Selective inactivation of NADPH-cytochrome P-450 (c) reductase by diazotized 3-aminopyridine adenine dinucleotide phosphate

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 211, No. 1, October 1, pp. 227-233, 1981

Selective inactivation of NADPH-Cytochrome P450 (c) Reductase Diazotized 3-Aminopyridine Adenine Dinucleotide Phosphate’ RICHARD Department

of Biochemistry

by

E. EBEL

and Nutrition, Virginia Polytechnic Institute and State University, Black&u& Virginia 24061 Received

February

6, 1981

3-Aminopyridine adenine dinucleotide phosphate was found to be a potent competitive inhibitor of NADPH-cytochrome P-450 (c) reductase. The reductase was rapidly and irreversibly inactivated at pH 7 and 4’C by diazotized 3-aminopyridine adenine dinucleotide phosphate. However, inactivation required the prereduction of the enzyme by NADPH. Spectral studies were consistent with the incorporation of 0.52 mol of nucleotide per mole of flavin or 1.04 mol per mole of enzyme.

NADPH-cytochrome P-450 (c) reductase (EC 1.6.2.4) is an integral component of the microsomal monooxygenase system. As such it is involved in the hydroxylation of a wide variety of chemicals including drugs, polycyclic hydrocarbons, pesticides, steroids, and other exogenous and endogenous compounds (2, 3). The reductase is responsible for the transfer of electrons from NADPH via its two flavin prosthetic groups (4) to the hemin iron of cytochrome P-450 where they are used to produce an active oxygen species involved in the hydroxylation reaction. The reductase and cytochrome interact in the two-dimensional plane of the microsomal membrane and their arrangement is such that a single reductase molecule can efficiently reduce about 20 mol of cytochrome (5, 6). The present study was initiated to investigate the interaction of the NADP analog, 3-aminopyridine adenine dinucleotide phosphate (AADP),2 and its diazonium i This work was supported in part by USPHS Grants CA24364 and ES02045 and by Hatch Project VA-0612304. A preliminary report of this work has been presented (1). ’ Abbreviations used: AADP, 3-aminopyridine adenine dinucleotide phosphate; AAD, 3-aminopyridine adenine dinucleotide; DCIP, 2,6-dichlorophenol-indophenol; 2’-PADPR,2’-monophosphoadenosine 5’disphosphoribose; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

derivative with NADPH-cytochrome P450 (c) reductase. An effective active sitedirected reagent would be of use in the study of the active site of the enzyme as well as a probe for investigating the interaction of the reductase and cytochrome. AADP and the corresponding NAD analog, 3-aminopyridine adenine dinucleotide (AAD), were developed by Anderson and co-workers (7-9) who demonstrated that they were competitive inhibitors versus the corresponding pyridine nucleotides for several NAD- or NADP-requiring enzymes. In addition, these workers showed that the diazonium derivative of the 3amino function behaved as a site-specific sulfhydryl reagent under suitable conditions (0-4”C, neutral pH) (7-10). MATERIALS

AND

METHODS

Phenobarbital, cytochrome c, 2,6-dichlorophenolindophenol (DCIP), DL-isocitrate, isocitrate dehydrogenase, and various nucleotides (Sigma Chemical Co.); Renex 690 (ICI Americas, Inc.); and I-dimethylaminoantipyrine (Aldrich Chemical Co.) were obtained from the indicated sources. 3-Aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP) were kindly provided by Dr. B. M. Anderson. All other chemicals used were of the highest quality commercially available and, with the exception of aniline which was redistilled, were used without further purification.

227

0003-9861/81/11022’7-07$02.00/O Copyright (B 1981 by Academic Press, All rights of reproduction in any form

Inc. reserved.

228

RICHARD

The microsomal fraction was prepared as previously described (11) from the livers of 200- to 250-g Sprague-Dawley rats (Flow Laboratories, Dublin, Va.) or from bovine liver obtained from the Department of Food Science and Technology. The rats received intraperitoneal injections of sodium phenobarbital (80 mg/kg body wt) for 4 days and were fasted for about 18 h prior to sacrifice by decapitation. NADPH-cytochrome P-450 (c) reductase was isolated from the microsomal fraction following proteolysis using the procedure of Prough and Masters (12) except that trypsin was used rather than steapsin. The microsomal fraction (lo-15 mg protein/ml) was incubated at 25°C with 0.1 mg trypsin/ml for 20 min to release the reductase. Chicken egg white trypsin inhibitor (0.2 mg/ml) was added after this 20-min incubation. The reductase was also isolated following detergent solubilization using a slight modification of published procedures (13-15). These procedures were used with the microsomal fractions obtained from both rat and bovine liver. Rabbit liver NADPHcytochrome c (P-450) reductase was generously provided by Dr. B. S. S. Masters. In these studies, comparable results were obtained for the reductases isolated from these three sources eliminating the possibility of species variation. Reductase activity was monitored by following the reduction of cytochrome c at 550 nm (e = 21 mM-’ cm-‘) (16) or DCIP at 600 nm (c = 21 mM-’ cm-i) (17) at 25°C using a Cary 219 spectrophotometer (Varian Instruments). Specific conditions for the enzymatic assays are described in the figure legends. I-Dimethylaminoantipyrine (aminopyrine) N-demethylase activity was assayed by formaldehyde release (18). Aniline phydroxylase activity was monitored by the method of Imai et al. (19). In both cases (aminopyrine N-demethylase and aniline phydroxylase), the assay was carried out at 25°C and consisted of 50 mM Hepes buffer, pH 7.4,150 mM KCl, 10 mM MgClc, 10 mM DLisocitrate, 0.4 unit/ml isocitrate dehydrogenase, 1 mg microsomal protein/ml, and 0.2 mM NADPH. the substrate concentrations were 1 mxe aminopyrine or 10 mM aniline. The diazotization of AAD or AADP was performed according to Fisher et al. (7). Incubation of reductase with the diazotized analogs at pH 7 was performed at 0-4°C. In kinetic studies, reductase, typically 20 pg/ml, was included in the incubation and 10 pl of this was added to a l-ml reaction mixture. The cytochrome (P-450 and 9) content of the microsomal preparations was determined by the method of Omura and Sato (20). RESULTS

Inhibition bv AADP AADP was a competitive inhibitor versus NADPH (Fig. 1). Using an NADPH-

E. EBEL

O-cl

I/NADPH

b/M)-”

FIG. 1. Competitive inhibition of trysin-solubilized rat liver reductase by AADP. The assay consisted of 50 rnM Hepes buffer, pH 7.7, 50 PM DCIP, 10 mM MgClc, 10 mMDL-isocitrate, 0.4 unit/ml isocitrate dehydrogenase, and variable concentrations of NADPH. In B, 250 mM KC1 was also present. The concentrations of AADP were 0 (O), 0.1 (A), 0.2 (m), and 1.0 PM (v) in A and 0 (a), 1.0 (A), 2.0 (m), and 4.0 NM (V) in B.

generating system consisting of 10 mM MgC&, 10 mM DL-isocitrate, and isocitrate dehydrogenase (0.4 unit/ml), the K,,, for NADPH was 0.3 and 2.7 PM while the Ki values for AADP were 0.2 and 1.8 PM with low and high salt concentrations, respectively. These conditions with regard to salt concentration are comparable to those commonly reported in the literature. Masters et al. (21) originally described a system employing 50 mM phosphate buffer while Vermilion and Coon (22) and Dignam and Strobe1 (23) used 300 mM phosphate buffer. Using the higher salt concentrations an approximate doubling of the reductase activity was obtained (13). Since in these studies an NADPH-generating system containing MgC12 was used, 50 mM Hepes and 50 InM Hepes plus 250 mM KC1 replaced 50 and 300 mM phosphate, respectively. Routinely, an assay system containing 50 mM Hepes was used. AAD was without effect on the NADPHdependent reduction reaction. Similar re-

LIVER

CYTOCHROME

P-450

sults were obtained whether the microsomal fraction or the various isolated reductases were used. AADP was among the most potent of the nucleotide inhibitors of the reductase (Table I). The concentration required for 50% inhibition estimated by the procedure of Job et al. (24) was 4 PM using 10 I.LM NADPH. Only 2’,5’-ADP and (thionicotinamide)ADP produced a comparable level of inhibition. While AADP was a potent inhibitor of NADPH-cytochrome c reductase activity of the microsomal fraction, AAD was a very poor inhibitor of the NADH-cytochrome c reductase activity. Approximately 1 mM AAD was required to produce a 50% inhibition of the NADH-dependent activity using 10 PM NADH. Inactivation

by Diazotixed

AADP

Incubation of the purified reductase with diazotized AADP resulted in no loss of activity. A time-dependent loss of activity was observed with isolated reductase only when the enzyme was reduced with NADPH prior to incubation with diazotized AADP (Fig. 2). Neither NADP nor NADH could substitute for NADPH. The rate of inactivation was dependent TABLE INHIBITION

I

BY VARIOUS NUCLEOTIDES NUCLEOTIDE ANAMGS

Concentration for 50% inhibition (PM)

Inhibitor” NADP 2’,5’-ADP 2’-PADPR 2’-AMP 5’-AMP (3-Aminopyridine)ADP (Thionicotinamide)ADP (3-Acetylpyridine)ADP

AND

(AADP)

15 4 21 155 2OmM 4 4 8

a Nicotinic acid, nicotinamide. nicotinamide riboside, nicotinamide mononucleotides, and 3-aminopyridine caused no inhibition at concentrations up to 10 mM.

REDUCTASE

229

INACTIVATION

0

, 0

I

‘. , 2

-----‘I,

( 4 1wmTl0~

I

, , , 6 8 TIME (MINI

I

., IO

FIG. 2. Inactivation of reductase as a function of diazotixed AADP concentration. The enzyme was preincubated with 100 WM NADPH before exposure to 46 (A), 93 (m), or 180 PM (v) diazotized AADP. There was no loss of activity over the time period if the enzyme was incubated with 93 MM diazotized AADP in the absence of NADPH (0).

upon both the concentration of diazotized AADP and NADPH. In the presence of 100 pM NADPH (Fig. 2), increasing concentrations of diazotized AADP resulted in an increase in both the rate and the extent of activity loss. At each concentration of diazotized AADP in the presence of NADPH, there was an initial rapid loss of activity. Following this initial loss of activity there was very little change in activity with time. At a fixed concentration of diazotized AADP, relatively low concentrations of NADPH allowed inactivation to occur but at higher concentrations of NADPH the enzyme was protected from inactivation (see below and Fig. 3).

IC

“2c=== 2

8

INCdTION

TiE

IO

(MN)

FIG. 3. NADPH protection of reductase from inactivation by diasotized AADP. The concentration of diazotised AADP was 30 PM. The concentrations of NADPH were 0 (O), 5 (‘I), 50 (o), 100 (A), and 300 PM w.

230

RICHARD

With the microsomal fraction, the effect of inactivation by diazotized AADP on drug turnover could be investigated as well as that on cytochrome c reductase activity. Incubation of the microsomal fraction for 5 min with 200 PM diazotized AADP resulted in a 20-25s decrease in aminopyrine N-demethylase and aniline p-hydroxylase activities. If the microsomal fraction was incubated with 100 ~.LM NADPH prior to incubation with 200 PM diazotized AADP, about ‘70-80s of these activities were lost in 5 min (Table II). These results parallel those for the loss of NADPH-cytochrome c reductase activity in the microsomal fraction. The content of cytochrome P-450 and bs was not affected by this treatment nor was cytochrome P-450 converted to P-420. NADHcytochrome c reductase activity was also not altered. Since reduction of the enzyme was required for inactivation, the obvious question remained as to which reduced species of the enzyme was required. In an attempt to address this question, the air-stable semiquinone form of the reductase was produced by incubation with a slight excess of NADPH. The formation of the air-stable semiquinone was monitored spectrally. Incubation of this form of the enzyme with 200 I.IM diazotized AADP resulted in a loss of 50% of the activity in 5 min and 65% in 10 min. If the reductase were reduced with 50 PM NADPH and rap-

TABLE

II

E. EBEL

idly mixed with 200 PM diazotized AADP before the enzyme could reoxidize to the semiquinone state, essentially all of the reductase activity was lost within 30 s. Thus, it appears that more than one reduced form of the enzyme can be inactivated by diazotized AADP but that the rates at which these species react are significantly different. The experimental conditions were not adequate to determine if both the two- and three-electron reduced forms of the enzyme react. The fully reduced (four-electron) form would not be produced under the conditions used. While NADPH-cytochrome c reductase activity was lost upon incubation with diazotized AADP using appropriate conditions, incubation of the microsomal fraction with up to 2 mM diazotized AAD resulted in no loss of NADPH- or NADHcytochrome c reductase activites even if the sample was prereduced with NADH or NADPH. NADPH and NADP Protection against Inactivation by Diaxotized AADP While inactivation of NADPH-cytochrome P-450 (c) reductase by diazotized AADP required that the enzyme be reduced by NADPH prior to incubation with the diazotized analog, increasing the concentration of NADPH relative to that of diazotized AADP resulted in protection from inactivation (Fig. 3 and Table III). NADP also protected the NADPH-reduced enzyme from inactivation by diazotized AADP (Table III). NADH and NAD provided no protection.

PERCENTAGE ACTIVITY RAT LIVER MICROSOMES

Preincubation

AminoAniline pyrine Np demethhydroxyylase lase

None 200 PM diazotized AADP 100 /LM NADPH + 200 PM diazotized AADP ‘The numbers in parentheses in nmol/min/mg protein.

100 (5.2)” 100 (0.81) 79 (4.1) 75 (0.61) 19 (1.0)

28 (0.23)

represent activity

Spectral Studies of Diaxotixed Modified Reductase

AADP-

Following reduction with NADPH and inactivation with diazotized AADP, the reductase was treated with 1 mM K3Fe(CN),, charcoal, and extensively dialyzed against 50 mM phosphate buffer, pH 7.4, 20% glycerol, 0.1 mM EDTA. The resulting uv-difference spectrum (Fig. 4) of this modified reductase versus a sample of reductase treated in the same manner

LIVER TABLE INACTIVATION

CYTOCHROME

PROTECTION

10 /.lM diazotized AADP

10 FM NADPH 10 PM NADPH None 100 PM NADPH 10 /AM NADPH + 75 /.LM NADP

Percentage activity after 5 min

+ + +

0 100 100 84

+

71

300

0.10 0.4 0.05

0.2

400 600 WAVELENGTH

600

400 WAVELENGTH

0.6

300

231

INACTIVATION

BY

except that AADP was not included was comparable to the known absorption spectrum of AAD (7). From this difference spectrum it was possible to calculate that 0.52 mol of AADP was bound to the enzyme per mole of flavin assuming that the extinction coefficient at about 250 nm of the bound AADP is the same as that of free diazotized AADP (t = 19.5 mM-’ cm-‘) (7). Flavin concentration was determined using an extinction coefficient of 10.6 mM-’ cm-‘. The uv spectrum following treatment with 6 M quanidine HCl and dialysis against 50 mM phosphate buffer, pH 7.4, 0.1 mM EDTA (Fig. 5) demonstrated that the modification was at the protein and not the flavin level.

0 200

REDUCTASE

III

BY DIAZOTIZED AADP NADPH AND NADP

Preincubation

P-450

0 700

FIG. 4. Spectra of detergent-solubilized and purified rat liver reductase. The spectrum indicated by the symbol (0) is that inactivated with diazotized AADP. The unlabeled spectrum is that of reductase treated in the same manner as the inactivated sample except that AADP was not included. Inset: Difference spectrum of inactivated reductase versus control (no AADP).

FIG. fied rat nidine (0) is AADP. treated except

600

600

(nm)

5. Spectra of detergent-solubilized and puriliver reductase following treatment with quaHCl. The spectrum indicated by the symbol that of reductase inactivated with diazotized The unlabeled spectrum is that of reduetase in the same manner as the inactivated sample that AADP was not included.

DISCUSSION

AADP is a potent competitive inhibitor versus NADPH of NADPH-cytochrome P450 (c) reductase both as it exists in the microsomal membrane and when isolated following detergent solubilization or trypsin treatment. The affinity of AADP for the reductase is comparable to that of 2’,5’-ADP and (thionicotinamide)ADP and is about four times better than that of NADP (Table I). The specificity is as expected in that AAD is without effect on the NADPH-dependent reductase. In contrast, AAD is a poor inhibitor of microsomal NADH-cytochrome c reductase activity requiring a loo-fold molar excess of AAD to NADH to produce 50% inhibition. However, AAD is a more potent inhibitor of this reaction than is NAD which produced no inhibition at a loo-fold molar excess (25). The diazonium derivative of AADP produces a time-dependent loss of reductase activity only when the enzyme has been prereduced with NADPH. While the oneelectron reduced (26) air-stable semiquinone form of the enzyme can be inactivated, a more reduced form of the enzyme (two or three electron) reacts faster. About 20% of the reductase activity is lost when diazotized AADP reacts with the reductase in the microsomal membrane in the

232

RICHARD

absence of added NADPH. This may be the result of reductase which remains partially reduced during the microsomal isolation procedure. This suggestion is consistent with the reported stability of the semiquinone state of the enzyme (27). Diazotized AAD is without effect on either NADPHor NADH-cytochrome c reductase activity independent of the prereduced state of the system. This is not an unexpected result when the poor affinity of NADH (or NAD) for the NADPH-dependent enzyme (F&n~nn - 25 IYIM) (28) and the weak inhibitory effect of AAD versus the NADH-dependent reductase are considered. The data are consistent with the conclusion that diazotized AADP is an active site-directed reagent for the reductase. AADP is a competitive inhibitor versus NADPH; the enzyme is protected from inactivation by NADPH and NADP but not NADH or NAD, and spectral data support the incorporation of 0.52 mol of AADP per mole of flavin or 1.04 mol of AADP per mole of enzyme. The interaction between diazotized AADP and reductase is probably through a sulfhydryl residue at the active site. This is consistent with the known chemistry of this derivative (7-10) and the sensitivity of the enzyme to sulfhydryl reagents (21). In addition, Lazar et al. (29) have demonstrated using 5,5’-dithiobis(2-nitrobenzoate) that one sulfhydryl residue of the protein is required for activity and that this residue can be protected with NADP. The requirement for prereduction of the reductase prior to inactivation by diazotized AADP has been reported in only one other system, Neurospora crassa nitrate reductase (30). In that instance, the authors invoked the reduction of a disulfide bridge by NADPH and suggested that one of the resulting sulfhydryl groups reacted with diazotized AADP. This explanation cannot be generalized to NADPH-cytochrome P-450 (c) reductase since all of the cysteine residues are present in the free sulfhydryl form (29). In conclusion, diazotized AADP has been shown to be an effective active site-

E. EBEL

directed reagent for NADPH-cytochrome P-450 (c) reductase. As such, it should prove useful in mapping the active site of this enzyme and in studies of the interaction between reductase and cytochrome P-450 in the microsomal membrane. ACKNOWLEDGMENT The author would for his encouragement

like to thank Dr. B. M. Anderson and helpful discussions.

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CYTOCHROME

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INACTIVATION

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eds.), Vol. 10, pp. 561-565, Academic Press, New York. 26. IYANAGI, T., AND MASON, H. S. (1973) Biochemistry 12.2297-2308. 27. MASTERS, B. S. S., KAMIN, H., GIBSON, Q. H., AND WILLIAMS, C. H., JR. (1965) J. BioZ. C&m. 240, 921-931. 28. FAN, L. L., AND MASTERS, B. S. S. (1974) Arch. Bioch~m. Bionhwx 165.665-671. L. (1977) Eur. 29. LAZAR, T., EHRI& H., ANDLUMPER,

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