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Purification and characterization of NADPH-cytochrome c (P450) reductase from the house fly, Musca domestica
Corap. Biochen~ PhysioL, 1977, Vol. 57B, pp. 81 to 87. Pergamon Press. Printed in Great Britain
PURIFICATION AND CHARACTERIZATION OF NADPH-CYTOCHROME c (P45o) REDUCTASE FROM THE HOUSE FLY, MUSCA DOMESTICA RICHARD T. MAYER AND RUSSELLA. PROU~H The Veterinary Toxicology and Entomology Research Laboratory, Agric. Res. Serv., U.S. Department of Agriculture, College Station, TX 77840, U.S.A. and The Biochemistry Department, The University of Texas Health Science Center, Dallas, TX 75235, U.S.A. (Received 1 September 1976)
Abstract--i. NADPH-cytochrome c (P4so) reductase was purified 460-fold from microsomes of whole house flies. 2. FAD and FMN were present in about equimolar amounts. 3. The molecular weight was in the range of 65,000-70,000. 4. The 2'-AMP binding site was very weak in the house fly reductase. 5. The enzyme appeared to be a necessary component of house fly cytochrome P45o catalyzed reactions.
INTRODUCTION The flavoprotein, NADPH-cytochrome c reductase, has been extensively studied and purified from many sources. Horecker (1950) first isolated this enzyme from pig liver acetone extracts. The flavoenzyme was shown to be a component of liver microsomes (Williams & Kamin, 1962; Phillips & Langdon, 1962) and • to have a close relationship with cytochrome Paso, which is also present in the microsomal membrane (Lu & Coon, 1968; Strobel et al., 1970; Omura & Sato, 1962; Omura et al., 1965). The relationship NADPH-cytochrome c reductase has with cytochrome P450 is that it provides reducing equivalents for cytochrome P~,5o in the presence of NADPH (Ichikawa & Yamano, 1969; Strobel et al., 1970; Masters et al., 1973). This fact has prompted the use of the term "NADPH-cytochrome Paso reductase" (Masters et al., 1973). Besides reducing cytochrome c and cytochrome Paso, the enzyme can transfer electrons to such electron acceptors as 2,6-dichloropheno= lindophenol, neotetrazolium, ferricyanide and menadione (Kamin et al., 1966). The NADPH-cytochrome c (Paso) reductase present in insects has not been well studied, and many questions concerning this enzyme in insects remain unresolved. Wilson & Hodgson (1971a, b) partially purified this enzyme from the abdomens of house flies, Musca domestica L., and studied the kinetic characteristics of the enzyme with different substrates and inhibitors. Also, Capdevilla et al. (1975) partially purified NADPH-cytochrome c reductase from house flies. To date, these papers report the only attempts to study this enzyme in detail from insects. In this paper, we describe the solubilization and purification of NADPH-cytochrome c reductase from house fly microsomes. Also, we have studied the kinetic properties of the purified enzyme and compared * Mention of a commercial or proprietary product does not constitute an endorsement of this product by the USDA. C.P.B. 57/1B--F
81
its activity with purified mammalian liver microsomal NADPH-cytochrome c reductase. Data on flavin content, molecular weight estimation, and immunochemical cross-reactivity are also given. MATERIALS AND METHODS Chemicals*
NADH, NADPH, NADP+, adrenalin, 2,6-dichlorophenolindophenol (DCIP), menadione (2-methyl-l,4-naphthoquinone), 2'-AMP and cytochrome c were purchased from the Sigma Chemical Co., St. Louis, MO. Aldrin (1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro- 1,4-endoexo-5,8-dimethanonaphthalene) and dieldrin (1,2,3,4,10,10hexachloro-6,7-epoxy-1, 4,4a,5,6,7,8,8a-octahydro-l,4-endoexo-5,8-dimethanonaphthalene) were gifts of the Shell Chemical Co., San Ramon, CA. Ethoxyresorufin was prepared according to the method of Mayer et al. (1976). All other chemicals were of the highest purity available from commercial sources. Biological material House flies resistant to diazinon (O,O-diethyl O-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate) were originally obtained from Dr. F. W. Plapp, Texas A&M University, College Station, TX. The house flies carried brown-body and ochre-eye phenotypic markers. Adults were maintained on a mixture of powdered milk, sucrose, and powdered egg (80:10:10, w/w). The flies were starved overnight prior to killing for preparation of microsomes. Preparation of microsomes Microsomes were prepared from whole flies as previously described by Mayer et al. (1976). Protein content was measured by the method of Lowry et al. (1951). Preparations of microsomes were pooled and stored at - 50°C for up to 3 months before solubilization and purification without any obvious loss of NADPH-cytochrome c reductase activity. Preparation of NADPH-cytochrome c reduetase from pig, rat, and house fly NADPH-cytochrome c reductase from porcine and rat liver microsomes was prepared according to Prough & Masters (1973). The specific activity of the enzyme was
82
RICHARD T. MAYER AND RUSSELL A. PROUGH
consistently between 1,000 and 1,200 m-mol cytochrome c reduced rain-1 m-mol-1 flavin. The house fly microsomal pellets that had been pooled and stored frozen over a 3-month period were diluted to about 15rag microsomal protein per ml of 50mM Tris buffer (pH 7.7) containing 1 mM EDTA. The suspension (ca. 140 ml) was wacmed to 37°C in a shaker bath, porcine pancreatic lipase prepared according to Masters et al. (1967) was added (0.2 ml lipase/10 ml suspension was determined to be optimum for solubilization of the reductase), and the mixture was incubated for 15 min at 37°C in the shaker bath. After incubation, the mixture was cooled and centrifuged at 100,000 y for 75 min. The pellets and supernatants were separated and assayed for NADPH-cytochrome c reductase activity. The supernatant (130 ml) was added to a DEAE-cellulose column (27 x 2.5cm) previously equilibrated with 50 mM Tris, 1 mM EDTA at pH 7.7. The column was eluted with a linear gradient of KC1 (0 to 0.25 M). The fractions were assayed for NADPHcytochrome c reductase activity; fractions with high activity were pooled and concentrated in an Amicon concentrator (Amicon Corp., Lexington, MA). After concentration, the sample containing the reductase was added to a G-200 Sephadex superfine column (27 x 2.5 cm) previously equilibrated with 50 mM Tris, 1 mM EDTA at pH 7.7; the flow was adjusted to 0.5 ml/min. The fractions were assayed for activity and concentrated as before. The concentrated sample was applied to a Whatman DE-52 column (20 x 2.5 cm) equilibrated with 50 mM Tris, 1 mM EDTA at pH 7.7. The column was eluted with a linear gradient of KC1 (0-0.25 M). The different fractions were assayed and pooled as before. The pooled fractions were concentrated in a collodion bag concentrator and stored frozen at - 20°C.
Enzyme assays NADPH-cytochrome c reductase activity was measured at 25°C by the method of Masters et al. (1967). Enzymatic reduction of DCIP, menadione, potassium ferricyanide, and adrenochrome formation was also measured at 25°C in 50mM phosphate buffer (pH 7.7) containing 0.1 mM EDTA. The reaction mixtures were 1 ml (final volume); the concentrations of DCIP, menadione, potassium ferricyanide, and adrenalin were 0.1, 0.1, 1.0 and 0.5 mM, respectively. The reactions were initiated by the addition of NADPH (0.1 mM final). The millimolar extinction coefficients (cm-1 raM-1) used in the calculation of enzyme activity were 21.0 at 600 nm for DCIP (Steyn-Parve & Beinert, 1958) and 4.02 for adrenochrome formation at 480 nm (Green et al., 1965). Reduction of menadione and potassium ferricyanide was measured indirectly by following the rate of NADPH oxidation at 340 nm (Masters et al., 1965a). Aldrin epoxidase activity of house fly microsomes was determined by measuring the conversion of aldrin into dieldrin at 30°C. The reaction mixture contained 15 ml of 50 mM Tris-HC1 buffer, 150 mM KC1, 7-8 mg microsomal protein, 0.5 mM glucose-6-phosphate, 3.75 units of glucose6-phosphate dehydrogenase, and 0.2 #M aldrin. The reaction was initiated by addition of NADPH (0.25 mM final concentration). Aliquots (1 ml) were removed at 0, 5, 10 and 15 rain and mixed with 1 ml acetone to terminate the reaction. Subsequent extraction and analysis of dieldrin followed the procedure of Krieger & Wilkinson (1969). O-deethylation of ethoxyresorufin catalyzed by insect microsomai monooxygenases was measured by the method of Burke & Mayer (1974). The sensitivity of the method was increased by utilizing an excitation wavelength of 560 nm. Ethoxyresorufin was dissolved in dimethyl-sulfoxide and added to the reaction mixtures in volumes of < 5 #1; volumes of dimethylsulfoxide > 5 #1 inhibited the O-deethylation reaction. Otherwise, the results were identi-
cal to those obtained using Tween 80 or Tris buffer solutions.
Determination of flavin content Total flavin was determined spectrophotometrically by measuring the absorbance at 450 nm and using a flavin extinction coefficient of 11.7 x 103 M -1 cm -1 for that wavelength (Masters et al., 1975). FAD and FMN content was determined fluorometricaily by using the method of Faeder & Siegel (1973). Standard samples of FAD and F M N were purified by chromatography on DEAE-cellulose by using a 0.1 M potassium phosphate- (pH 6.8) eluting buffer. Fluorescence spectra were recorded with an Aminco spectrofluorometer. All absorption spectra were recorded on either Aminco DW-2 or Cary 14R spectrophotometers. Sodium dodecylsulfate-polyacrylamide gel electrophoresis Purified NADPH-cytochrome c reductase was run on 1~o sodium dodecylsulfate-polyacrylamide gel electrophoresis according to the methods of Fairbanks et al. (1971). After running, the gels were stained with Coomassie blue to demarcate the protein bands. The molecular weight of the NADPH-cytochrome c reductase was determined by comparison with proteins of known molecular weight run independently in a similar fashion. The protein standards were bovine serum albumin, glutamate dehydrogenase, fumarase, alcohol dehydrogenase and catalase. Preparation of goat-antibody against rat liver NADPHcytochrome c reductase NADPH-cytochrome c reductase was purified from hepatic microsomes of Sprague-Dawley rats with pancreatic lipase (Prough & Masters, 1973). Polyacrylamide gel electrophoresis revealed the reductase to be a single major protein band that was stable on storage at -5°C. The reductase was excised from the gel and used as the challenging antigen. Goats were given three weekly injections of 1 mg of purified reductase in Freund's Adjuvant (Difco Co.) in the nape of the neck. Blood was obtained every other week from neck veins and tested for specific antibody titer against rat liver NADPH-cytochrome c reductase by measuring the percentage of inhibition (titer) of rat liver microsomal NADPH-cytochrome c reductase activity. Sera with high antibody titer from several bleedings were pooled, and the immunoglobulins were precipitated from the serum by addition of ammonium sulfate (1.75 M), redissolved in 0.05 M potassium phosphate buffer, pH 7.7, and dialyzed against the same buffer. These globulin solutions were stored frozen. Further purification of the globulin solutions and specificity tests and results of the specificity tests were essentially the same as those reported for rabbit antibody against rat liver NADPH-cytochrome c reductase (Prough & Burke, 1975).
RESULTS
Purification The N A D P H - c y t o c h r o m e c reductase was solubilized by lipase treatment from a b o u t 1250rag of house fly microsomal protein. D u r i n g the solubilization process, the suspension changed from a light pink to a black color. Table 1 is a summary of the purification procedure, and Fig. 1 shows the elution pattern of the enzyme from the various columns used in purification. The supernatant from the lipase solubilization contained most of the N A D P H - c y t o c h r o m e c reductase with a 162% yield. The supernatant from the lipase solubilization was applied directly to a
NADPH-cytochrome c reductase from Musca domestica
83
Table 1. Purification of house fly microsomal NADPHcytochrome c reductase* Total Activity (/zmol cytochrome c reduced rain- z) NADH NADPH % yield
Preparation Microsomes Lipase pellets Lipase supernatant DEAE-cellulose column eluate G-200 Sephadex column eluate DE-52 column eluate
355.0 114.0 15.4
103.0 15.9 167.0
100.0 15.4 162.1
--
102.7
99.7
---
94.8 53.4
92.0 51.8
* The specific activity of the NADPH-cytochrome c reductase in the original microsomal suspension was 83 nmol cytochrome c reduced min- ~ mg- 1. The flavin content was 22-24 nmoles total per mg protein. DEAE cellulose column. Fractions with high reductase activity were pooled, concentrated, and applied to a Sephadex G-200 superfine column. About 92~o of the original activity was recovered in this step, but a large amount of black coloration from the lipase solubilization remained. In an attempt to remove this A. DEAE-CELLULOSE
0
O0
150 "
B. G-2OO $EPHADEX
15
t> e,i00.
I-0
:E
m a. O-5
0.0
~
0
G.D E - 5 2
20 ~
O-Z
rO
0"1
0
I0
2O
i 3O 4O 50 60 7O 8O FRACTIONNUMBER
0-0
Fig. 1. Elution profiles of NADPH-cytochrome c reductase from: (A) a DEAE-cellulose column, (B) a G-200 superfine Sephadex column, and (C) a Whatman DE-52 column. A unit corresponds to 0.0476 micromole of cytochrome c reduced per min per ml of reaction mixture (Masters et al., 1967).
'E°°-°
I
I
I
400
440
480
I
I
I
520 560 600 WAVELENGTH (nm)
I
640
0
Fig. 2. Spectrum of the oxidized house fly NADPHcytochrome c reductase. The reference cuvette contained buffer only. coloration, we applied the concentrated reductase sample to a Whatman DE-52 column. The result of this step was that most of the black-colored contaminant was lost with a little less than half of the original activity remaining. U p o n concentrating the fractions with NADPH-cytochrome c reductase activity, the enzyme solution took on a strong yellow hue, characteristic of this flavin-contalning enzyme (Horecker, 1950; Williams & Kamin, 1962; Phillips & Langdon, 1962).
Determination of flavin content of NADPH-cytochrome c reductase Some of the black-colored contaminant remained with the purified enzyme and interfered with the absorption spectrum of the oxidized enzyme. Because of this problem, we recorded the difference spectrum of the oxidized minus the reduced NADPH-cytochrome c reductase (Fig. 2, curve 1). By allowing the reduced enzyme (reference cuvette) to reoxidize, it was possible to record the difference spectrum of the halfreduced enzyme (i.e. oxidized minus half-reduced NADPH-cytochrome c reductase). Both spectra are similar to those reported for NADPH-cytochrome c reductase isolated from other sources (Masters et al., 1965b). Fluorometric determinations of the flavin content revealed that FAD and F M N were present in nearly equimolar amounts, i.e. 41 and 59~ respectively, and were comparable to the values obtained with rat liver microsomal NADPH-cytochrome c reductase (Table 2). These values are very close to those reported elsewhere for the rat, rabbit, a n d pig liver N A D P H cytochrome c reductase (Iyanagi & Mason, 1973; Masters et al., 1975). Molecular weight determinations Figure 3 shows the electrophoretic pattern of the purified house fly NADPH-cytochrome c reductase on 1~ SDS-polyacrylamide gel. Two gels run similarly with molecular weight marker proteins are shown for comparison. The protein band containing the~ house fly NADPH-cytochrome c reductase migrates approximately the same distance as bovine serum albumin, i.e. the house fly enzyme has a molecular weight between 65,000 and 70,000 daltons. Moreover, house fly NADPH-cytochrome c reductase is in the same molecular weight range as porcine liver NADPH-cytochrome c reductase purified by lipase
84
RICHARDT. MAYERAND RUSSELLA. PP,OUG8
Table 2. FAD and FMN contents of house fly microsomal and rat liver microsomal NADPH-cytoehrome c reductase* Flavoprotein source Rat FAD FMN House fly FAD FMN
% flavin ~o flavin recoveryt
55 45 41 59
Salts (KC1, 0-1 M) and pH affected enzyme activity much the same as reported previously (Wilson & Hodgson, 1971b). The pH optimum for reduction of cytochrome c was 7.8. Salts had little effect on cytochrome c reductase activity except that at KC1 concentrations >0.4 M, activity decreased slightly.
99.8 A. NADPH
71.0
55~M
* Flavin determined by the method of Faeder & Siegel (1973). t Recovery calculated by % flavin concentrated (FMN + FAD) determined fluorometrically compared > 3 with the total flavin concentration determined by absor- bance at 450 nm. The absorbance at 450 nm may have been exaggerated by the presence Of the dark colored pigment 2 formed during lipase treatment. Total flavin content of the rat and house fly reductases was 6.0 x 10-SM and I 4.2 x 10-s M, respectively. 0
solubilization (Masters & Ziegler, 1971); it is of higher estimated molecular weight than the value of 57,000 previously reported for the house fly NADPH-cytochrome c reductase prepared by isobutanol solubilization (Wilson & Hodgson, 1971a).
~
0
IIO,,uM 220~uM
1670/aM
¢
0
|
20
i
|
40
|
i
|
i
t
,
60 80 I00 I X 103 [CYT _c]
I
i
R
120
V m a x - 1 6 6 0 n m o l e s c y t . _c r e d . / m i n / n m o f e
4
flovin
Km • 23.5~uM
Kinetic analysis Porcine liver NADPH-cytochrome c reductase has been previously shown to operate through a bi bi ping-pong mechanism (Masters et al., 1965b; Segal, 1975). The series of parallel lines shown in Fig. 4 are characteristic of the ping-pong kinetic mechanism and confirm the earlier observations of Wilson & Hodgson (1971b) that the house fly reductase followed this scheme. Replotting the ordinate intercepts in Fig. 4a against NADPH concentration, Fig. 4b, allowed the determination of Vmax at infinite concentrations of NADPH and cytochrome c. The specific activity calculated from Vmax data was consistently between 1,500 and 1,600tool cytochrome c reduced min -1 mol- 1 flavin. This value was significantly higher than that reported for porcine liver NADPH-cytochrome c reductase (1,100--1,200mol cytochrome c reduced min -1 mol -~ flavin, Masters et al., 1965b). The apparent Km's for NADPH and cytochrome c were calculated as 23.5 #M and 16.6 pM, respectively.
OUTSIDE GELS UREASE BOVINE SERUM A L B U M I N CATALASE GLUTAMATE DEHYDROGENASE F UMARASE,
3 -2
I
-4
0
i
i
4
8
)
12
I
16
|
20
[NA/PH] X 104
Fig. 4. Kinetic plots of NADPH-cytochrome c reductase activity. (A) Lineweaver-Burk plots with varying NADPH and cytochrome c. (B) Secondary reciprocal plot of the Vmax values obtained in (A) versus NADPH concentration. The cuvette contained NADPH-cytochrome c reductase (7.2 x 10-s M flavin), 50 mM potassium phosphate buffer (pH 7.7), and 10-4 M EDTA. V = #mol cytochrome c reduced rain-1 nmol-z flavin.
CENTER GEL
NADPH-CYTOCHROME c REDUCTASE
ALCOHOL DEHYDROGENASE.
Fig. 3. Sodium dodecylsulfate-polyacrylamidegel electrophoresis of the purified NADPH-cytochrome c reductase (center gel).
NADPH-cytochrome c reductase from Musca domestica
Other electron acceptors NADPH-cytochrome c reductase is capable of oxidizing and reducing a number of substrates. Several activities of purified house fly and porcine liver NADPH-cytochrome c reductases with various acceptors were compared, and the results of this study are shown in Table 3. There are essentially no differences in the activities of these two enzymes toward these substrates. Although the maximal activities were identical for adrenochrome formation, we observed that with the porcine liver enzyme, there was a slight lag time (Prough & Masters, 1973); with house fly enzyme, there was no lag.
85
Table 4. Effects of inhibitors on house fly, rat liver, and pig liver microsomal NADPH-cytochrome c reductases* Enzyme source
Ki (2'-AMP) #M
Ki (NADP) /~M
640 105 145
14 10 12
House fly Rat liver Pig livert
* The assay medium contained 50 mM potassium phosphate buffer (pH 7.7), 1 x 10-4 M EDTA, 45 #M cytochrome c, 10-200 /~M NADPH, 0-1 mM 2'-AMP, and 0-0.2 mM NADP. t Inhibition constants reported for pig liver NADPHcytochrome c reductase were taken from Phillips & Langdon (1962).
Inhibitors Both NADP and T-AMP have been shown to be competitive inhibitors of pig liver NADPH-cytochrome c reductase (Phillips & Langdon, 1962); only somal systems (Ray, 1967; Mayer et al., 1976). When NADP has been reported as an inhibitor of the house the antibody was added to the reaction mixtures at fly reductase (Wilson & Hodgson, 1971b). We tested a ratio of 50 mg antibody protein to 1 mg microsomal NADP and 2'-AMP as inhibitors of the house fly protein, the O-dealkylation of ethoxyresorufin and the reductase and compared the Ki's to those obtained epoxidation of aldrin were inhibited by 75 and 50~o, with purified rat and pig liver enzymes by using the respectively. Nonspecific globulin fractions (i.e. globumethod of Dixon & Webb (1964). The results (listed lins collected from animals not challenged with the in Table 4) show that the Ki of NADP is of equal reductase) had little effect on any of the enzyme reactions at similar ratios. magnitude, whatever the source of enzyme. However, the Ki for 2'-AMP varies considerably depending on the enzyme source. The 2'-AMP is a 4- to 6-fold DISCUSSION weaker inhibitor of the house fly enzyme than it is We have purified NADPH-cytochrome c (P, so) for the rat and pig liver enzymes. Kinetic plots showed both inhibitors to be competitive in all cases. • reductase from whole house flies by utilizing a lipase solubilization technique. The purity of the prepFunction of house fly cytochrome c reductase in insect aration can be estimated by utilizing the total flavin microsomal cytochrome P , so reactions content, assuming there are 2 moles of flavin per mole We tested the function of NADPH-cytochrome c of enzyme (as is the case in NADPH-cytochrome c reduetase in insect microsomal cytochrome P45o-sup- reductase purified from other sources (Masters et al., ported reactions immunoehemically. Purified anti- 1965b) and a mol. wt of 68,000. On the basis of the body to rat liver microsomal NADPH-cytochrome c specific activity (1,290 mol m i n - 1 mol- ~ flavin) of the reductase reacts with house fly microsomal NADPH- pure enzyme, the purification factor can be estimated cytocffrome c reductase to inhibit the rate of reduc- to be about 460. This is approximately 11 times tion of cytochrome c by 80% at a ratio of 50 mg anti- greater purity than has been reported previously body protein to 1 mg microsomal protein (Fig. 5). The (Wilson & Hodgson, 1971a). Electrophoresis of the epoxidation of aldrin to dieldrin and the O-dealkyla- enzyme on sodium dodecylsulfate-acrylamide gel tion of ethoxyresorufin have both been shown to be revealed little contamination by other proteins. cytochrome P,50-catalyzed reactions in insect microTable 3. Comparison of purified house fly and pig liver NADPH-cytochrome c reductase activities* Substrate Cytochrome c DCIP Adrenalin Potassium ferricyanide Oxygen Menadione
Specific activityt House fly Pig liver 1290 510 170 1680 9.2 426
1190 523 170 1740 2.0 420
* Assay medium contained either 7.2 x 10-s M flavin of the house fly enzyme or 5 x 10-s M flavin for the pig liver enzyme, 50 mM potassium phosphate buffer (pH 7.7), 1 x 10-4 M EDTA, and 0.1 mM NADPH. Final concentrations of the substrates were 45#M cytochrome c, 0.I mM DCIP, 0.5 mM adrenalin, I mM potassium ferricyanide, and 0.1 mM menadione. t nMol of substrate oxidized or reduced min- 1 nmol- t flavin.
,oof A-NADPH CYTOCHROME ~ REDUCTASE r-I-ALDRIN EPOXIOASE O-ETHOXYRESORUFIN D E A L K Y L A S E OPEN - IMMUNE SOLID-NON-IMMUNE
4o~
I
o o
I
;o
I
go
I
go
I
do
ANTIBODY/MICROSOMES
Fig. 5. Response of house fly NADPH-cytochrome c reductase to increased titers of antibody to the rat liver NADPH-cytochrome c reductase. Assay conditions were as described in Methods.
86
RICHARD T. MAYER AND RUSSELLA. PROUGH
The yellow color of the pure enzyme solution indicated flavin content. Fluorometric analysis revealed that the enzyme contained nearly equimolar amounts of F M N and FAD. This fact was not surprising since an identical result has been reported for enzyme preparations from the rat and pig (Iyanagi & Mason, 1973; Masters et al., 1975). Moreover, Wilson & Hodgson (1971a) found that when either FAD or F M N were added back to the apoenzyme, partial activity was restored. The NADPH-cytochrome c reductase isolated from house flies is similar in more ways to the N A D P H cytochrome c reductase isolated from mammalian liver than just in flavin content. The estimated molecular weight, 68,000, is approximately the same as that reported by Masters & Ziegler (1971) for mammalian liver enzyme. Data reported here show the activities of the two enzymes to be very nearly the same for every substrate tried. Kinetic analysis indicated that house fly NADPH-cytochrome c reductase followed the same sort of ping-pong mechanism previously reported for the pig liver enzyme (Masters et al., 1965b). Although mammalian liver and house fly N A D P H cytochrome c reductase appear to be very similar, there are major differences. Our studies with the inhibitor T - A M P indicate that 2'-AMP is an extremely poor inhibitor of the house fly enzyme. We believe this indicates that the T - A M P binding site is almost nonexistent in the house fly enzyme. Also, immunochemical data (unreported results) suggest some differences between the various NADPH-cytochrome c reductases. For example, we were unable to elicit a reaction between the antibody to the pig liver enzyme and the house fly enzyme; the antibody to the rat liver NADPH-cytochrome c reductase was capable of inhibiting reduction by the house fly enzyme by
REFERENCES
which is preferentially inducible by 3-methylcholanthrene. Drug Metab. Dispos. 2, 583-588. CAPDEVmLA J., Ar~IAD N. & AGOSIN M. (1975) Soluble cytochrome P-450 from housefly microsomes: Partial purification and characterization of two hemoprotein forms. J. biol. Chem. 250, 1048-1060. DIxoN M. & WEBB E. C. (1964) Enzymes. pp. 327-331. Academic Press, New York. FAEDm~E. J. & SIEGELL. M. (1973) A rapid micromethod for determination of FMN and FAD in mixtures. Anal. Biochem. 53, 332-336. FAmaANKS G., STECKT. L. & WALLACnD. F. H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2606--2617. GREEN S., MAZAR A. & S H ~ E. (1956) Mechanism of the catalytic oxidation of adrenalin by ferritin. J. biol. Chem. 220, 237-255. HOR.ECrd~R B. L. (1950) Triphosphopyridine nucleotidecytochrome c reductase in liver. J. biol. Chem. 183, 593-600. ICmKAWAY. & YAMANOT. (1969) Studies on the microsoreal reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase from rabbit liver. J. Biochem., Tokyo 66, 351-359. IYANAGIT. & MASONH. S. (1973) Some properties of hepatic reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase. Biochemistry 12, 2297-2308. KAMI~ H., MASTERS B. S. S. & GmSON Q. H. (1966) NADPH-cytochrome c oxidoreductase. In Flavins and Flavoproteins (Edited by SLATER E. C.). pp. 306-324. Elsevier, New York. KRmO~ R. I. & WILKINSONC. F. (1969) Microsomal mixed-function oxidases in insects. I Localization and properties of an enzyme system effecting aldrin epoxidation in larvae of the southern armyworm (Prodenia eridania). Biochem. Pharmacol. 18, 1403-1415. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. Lu A. Y. & COON M. J. (1968) Role of hemoprotein P-450 in fatty acid o-hydroxylation in a soluble enzyme system from liver microsomes. J. biol. Chem. 243, 1331-1332. MASTERS B. S. S. & Zr~GLEr, D. M. (1971) The distinct nature and function of NADPH-cytochrome c reductase and the NADPH-dependent mixed-function amine oxidase of porcine liver microsomes. Arch. Biochem. Biophys. 145, 358-364. MASTERs B. S. S., BIL~ORA M. H., KAMrN H. & GIBSON Q. H. (1965a) The mechanism of 1- and 2-electron transfers catalyzed by reduced triphosphopyridine nucleotide-cytochrome c reductase. J. biol. Chem. 240, 4081-4088. MASTERSB. S. S., KAM~NH., GIBSON Q. H. & WILLIAMS C. H. (1965b) Studies on the mechanisms of microsomal triphosphopyridine nucleotide-cytochrome c reductase. J. biol. Chem. 240, 921-930. MASTERSB. S. S., NELSON E. B., SCHACTERB. A., BARON J. & ISAACSON E. L. (1973) NADPH-cytochrome c reduetase and its role in microsomal cytochrome P-450-dependent reactions. Druo Metab. Dispos. 1, 121128. MASTERS B. S. S., PROUGH R. A. & KAMIN H. (1975) Properties of the stable aerobic and anaerobic halfreduced states of NADPH-cytochrome c reductase. Biochemistry 14, 607-613. MASTERS B. S. S., WILLIAMSC. H. & KAmN H. (1967) The preparation and properties of microsomal TPNHcytochrome c reductase from pig liver, pp. 565-573. In Methods in Enzymolooy (Edited by ESTABROOKR. W.
BURKEM. D. & MAYERR. T. (1974) Ethoxyresorufin: Direct fluorimetric assay of a microsomal O-dealkylation
R. A. (1976) The use of methoxyresorufin as a substrate
8070. By means of immunochemical experiments, we provided information as to the function of N A D P H cytochrome c reductase in house fly microsomal cytochrome P45o-catalyzed reactions. NADPH-cytochrome c reductase has been shown to be an obligatory component in cytochrome P45o catalyzed reactions (Ichikawa & Yamano, 1969; Strobel et al., 1970; Masters et al., 1973). However, its presence in insect microsomal cytochrome P , so-catalyzed reactions has never been shown to be necessary. The experiments performed here indicate that antibody to the rat liver NADPH-cytochrome c reductase also inhibited the house fly reductase in microsomal suspensions. Addition of the antibody to reactions catalyzed by house fly microsomal cytochrome P45o significantly inhibited these reactions as well. We conclude from these data that the house fly cytochrome c (Paso) reductase is an obligatory component in cytochrome P,socatalyzed reactions and that it functions to transfer electrons to cytochrome P4so. Acknowledgement--This work was supported in part by grant BC153 from the American Cancer Society.
& PULLMANM.) Vol. 10. Academic Press, New York. MAYER R. T., JERMYN J. W., BURKE M. D. & PROUGH
NADPH-cytochrome c reductase from Musca domestica for the direct fluorometric assay of insect microsomal O-dealkylases. Pestic. Bioehem. Physiol. (In press.) OMURA T. & SATO R. (1962) A new cytochrome in liver microsomes. J. biol. Chem. 237, 1375. OMURAT., SATOR., COOPERD. Y., ROSENTHALO. & ESTABROOK R. W. (1965) Function of cytochrome P-450 of microsomes. Fedn Proc. Fedn Am. Soc. exp. Biol. 24, 1181-1189. PHILLIPS A. H. & LANGDONR. G. (1962) Hepatic triph0sphopyridine nucleotide-cytochrome c reductase: Isolation, characterization, and kinetic studies, d. biol. Chem. 237, 2652-2660. PROUGH R. A. & BURKEM. n. (1975) The role of NADPHcytochrome c reductase in microsomal hydroxylation reactions. Arch. Biochem. Biophys. 170, 160-168. PROUGH R. A. & MASTERSB. S. S. (1973) Studies on the NADPH oxidase reaction of NADPH-cytochrome c reductase. I. The role of superoxide anion. Ann. N. Y Aead. Sci. 212, 89-93. RAY J. W. (1967) The epoxidation of aldrin by house fly microsomes and its inhibition by carbon monoxide. Biochem. Pharmacol. 16, 99-107.
87
SEGAL I. H. (1975) Enzyme Kinetics. pp. 606q512. John Wiley & Sons, New York. STEYN-PARVE E. P. & BEINERT H. (1958) On the - mechanism of dehydrogenation of fatty acyl derivatives of coenzyme A. VI. Isolation and properties of stable enzyme-substrate complexes. J. biol. Chem. 233, 843-852. STROBELH. W., LU A. Y., HEIOEMAJ. & COON M. J. (1970) Phosphatidylcholine requirement in enzymatic reduction of hemoprotein P-450 and in fatty acid, hydrocarbon, and drug hydroxylation. J. biol. Chem. 245, 4851-4854. WILLIAMSC. H. & KAMIN H. (1962) Microsomal triphosphopyridine nucleotide-cytochrome c reductase: Isolation, characterization, and kinetic studies. J. biol. Chem. 237, 587-595. WILSON T. G. & HODGSON E. (1971a) Microsomal NADPH-cytochrome c reductase from the housefly, Musca domestica: Solubilization and purification. Insect Biochem. 1, 19-26. WILSON T. G. & HODGSON E. (1971b) Microsomal NADPH-cytochrome c reductase from the housefly, Musca domestica: Properties of the purified enzyme. Insect Biochem. 1, 171-180.
PURIFICATION AND CHARACTERIZATION OF NADPH-CYTOCHROME c (P45o) REDUCTASE FROM THE HOUSE FLY, MUSCA DOMESTICA RICHARD T. MAYER AND RUSSELLA. PROU~H The Veterinary Toxicology and Entomology Research Laboratory, Agric. Res. Serv., U.S. Department of Agriculture, College Station, TX 77840, U.S.A. and The Biochemistry Department, The University of Texas Health Science Center, Dallas, TX 75235, U.S.A. (Received 1 September 1976)
Abstract--i. NADPH-cytochrome c (P4so) reductase was purified 460-fold from microsomes of whole house flies. 2. FAD and FMN were present in about equimolar amounts. 3. The molecular weight was in the range of 65,000-70,000. 4. The 2'-AMP binding site was very weak in the house fly reductase. 5. The enzyme appeared to be a necessary component of house fly cytochrome P45o catalyzed reactions.
INTRODUCTION The flavoprotein, NADPH-cytochrome c reductase, has been extensively studied and purified from many sources. Horecker (1950) first isolated this enzyme from pig liver acetone extracts. The flavoenzyme was shown to be a component of liver microsomes (Williams & Kamin, 1962; Phillips & Langdon, 1962) and • to have a close relationship with cytochrome Paso, which is also present in the microsomal membrane (Lu & Coon, 1968; Strobel et al., 1970; Omura & Sato, 1962; Omura et al., 1965). The relationship NADPH-cytochrome c reductase has with cytochrome P450 is that it provides reducing equivalents for cytochrome P~,5o in the presence of NADPH (Ichikawa & Yamano, 1969; Strobel et al., 1970; Masters et al., 1973). This fact has prompted the use of the term "NADPH-cytochrome Paso reductase" (Masters et al., 1973). Besides reducing cytochrome c and cytochrome Paso, the enzyme can transfer electrons to such electron acceptors as 2,6-dichloropheno= lindophenol, neotetrazolium, ferricyanide and menadione (Kamin et al., 1966). The NADPH-cytochrome c (Paso) reductase present in insects has not been well studied, and many questions concerning this enzyme in insects remain unresolved. Wilson & Hodgson (1971a, b) partially purified this enzyme from the abdomens of house flies, Musca domestica L., and studied the kinetic characteristics of the enzyme with different substrates and inhibitors. Also, Capdevilla et al. (1975) partially purified NADPH-cytochrome c reductase from house flies. To date, these papers report the only attempts to study this enzyme in detail from insects. In this paper, we describe the solubilization and purification of NADPH-cytochrome c reductase from house fly microsomes. Also, we have studied the kinetic properties of the purified enzyme and compared * Mention of a commercial or proprietary product does not constitute an endorsement of this product by the USDA. C.P.B. 57/1B--F
81
its activity with purified mammalian liver microsomal NADPH-cytochrome c reductase. Data on flavin content, molecular weight estimation, and immunochemical cross-reactivity are also given. MATERIALS AND METHODS Chemicals*
NADH, NADPH, NADP+, adrenalin, 2,6-dichlorophenolindophenol (DCIP), menadione (2-methyl-l,4-naphthoquinone), 2'-AMP and cytochrome c were purchased from the Sigma Chemical Co., St. Louis, MO. Aldrin (1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro- 1,4-endoexo-5,8-dimethanonaphthalene) and dieldrin (1,2,3,4,10,10hexachloro-6,7-epoxy-1, 4,4a,5,6,7,8,8a-octahydro-l,4-endoexo-5,8-dimethanonaphthalene) were gifts of the Shell Chemical Co., San Ramon, CA. Ethoxyresorufin was prepared according to the method of Mayer et al. (1976). All other chemicals were of the highest purity available from commercial sources. Biological material House flies resistant to diazinon (O,O-diethyl O-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate) were originally obtained from Dr. F. W. Plapp, Texas A&M University, College Station, TX. The house flies carried brown-body and ochre-eye phenotypic markers. Adults were maintained on a mixture of powdered milk, sucrose, and powdered egg (80:10:10, w/w). The flies were starved overnight prior to killing for preparation of microsomes. Preparation of microsomes Microsomes were prepared from whole flies as previously described by Mayer et al. (1976). Protein content was measured by the method of Lowry et al. (1951). Preparations of microsomes were pooled and stored at - 50°C for up to 3 months before solubilization and purification without any obvious loss of NADPH-cytochrome c reductase activity. Preparation of NADPH-cytochrome c reduetase from pig, rat, and house fly NADPH-cytochrome c reductase from porcine and rat liver microsomes was prepared according to Prough & Masters (1973). The specific activity of the enzyme was
82
RICHARD T. MAYER AND RUSSELL A. PROUGH
consistently between 1,000 and 1,200 m-mol cytochrome c reduced rain-1 m-mol-1 flavin. The house fly microsomal pellets that had been pooled and stored frozen over a 3-month period were diluted to about 15rag microsomal protein per ml of 50mM Tris buffer (pH 7.7) containing 1 mM EDTA. The suspension (ca. 140 ml) was wacmed to 37°C in a shaker bath, porcine pancreatic lipase prepared according to Masters et al. (1967) was added (0.2 ml lipase/10 ml suspension was determined to be optimum for solubilization of the reductase), and the mixture was incubated for 15 min at 37°C in the shaker bath. After incubation, the mixture was cooled and centrifuged at 100,000 y for 75 min. The pellets and supernatants were separated and assayed for NADPH-cytochrome c reductase activity. The supernatant (130 ml) was added to a DEAE-cellulose column (27 x 2.5cm) previously equilibrated with 50 mM Tris, 1 mM EDTA at pH 7.7. The column was eluted with a linear gradient of KC1 (0 to 0.25 M). The fractions were assayed for NADPHcytochrome c reductase activity; fractions with high activity were pooled and concentrated in an Amicon concentrator (Amicon Corp., Lexington, MA). After concentration, the sample containing the reductase was added to a G-200 Sephadex superfine column (27 x 2.5 cm) previously equilibrated with 50 mM Tris, 1 mM EDTA at pH 7.7; the flow was adjusted to 0.5 ml/min. The fractions were assayed for activity and concentrated as before. The concentrated sample was applied to a Whatman DE-52 column (20 x 2.5 cm) equilibrated with 50 mM Tris, 1 mM EDTA at pH 7.7. The column was eluted with a linear gradient of KC1 (0-0.25 M). The different fractions were assayed and pooled as before. The pooled fractions were concentrated in a collodion bag concentrator and stored frozen at - 20°C.
Enzyme assays NADPH-cytochrome c reductase activity was measured at 25°C by the method of Masters et al. (1967). Enzymatic reduction of DCIP, menadione, potassium ferricyanide, and adrenochrome formation was also measured at 25°C in 50mM phosphate buffer (pH 7.7) containing 0.1 mM EDTA. The reaction mixtures were 1 ml (final volume); the concentrations of DCIP, menadione, potassium ferricyanide, and adrenalin were 0.1, 0.1, 1.0 and 0.5 mM, respectively. The reactions were initiated by the addition of NADPH (0.1 mM final). The millimolar extinction coefficients (cm-1 raM-1) used in the calculation of enzyme activity were 21.0 at 600 nm for DCIP (Steyn-Parve & Beinert, 1958) and 4.02 for adrenochrome formation at 480 nm (Green et al., 1965). Reduction of menadione and potassium ferricyanide was measured indirectly by following the rate of NADPH oxidation at 340 nm (Masters et al., 1965a). Aldrin epoxidase activity of house fly microsomes was determined by measuring the conversion of aldrin into dieldrin at 30°C. The reaction mixture contained 15 ml of 50 mM Tris-HC1 buffer, 150 mM KC1, 7-8 mg microsomal protein, 0.5 mM glucose-6-phosphate, 3.75 units of glucose6-phosphate dehydrogenase, and 0.2 #M aldrin. The reaction was initiated by addition of NADPH (0.25 mM final concentration). Aliquots (1 ml) were removed at 0, 5, 10 and 15 rain and mixed with 1 ml acetone to terminate the reaction. Subsequent extraction and analysis of dieldrin followed the procedure of Krieger & Wilkinson (1969). O-deethylation of ethoxyresorufin catalyzed by insect microsomai monooxygenases was measured by the method of Burke & Mayer (1974). The sensitivity of the method was increased by utilizing an excitation wavelength of 560 nm. Ethoxyresorufin was dissolved in dimethyl-sulfoxide and added to the reaction mixtures in volumes of < 5 #1; volumes of dimethylsulfoxide > 5 #1 inhibited the O-deethylation reaction. Otherwise, the results were identi-
cal to those obtained using Tween 80 or Tris buffer solutions.
Determination of flavin content Total flavin was determined spectrophotometrically by measuring the absorbance at 450 nm and using a flavin extinction coefficient of 11.7 x 103 M -1 cm -1 for that wavelength (Masters et al., 1975). FAD and FMN content was determined fluorometricaily by using the method of Faeder & Siegel (1973). Standard samples of FAD and F M N were purified by chromatography on DEAE-cellulose by using a 0.1 M potassium phosphate- (pH 6.8) eluting buffer. Fluorescence spectra were recorded with an Aminco spectrofluorometer. All absorption spectra were recorded on either Aminco DW-2 or Cary 14R spectrophotometers. Sodium dodecylsulfate-polyacrylamide gel electrophoresis Purified NADPH-cytochrome c reductase was run on 1~o sodium dodecylsulfate-polyacrylamide gel electrophoresis according to the methods of Fairbanks et al. (1971). After running, the gels were stained with Coomassie blue to demarcate the protein bands. The molecular weight of the NADPH-cytochrome c reductase was determined by comparison with proteins of known molecular weight run independently in a similar fashion. The protein standards were bovine serum albumin, glutamate dehydrogenase, fumarase, alcohol dehydrogenase and catalase. Preparation of goat-antibody against rat liver NADPHcytochrome c reductase NADPH-cytochrome c reductase was purified from hepatic microsomes of Sprague-Dawley rats with pancreatic lipase (Prough & Masters, 1973). Polyacrylamide gel electrophoresis revealed the reductase to be a single major protein band that was stable on storage at -5°C. The reductase was excised from the gel and used as the challenging antigen. Goats were given three weekly injections of 1 mg of purified reductase in Freund's Adjuvant (Difco Co.) in the nape of the neck. Blood was obtained every other week from neck veins and tested for specific antibody titer against rat liver NADPH-cytochrome c reductase by measuring the percentage of inhibition (titer) of rat liver microsomal NADPH-cytochrome c reductase activity. Sera with high antibody titer from several bleedings were pooled, and the immunoglobulins were precipitated from the serum by addition of ammonium sulfate (1.75 M), redissolved in 0.05 M potassium phosphate buffer, pH 7.7, and dialyzed against the same buffer. These globulin solutions were stored frozen. Further purification of the globulin solutions and specificity tests and results of the specificity tests were essentially the same as those reported for rabbit antibody against rat liver NADPH-cytochrome c reductase (Prough & Burke, 1975).
RESULTS
Purification The N A D P H - c y t o c h r o m e c reductase was solubilized by lipase treatment from a b o u t 1250rag of house fly microsomal protein. D u r i n g the solubilization process, the suspension changed from a light pink to a black color. Table 1 is a summary of the purification procedure, and Fig. 1 shows the elution pattern of the enzyme from the various columns used in purification. The supernatant from the lipase solubilization contained most of the N A D P H - c y t o c h r o m e c reductase with a 162% yield. The supernatant from the lipase solubilization was applied directly to a
NADPH-cytochrome c reductase from Musca domestica
83
Table 1. Purification of house fly microsomal NADPHcytochrome c reductase* Total Activity (/zmol cytochrome c reduced rain- z) NADH NADPH % yield
Preparation Microsomes Lipase pellets Lipase supernatant DEAE-cellulose column eluate G-200 Sephadex column eluate DE-52 column eluate
355.0 114.0 15.4
103.0 15.9 167.0
100.0 15.4 162.1
--
102.7
99.7
---
94.8 53.4
92.0 51.8
* The specific activity of the NADPH-cytochrome c reductase in the original microsomal suspension was 83 nmol cytochrome c reduced min- ~ mg- 1. The flavin content was 22-24 nmoles total per mg protein. DEAE cellulose column. Fractions with high reductase activity were pooled, concentrated, and applied to a Sephadex G-200 superfine column. About 92~o of the original activity was recovered in this step, but a large amount of black coloration from the lipase solubilization remained. In an attempt to remove this A. DEAE-CELLULOSE
0
O0
150 "
B. G-2OO $EPHADEX
15
t> e,i00.
I-0
:E
m a. O-5
0.0
~
0
G.D E - 5 2
20 ~
O-Z
rO
0"1
0
I0
2O
i 3O 4O 50 60 7O 8O FRACTIONNUMBER
0-0
Fig. 1. Elution profiles of NADPH-cytochrome c reductase from: (A) a DEAE-cellulose column, (B) a G-200 superfine Sephadex column, and (C) a Whatman DE-52 column. A unit corresponds to 0.0476 micromole of cytochrome c reduced per min per ml of reaction mixture (Masters et al., 1967).
'E°°-°
I
I
I
400
440
480
I
I
I
520 560 600 WAVELENGTH (nm)
I
640
0
Fig. 2. Spectrum of the oxidized house fly NADPHcytochrome c reductase. The reference cuvette contained buffer only. coloration, we applied the concentrated reductase sample to a Whatman DE-52 column. The result of this step was that most of the black-colored contaminant was lost with a little less than half of the original activity remaining. U p o n concentrating the fractions with NADPH-cytochrome c reductase activity, the enzyme solution took on a strong yellow hue, characteristic of this flavin-contalning enzyme (Horecker, 1950; Williams & Kamin, 1962; Phillips & Langdon, 1962).
Determination of flavin content of NADPH-cytochrome c reductase Some of the black-colored contaminant remained with the purified enzyme and interfered with the absorption spectrum of the oxidized enzyme. Because of this problem, we recorded the difference spectrum of the oxidized minus the reduced NADPH-cytochrome c reductase (Fig. 2, curve 1). By allowing the reduced enzyme (reference cuvette) to reoxidize, it was possible to record the difference spectrum of the halfreduced enzyme (i.e. oxidized minus half-reduced NADPH-cytochrome c reductase). Both spectra are similar to those reported for NADPH-cytochrome c reductase isolated from other sources (Masters et al., 1965b). Fluorometric determinations of the flavin content revealed that FAD and F M N were present in nearly equimolar amounts, i.e. 41 and 59~ respectively, and were comparable to the values obtained with rat liver microsomal NADPH-cytochrome c reductase (Table 2). These values are very close to those reported elsewhere for the rat, rabbit, a n d pig liver N A D P H cytochrome c reductase (Iyanagi & Mason, 1973; Masters et al., 1975). Molecular weight determinations Figure 3 shows the electrophoretic pattern of the purified house fly NADPH-cytochrome c reductase on 1~ SDS-polyacrylamide gel. Two gels run similarly with molecular weight marker proteins are shown for comparison. The protein band containing the~ house fly NADPH-cytochrome c reductase migrates approximately the same distance as bovine serum albumin, i.e. the house fly enzyme has a molecular weight between 65,000 and 70,000 daltons. Moreover, house fly NADPH-cytochrome c reductase is in the same molecular weight range as porcine liver NADPH-cytochrome c reductase purified by lipase
84
RICHARDT. MAYERAND RUSSELLA. PP,OUG8
Table 2. FAD and FMN contents of house fly microsomal and rat liver microsomal NADPH-cytoehrome c reductase* Flavoprotein source Rat FAD FMN House fly FAD FMN
% flavin ~o flavin recoveryt
55 45 41 59
Salts (KC1, 0-1 M) and pH affected enzyme activity much the same as reported previously (Wilson & Hodgson, 1971b). The pH optimum for reduction of cytochrome c was 7.8. Salts had little effect on cytochrome c reductase activity except that at KC1 concentrations >0.4 M, activity decreased slightly.
99.8 A. NADPH
71.0
55~M
* Flavin determined by the method of Faeder & Siegel (1973). t Recovery calculated by % flavin concentrated (FMN + FAD) determined fluorometrically compared > 3 with the total flavin concentration determined by absor- bance at 450 nm. The absorbance at 450 nm may have been exaggerated by the presence Of the dark colored pigment 2 formed during lipase treatment. Total flavin content of the rat and house fly reductases was 6.0 x 10-SM and I 4.2 x 10-s M, respectively. 0
solubilization (Masters & Ziegler, 1971); it is of higher estimated molecular weight than the value of 57,000 previously reported for the house fly NADPH-cytochrome c reductase prepared by isobutanol solubilization (Wilson & Hodgson, 1971a).
~
0
IIO,,uM 220~uM
1670/aM
¢
0
|
20
i
|
40
|
i
|
i
t
,
60 80 I00 I X 103 [CYT _c]
I
i
R
120
V m a x - 1 6 6 0 n m o l e s c y t . _c r e d . / m i n / n m o f e
4
flovin
Km • 23.5~uM
Kinetic analysis Porcine liver NADPH-cytochrome c reductase has been previously shown to operate through a bi bi ping-pong mechanism (Masters et al., 1965b; Segal, 1975). The series of parallel lines shown in Fig. 4 are characteristic of the ping-pong kinetic mechanism and confirm the earlier observations of Wilson & Hodgson (1971b) that the house fly reductase followed this scheme. Replotting the ordinate intercepts in Fig. 4a against NADPH concentration, Fig. 4b, allowed the determination of Vmax at infinite concentrations of NADPH and cytochrome c. The specific activity calculated from Vmax data was consistently between 1,500 and 1,600tool cytochrome c reduced min -1 mol- 1 flavin. This value was significantly higher than that reported for porcine liver NADPH-cytochrome c reductase (1,100--1,200mol cytochrome c reduced min -1 mol -~ flavin, Masters et al., 1965b). The apparent Km's for NADPH and cytochrome c were calculated as 23.5 #M and 16.6 pM, respectively.
OUTSIDE GELS UREASE BOVINE SERUM A L B U M I N CATALASE GLUTAMATE DEHYDROGENASE F UMARASE,
3 -2
I
-4
0
i
i
4
8
)
12
I
16
|
20
[NA/PH] X 104
Fig. 4. Kinetic plots of NADPH-cytochrome c reductase activity. (A) Lineweaver-Burk plots with varying NADPH and cytochrome c. (B) Secondary reciprocal plot of the Vmax values obtained in (A) versus NADPH concentration. The cuvette contained NADPH-cytochrome c reductase (7.2 x 10-s M flavin), 50 mM potassium phosphate buffer (pH 7.7), and 10-4 M EDTA. V = #mol cytochrome c reduced rain-1 nmol-z flavin.
CENTER GEL
NADPH-CYTOCHROME c REDUCTASE
ALCOHOL DEHYDROGENASE.
Fig. 3. Sodium dodecylsulfate-polyacrylamidegel electrophoresis of the purified NADPH-cytochrome c reductase (center gel).
NADPH-cytochrome c reductase from Musca domestica
Other electron acceptors NADPH-cytochrome c reductase is capable of oxidizing and reducing a number of substrates. Several activities of purified house fly and porcine liver NADPH-cytochrome c reductases with various acceptors were compared, and the results of this study are shown in Table 3. There are essentially no differences in the activities of these two enzymes toward these substrates. Although the maximal activities were identical for adrenochrome formation, we observed that with the porcine liver enzyme, there was a slight lag time (Prough & Masters, 1973); with house fly enzyme, there was no lag.
85
Table 4. Effects of inhibitors on house fly, rat liver, and pig liver microsomal NADPH-cytochrome c reductases* Enzyme source
Ki (2'-AMP) #M
Ki (NADP) /~M
640 105 145
14 10 12
House fly Rat liver Pig livert
* The assay medium contained 50 mM potassium phosphate buffer (pH 7.7), 1 x 10-4 M EDTA, 45 #M cytochrome c, 10-200 /~M NADPH, 0-1 mM 2'-AMP, and 0-0.2 mM NADP. t Inhibition constants reported for pig liver NADPHcytochrome c reductase were taken from Phillips & Langdon (1962).
Inhibitors Both NADP and T-AMP have been shown to be competitive inhibitors of pig liver NADPH-cytochrome c reductase (Phillips & Langdon, 1962); only somal systems (Ray, 1967; Mayer et al., 1976). When NADP has been reported as an inhibitor of the house the antibody was added to the reaction mixtures at fly reductase (Wilson & Hodgson, 1971b). We tested a ratio of 50 mg antibody protein to 1 mg microsomal NADP and 2'-AMP as inhibitors of the house fly protein, the O-dealkylation of ethoxyresorufin and the reductase and compared the Ki's to those obtained epoxidation of aldrin were inhibited by 75 and 50~o, with purified rat and pig liver enzymes by using the respectively. Nonspecific globulin fractions (i.e. globumethod of Dixon & Webb (1964). The results (listed lins collected from animals not challenged with the in Table 4) show that the Ki of NADP is of equal reductase) had little effect on any of the enzyme reactions at similar ratios. magnitude, whatever the source of enzyme. However, the Ki for 2'-AMP varies considerably depending on the enzyme source. The 2'-AMP is a 4- to 6-fold DISCUSSION weaker inhibitor of the house fly enzyme than it is We have purified NADPH-cytochrome c (P, so) for the rat and pig liver enzymes. Kinetic plots showed both inhibitors to be competitive in all cases. • reductase from whole house flies by utilizing a lipase solubilization technique. The purity of the prepFunction of house fly cytochrome c reductase in insect aration can be estimated by utilizing the total flavin microsomal cytochrome P , so reactions content, assuming there are 2 moles of flavin per mole We tested the function of NADPH-cytochrome c of enzyme (as is the case in NADPH-cytochrome c reduetase in insect microsomal cytochrome P45o-sup- reductase purified from other sources (Masters et al., ported reactions immunoehemically. Purified anti- 1965b) and a mol. wt of 68,000. On the basis of the body to rat liver microsomal NADPH-cytochrome c specific activity (1,290 mol m i n - 1 mol- ~ flavin) of the reductase reacts with house fly microsomal NADPH- pure enzyme, the purification factor can be estimated cytocffrome c reductase to inhibit the rate of reduc- to be about 460. This is approximately 11 times tion of cytochrome c by 80% at a ratio of 50 mg anti- greater purity than has been reported previously body protein to 1 mg microsomal protein (Fig. 5). The (Wilson & Hodgson, 1971a). Electrophoresis of the epoxidation of aldrin to dieldrin and the O-dealkyla- enzyme on sodium dodecylsulfate-acrylamide gel tion of ethoxyresorufin have both been shown to be revealed little contamination by other proteins. cytochrome P,50-catalyzed reactions in insect microTable 3. Comparison of purified house fly and pig liver NADPH-cytochrome c reductase activities* Substrate Cytochrome c DCIP Adrenalin Potassium ferricyanide Oxygen Menadione
Specific activityt House fly Pig liver 1290 510 170 1680 9.2 426
1190 523 170 1740 2.0 420
* Assay medium contained either 7.2 x 10-s M flavin of the house fly enzyme or 5 x 10-s M flavin for the pig liver enzyme, 50 mM potassium phosphate buffer (pH 7.7), 1 x 10-4 M EDTA, and 0.1 mM NADPH. Final concentrations of the substrates were 45#M cytochrome c, 0.I mM DCIP, 0.5 mM adrenalin, I mM potassium ferricyanide, and 0.1 mM menadione. t nMol of substrate oxidized or reduced min- 1 nmol- t flavin.
,oof A-NADPH CYTOCHROME ~ REDUCTASE r-I-ALDRIN EPOXIOASE O-ETHOXYRESORUFIN D E A L K Y L A S E OPEN - IMMUNE SOLID-NON-IMMUNE
4o~
I
o o
I
;o
I
go
I
go
I
do
ANTIBODY/MICROSOMES
Fig. 5. Response of house fly NADPH-cytochrome c reductase to increased titers of antibody to the rat liver NADPH-cytochrome c reductase. Assay conditions were as described in Methods.
86
RICHARD T. MAYER AND RUSSELLA. PROUGH
The yellow color of the pure enzyme solution indicated flavin content. Fluorometric analysis revealed that the enzyme contained nearly equimolar amounts of F M N and FAD. This fact was not surprising since an identical result has been reported for enzyme preparations from the rat and pig (Iyanagi & Mason, 1973; Masters et al., 1975). Moreover, Wilson & Hodgson (1971a) found that when either FAD or F M N were added back to the apoenzyme, partial activity was restored. The NADPH-cytochrome c reductase isolated from house flies is similar in more ways to the N A D P H cytochrome c reductase isolated from mammalian liver than just in flavin content. The estimated molecular weight, 68,000, is approximately the same as that reported by Masters & Ziegler (1971) for mammalian liver enzyme. Data reported here show the activities of the two enzymes to be very nearly the same for every substrate tried. Kinetic analysis indicated that house fly NADPH-cytochrome c reductase followed the same sort of ping-pong mechanism previously reported for the pig liver enzyme (Masters et al., 1965b). Although mammalian liver and house fly N A D P H cytochrome c reductase appear to be very similar, there are major differences. Our studies with the inhibitor T - A M P indicate that 2'-AMP is an extremely poor inhibitor of the house fly enzyme. We believe this indicates that the T - A M P binding site is almost nonexistent in the house fly enzyme. Also, immunochemical data (unreported results) suggest some differences between the various NADPH-cytochrome c reductases. For example, we were unable to elicit a reaction between the antibody to the pig liver enzyme and the house fly enzyme; the antibody to the rat liver NADPH-cytochrome c reductase was capable of inhibiting reduction by the house fly enzyme by
REFERENCES
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8070. By means of immunochemical experiments, we provided information as to the function of N A D P H cytochrome c reductase in house fly microsomal cytochrome P45o-catalyzed reactions. NADPH-cytochrome c reductase has been shown to be an obligatory component in cytochrome P45o catalyzed reactions (Ichikawa & Yamano, 1969; Strobel et al., 1970; Masters et al., 1973). However, its presence in insect microsomal cytochrome P , so-catalyzed reactions has never been shown to be necessary. The experiments performed here indicate that antibody to the rat liver NADPH-cytochrome c reductase also inhibited the house fly reductase in microsomal suspensions. Addition of the antibody to reactions catalyzed by house fly microsomal cytochrome P45o significantly inhibited these reactions as well. We conclude from these data that the house fly cytochrome c (Paso) reductase is an obligatory component in cytochrome P,socatalyzed reactions and that it functions to transfer electrons to cytochrome P4so. Acknowledgement--This work was supported in part by grant BC153 from the American Cancer Society.
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