in the range l-300 nm were
398
KANAEVA
ET
TABLE Parameters
of Cytochrome
P450
Difference
Microsomes LM2 oligomers LMP monomers
&in (nm)
Lx bm)
421 423
385 383 385
421
Note. The incubation mixture contained 0.25 g/liter Emulgen
wnax (X103) 43 f 41 t 41 f
0.5 0.4 0.3
II
Binding
Benzphetamine
System
AL.
Spectra
with
Type
I and
II Substrates
Dimethylaniline &in Kd
0.03 0.3 0.6
bM)
f
0.003 + 0.05
+ 0.1
(nm)
425 418 423
Lx
Aniline
uwu (X103)
bm)
389 379 383
&
28 f 0.3 21 f 0.1 15 2 0.2
6-d
Substrate binding to cytochrome P450. The binding spectra of cytochrome P450 with type I and II substrates were recorded by the differential scheme using a Hewlett-Packard 8451A diode array spectrophotometer. The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5, 1 fiM LM2 in the oligomeric or monomeric state or the same concentration of cytochrome P450 in phenobarbital-induced rabbit liver microsomes and various concentrations of benxphetamine, dimethylaniline, or aniline. Spectral binding constants (&) and maximum absorbance change (AA,) were calculated by the Eadie-Hofstee equation using a computer program based on the linear regression procedure.
Hemoprotein peroridase activity measurements. The rates of HzOzand CHP-dependent benzphetamine N-demethylation and aniline phydroxylation were estimated by measuring the amounts of formaldehyde and p-aminophenol formed, respectively. The incubation mixture (0.5 ml) contained 100 mM K-phosphate buffer, pH 7.5, and various concentrations of benzphetamine or aniline. The concentration of LM2 or microsomal cytochrome P450 was 1.0 pM. The reactions were started by the addition of various amounts of HzOz or CHP and carried out at 30°C for 1 min. In the case of H,O,-supported reactions, unreacted HzOz was removed by addition of 16,000-48,000 U/ml catalase to the incubation mixture. The reactions were stopped by the addition of trichloroacetic acid to a final concentration of 7.5%. For formaldehyde determination 0.5 ml of Nash reagent consisting of 4 M ammonium acetate, 0.1 M glacial acetic acid, and 0.04 M acetylacetone was added to 0.5 ml of the supernatant and incubated at 37°C for 30 min. The extinction coefficient of the colored product at 412 nm was 4 mM-’ cm-‘. For paminophenol determination, 0.25 ml of 20% Na,CO, and 0.75 ml of 2% phenol were added to 0.5 ml of the supernatant and incubated at 37’C for 30 min. The extinction coefficient of the colored product at 630 nm was 10 mM-’ cm-i. K,,, and V,, values for the substrates and cosubstrates were calculated by the nonlinear regression technique using a modified KINFIT program (28). Other assays. The concentration of cytochrome P450 was determined by the method of Omura and Sato (29) in a Hewlett-Packard 8451A spectrophotometer (USA) from the CO-difference spectrum of dithionitereduced microsomes or LMp based on an extinction coefficient of 91 mM-’ cm-i. The concentration of the purified reductase was determined from its absolute absorption spectrum based on an extinction coefficient of 21.4 mM-’ cm-’ (30). NADPH-cytochrome c reductase activity was determined in an Ultraspec spectrophotometer (Sweden) from the cytochrome c reduction rate at 3O”C, based on the extinction coefficient of reduced cytochrome c, 21.1 mM-’ cm-’ for 550 nm (31). Inactivation of LM, oligomers and monomers was evaluated by recording difference absorption spectra of the reduced CO-LM2 complex. Heat resistance of
Lax (nm)
unax (XlOs)
439 441 450
34 + 0.4 25 + 0.3 16 I!Z 0.4
0.05 0.6
+ 0.01 + 0.03
399 407
0.9
+ 0.1
413
contained 100 mM K-phosphate buffer, pH 7.5, and 1 pM cytochrome 913. 7’ = 30°C. Means of five or six measurements are presented *SE.
taken into consideration. Data presented are means from four or five measurements f SE. The Coulter N4 software uses the Stokes-Einstein equation for spherical particles to derive particle diameter as an estimation of particle size.
Lain (nm)
P450.
&
0.8 f 0.1 1.3 k 0.1 3.8 f 0.3
In the case of monomers
the reductase oligomers and monomers NADPH-cytochrome c reductase activity.
was estimated
bM)
the buffer
by measuring
RESULTS AND DISCUSSION Particle Sizes of the Purified Reductase and LA!, It was shown earlier in this laboratory that the incubation of purified 5 PM reductase and 5 PM LM2 with 0.25 g/liter Emulgen 913 in 100 mM K-phosphate buffer led to the dissociation of protein oligomers. The molecular weights of the reductase and LM2 monomers estimated by gel filtration were 135 and 65 kDa, respectively (19, 20,22). Such great values may be due to the large amounts of detergent tightly associated with the proteins. Measurement of amount of detergent tightly bound (27) to the proteins showed that lo-20 and 50-60 detergent molecules are associated with 1 molecule of LMP and reductase, respectively (19, 20, 22). Taking into account the molecular weight of Emulgen 913, which is equal 792 Da,
1.25
I
I,
I.,
,
I. l
.
1.05
f
.
0.85 l
.5 -0 y
:/~
0.65
0.45
Ii Ii
0.25
" 0.00
" 0.01
a 0.02
IEmulgenl,
1 0.03
" 0.04
0.05
%
FIG. 4. Dependence of spectral binding constants Kd for benxphetamine on Emulgen 913 concentration. The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5,1 pM LM,, and various concentrations of the detergent. T = 3OOC. The spectral binding constants of LMP with benzphetamine were determined at the detergent concentrations indicated under Experimental Procedures.
RECONSTITUTION
OF
MONOMERIC
MICROSOMAL TABLE
Kinetic Parameters of H,O,-Dependent
MONOOXYGENASE
For HzOz
III
Microsomes LM2 oligomers LMx monomers
110 + 13 420 f 22 340 ?I 61
VJK,,,
For
399
I
Benzphetamine (BP) N-Demethylation
Km (mM) System
SYSTEM,
BP
V,..
0.23 f 0.04 0.54 + 0.08 0.61 + 0.08
(mM
min-‘)
0.16 + 0.03 0.10 f 0.02 0.090 f 0.001
(X
10x mini)
For H202
For BP
0.15 0.02 0.03
70 19 15
Note. The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5, and 1 PM cytochrome P450. In the case of monomers the buffer contained 0.25 g/liter Emulgen 913. The reaction was initiated by the addition of various concentrations of H202. When evaluating Km for H202 and BP the concentrations of BP and HzOz were 2 mM and 1 M, respectively. The incubation was carried out for 1 min at 30°C. Means of three or four measurements are presented *SE.
the aforementioned molecular weights of the protein monomers seem reasonable. Thus, Emulgen 913 induced monomer formation under experimental conditions. To prove that monomerization actually takes place under the above conditions the sizes of the reductase and LMB particles were measured in the absence and presence of Emulgen 913 using laser correlation spectroscopy. From Table I it is seen that incubation of oligomers with 0.25 g/liter Emulgen 913 leads to the dissociation of the oligomers, with particle sizes decreasing to 6.1 + 1.3 and 5.2 + 0.4 nm, respectively, which agrees well with the values of 5.6 and 4.7 nm we estimated from earlier reported molecular weights for the monomeric reductase and LM2. Apparent monomeric values derived from SDS-PAGE analysis for NADPH-cytochrome P450 reductase and LM, purified from rabbit liver microsomes were reported by Muller-Enoch et al. (18) and corresponded to 74,000 and 49,000 respectively. These values were used for estimation of the diameters of the proteins, assuming a globular shape of these molecules and a density of proteins of 1.35 X lo3 kg rnp3 (32). Thus, the results obtained by two independent methods provide convincing evidence that under these conditions the dissociation reveals protein particles corresponding to monomeric ones.
TABLE Kinetic
Parmeters
Thermal Stability of the Reductase and LM, Oligomers and Monomers To study how the change from protein-protein interactions in oligomers to protein-detergent interactions in monomers influences the protein thermal stability, LMz content, and cytochrome c reductase activity of the reductase were estimated before and after the incubation of oligomers and monomers at 25, 30, and 37°C for 10 min. The oligomers and monomers of ferric LM2 were not inactivated at 25°C. At 3O”C, insignificant inactivation (by as much as 10%) of only LMz monomers was observed (Fig. 1). At 37”C, LM, oligomers were inactivated by 10% and monomers by 40% (Fig. 1). The addition of benzphetamine to ferric LM2 oligomers and monomers had no effect on their thermal stability (Fig. 1). The inactivation of dithionite-reduced LMz oligomers occurred at 37°C only (by 40%) (Fig. 2). The reduced form of LM2 monomers was inactivated by 30% at 25°C and by about 45-50% at 30 and 37°C (Fig. 2). In the case of ferrous LM2, inactivation was slowed down by benzphetamine so that oligomeric LM2 was inactivated at 37°C by only 10% (Fig. 2). With monomers, the greatest protective effect was observed at 25 and 30°C. Unlike LM2, the reductase showed considerably higher thermal sta-
IV
of CHP-Dependent Benzphetamine (BP) N-Demethylation KmbM)
V,,/K,
System
For CHP
For BP
Microsomes LMx oligomers LM2 monomers
0.12 f 0.01 0.11 + 0.03 0.28 + 0.04
0.50 + 0.08 1.8 -c 0.2 0.90 f 0.15
V,,,
(mM
mini)
0.013 + 0.004 0.010 + 0.001 0.010 f 0.002
(min-*)
For CHP
For BP
0.11 0.09 0.04
0.026 0.006 0.011
Note. The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5, and 1 PM cytochrome P450. In the case of monomers the buffer contained 0.25 g/liter Emulgen 913. The reaction was initiated by addition of various concentrations of CHP. When evaluating K,,, for CHP and BP the concentrations of BP and CHP, respectively, were 2 mM. The incubation was carried out for 1 min at 30°C. Means of three or four measurements are presented +SE.
400
KANAEVA TABLE Kinetic
Parameters
of HeOe-Dependent
ET
AL.
V Aniline
(AN)
p-Hydroxylation V&K,,,
Km (mM) System
For H202
For AN
Microsomes LM2 oligomers LM2 monomers
66k a 80+ 10 800 + 105
0.32 + 0.06 0.40 f 0.05 3.4 f 0.6
V,,
(mM
0.038 0.024 0.020
mini)
+ 0.005 f 0.003 f 0.004
(X
10’
mini)
For HzOz
For AN
0.06 0.03 0.003
12 5.6 0.6
Note. The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5, and 1 pM cytochrome P-450. In the case of monomers the buffer contained 0.25 g/liter Emulgen 913. The reaction was initiated by the addition of various concentrations of HzOz. When evaluating Km for HzO, and AN, the concentrations of H202 and AN were 1 M and 20 mM, respectively. The incubation was carried out for 1 min at 30°C. Means of three or four measurements are presented -+SE.
bility, and the NADPH-cytochrome c reductase activity of reductase oligomers and monomers did not change after incubation for 10 min at 25, 30, and 37°C (data not shown). Therefore, in all cases LM2 oligomers were more thermostable than monomers. In other words, the substitution of protein-protein interactions in oligomers with proteindetergent interactions in monomers reduces hemoprotein stability. In rabbit liver phenobarbital-induced microsomes the oxidized and reduced forms of cytochrome P450 were not inactivated at the above temperatures (33). Substrate Binding to LM, Oligomers, LM, Monomers, and Cytochrome P450 of Phenobarbital-Induced Rabbit Liver Microsomes The binding spectra of microsomal cytochrome P450 and isolated oligomeric and monomeric LM2 with benzphetamine, dimethylaniline, and aniline are presented in Fig. 3 and their parameters in Table II. It is seen that similar to microsomal hemoprotein, LM2 oligomers and monomers are capable of binding both type I and II substrates. With benzphetamine and dimethylaniline the positions of the absorption maxima (379-385 nm) and minima (418-427 nm) of the microsomal hemoprotein, oligomers, and monomers of LM2 were typical of type I substrates. The binding of the type II substrate aniline to monomers produced a maximum at 450 nm, whereas with oligomers and microsomes the maxima were at 441 and 439 nm, respectively. The magnitudes of maximum absorbance changes of LMz-benzphetamine complexes were the same for LM2 oligomers, LM2 monomers, and the hemoprotein of the microsomal fraction. With dimethylaniline and aniline these values for microsomal cytochrome P450 were 1.32 times those for LM2 oligomers and monomers. As seen from Table II, spectral binding constants (&) decreased in the order monomers > oligomers > microsomes. With monomers, Kd values for benzphetamine and dimethylaniline were 1.5-2 times those with oligomers and an order of magnitude greater than those with microsomes. With
aniline, the Kd values for monomers was 3 times that for oligomers and 5 times that for microsomal cytochrome P450. The different spectral binding constants but similar maximum absorbance changes may be due to the presence of the detergent, either in the tightly protein-bound form in the case of LM2 oligomers or in solution (0.25 g/liter Emulgen 913) of LM2 monomers. This assumption was confirmed by experiments on the influence of various concentrations of Emulgen 913 on the Kd for benzphetamine. The Kd increased with detergent concentration (Fig. 4), indicating the possibility of competition between the detergent and the substrate for binding sites on the hemoprotein. Peroxidase Activities of LM2 Oligomers, LM2 Monomers, and Cytochrome of Phenobarbital-Induced Rabbit Liver Microsomes To elucidate the influence of protein-protein and protein-detergent interactions on the catalytic activity of LM2 the oxidation rates of type I and II substrates were measured in the presence of the active oxygen donors HzOz and CHP which differ substantially in hydrophobicity. These studies were of particular interest since some authors believe that the quarternary structure of LM2 plays an important role in its catalytic activity (34). As seen from Table III, in the case of benzphetamine (type I substrate), K,,, values for the substrate and for hydrogen peroxide, as well as V,,, values, were close to those for the oligomers and monomers. CHP-dependent benzphetamine oxidation (Table IV) was characterized by the same V,, values for oligomers and monomers but different K,,, values for the substrate and cosubstrate. With monomers, the K, for CHP was 2.5 times higher and the K,,, for benzphetamine 2 times lower than that for oligomers. As a consequence, the V,,,/K, ratio for CHP was greater in the case of oligomers and that for benzphetamine was greater in the case of monomers. Thus, the substitution of protein-protein with proteindetergent interactions has no effect on the ability of LM2 to catalyze type I substrate oxidation as evidenced by vir-
RECONSTITUTION
OF
MONOMERIC
MICROSOMAL TABLE
Kinetic
Parameters
of CHP-Dependent
MONOOXYGENASE
SYSTEM,
VI
Aniline
(AN) p-Hydroxylation
Km bM) System Microsomes LM2 oligomers LMP monomers
401
I
(min-‘)
V,,/K,
For CHP
For AN
0.15 f 0.02 0.28 f 0.04 1.2 + 0.3
1.6 f 0.2 4.4 +0.6 6.8 f 0.8
V,,
(mM
0.021 0.016 0.015
min-‘)
+ 0.003 f 0.002 f 0.003
For CHP
For AN
0.14 0.06 0.013
0.013 0.004 0.002
Note. The incubation mixture contained 100 mM K-phosphate buffer, pH 7.5,l pM, hemoprotein. In the case of monomers the buffer contained 0.25 g/liter Emulgen 913. The reaction was initiated by the addition of various concentrations of CHP. When evaluating K, for CHP and AN, the concentrations of CHP and AN were 2 and 20 mM, respectively. The incubation was carried out for 1 min at 30°C. Means of three or four measurements are presented +SE.
tually identical V,,, values in both systems. It may be concluded therefore that the quarternary (oligomeric) structure of LM2 does not affect its peroxidase activity. On the other hand the activity of the hemoprotein in the microsomal membrane was 1.5 times higher than in solution, which confirms our previous conclusion that the “liquid” membrane fixes LM2 in the active state (17, 35, 36). With the type II substrate aniline no differences were found between LM2 oligomers and monomers with respect to V,,, (Tables V and VI). However, the K,,, values for HzOz, CHP, and aniline did differ. In the H202-dependent system, the K,,, values for LM2 monomers were one order of magnitude higher than those for oligomers, which resulted in different V,,,/K, ratios. With the CHP-dependent system the K,,, values for the substrate and cosubstrate were also higher in the case of monomers. The differences can be due to competition for the binding site(s) in the active center of LM2 monomers between the substrate or cofactor, on the one hand, and the nonionic detergent Emulgen 913, on the other hand. The HzOzand CHP-dependent aniline hydroxylase activities of cytochrome P450 in microsomes were higher than in solution. It may be concluded therefore that the peroxidase activity of LM2 is independent of its aggregation state. LM2 monomers, like the oligomers and microsomes, can utilize hydrogen peroxide and CHP as active oxygen donors. The substitution of protein-protein interactions in LM2 oligomers by protein-detergent interactions in monomers affects only the Km values for the cosubstrates and for type II substrate aniline, but not for benzphetamine. The microsomal hemoprotein has higher thermal stability and catalytic activity as compared to LM2 oligomers and monomers, which can be accounted for by the stabilizing action of the membrane on the hemoprotein and the ability of the phospholipid bilayer to maintain the enzyme in a conformational state close to the transitional one (17, 35, 36).
ACKNOWLEDGMENT The authors of the English
thank Dr. A. A. Zhukov for useful version of the manuscript.
discussion
and revision
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