Insect Bioehem., z97z, I, 171-180. [Scientechnica (Publishers) Ltd.] z7x

MICROSOMAL NADPH-CYTOCHROME C REDUCTASE FROM

THE HOUSEFLY, M U S C A DOMESTICA: PROPERTIES

OF THE PURIFIED ENZYME*

T. G. WILSON AND ERNEST HODGSON

Department of Entomology, North Carolina State University, Raleigh, North Carolina 27607, U.S.A.

(Received I2 Dec., 197o)

ABSTRACT

The characteristics of purified NADPH-cytochrome c reductase (E.C.x.2.6.3) from housefly microsomes have been studied. Like the mammalian reductase, the housefly reductase is specific for NADPH, utilizes 2,6-dichlorophenol- indophenol and ferricyanide as electron acceptor substrates, has a ping-pong reaction mechanism, and is competitively inhibited by NADP. Unlike the mammalian enzyme, the housefly reductase is much less sensitive to changes in ionic strength, has a lower affinity for substrates, and is more sensitive to sulphydryl inhibitors such as p-chloromercuribenzoate and N-ethyl maleimide.

REDUCED nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome c reductase (E.C.I.6.z.3), has been purified as a soluble flavoprotein from yeast (Haas, Horecker, and Hogness, 194 o) and from a bacterium, Thiobacillus thiooxidam (Tano and Imai, I968 ). T h e reductase occurs as a microsomal flavoprotein in mammals (Phillips and Langdon, I962; Williams and Kamin, 1962; Nishibayashi Omura and Sato, 1963). Recently, the enzyme has been isolated and characterized from human liver microsomes (Kennedy and Soyka, 197o ).

In a recent communication (Wilson and Hodgson, 1971 ) NADPH-cytochrome c reductase was solubilized and partially purified from housefly abdomen microsomes. We now wish to report some further characteristics of the insect reductase.

MATERIALS AND METHODS CHEMICALS

NADPH, p-chloromercuribenzoate (PCMB), N-ethyl maleimicle (NEM), and 2,6-dichloro- pheno-indophenol (DCIP) were obtained from Nutritional Biochemicals Co. NADP and 'type I I I ' cytochrome c were obtained from Sigma Biochemicals Co. Bovine serum albumin fraction V (BSA) was obtained from Armour Pharmaceutical Co. All other reagents were ACS or analytical grade.

ENZYME PREPARATION

Both housefly microsomes and microsomal NADPH-cytochrome c reductase were prepared as previously described (Wilson and Hodgson, I97X).

ASSAYS All assays were performed in a Beckman DB spectrophotometer at room temperature

(25-27 ° C.), except for the temperature study; in that case a Thermo-Cool and a Haacke

*Paper number 3354 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina, U.S.A.

172 WILSON AND HODGSON Insect Biochem.

Thermo-circulator (Brinkman Instrument Co.) water-bath were connected to the cooling jacket of the cuvette sample compartment. Absorbance was read directly from the spectrophotometer, usually for the first 1.5 minutes of the reaction. All assays were initiated by addition of NADPH, except for the temperature study; since enzyme degradation was rapid at about 4 °0 C., the cuvette, with all components present except enzyme, was brought to the desired temperature in a water- bath. Enzyme was then added to initiate the reaction. No reduction of an electron acceptor sub- strate occurred in the absence of enzyme or NADPH, except in the case of ferricyanide, which is slowly reduced non-enzymatically by NADPH. The rate of cytochrome c reduction was linear for the first 3 minutes of the reaction; DCIP reduction began to lose linearity after the first 1.5-2 minutes. The reduction of ferricyanide is not linear; this may be due in part to the low extinction coefficient and also to the non-enzymatic reduction of ferricyanide by NADPH. The rate of cyto- chrome c reduction as a function of enzyme concentration was linear beyond the amounts of enzyme used in the experiments.

Potassium phosphate buffer (pH 7'8, ionic strength o" Io) was used in all experiments. NADPH-cytochrome c reductase and protein were assayed as described previously (Wilson and

Hodgson, x97I). DCIP reduction was assayed by measuring the decrease in absorbance at 6oo nm. The cuvette

contained 3"o× io -7 moles NADPH, 2"9 × IO -7 moles DCIP, and enzyme in a total volume of 2"4 ml. of assay buffer. The blank contained all components except NADPH. One unit of activity is defined in an analogous manner to NADPH-cytochrome c reductase.

Ferricyanide reductase was assayed by measuring the decrease in absorbance at 42o nm. The cuvette contained 3"o × lO -7 moles NADPH, 2"4 × IO -6 moles KsFe(CN)e, and enzyme in a total volume of 2"4 ml. of assay buffer. The control contained all components except enzyme.

The molar extinction coefficients used were: 21.o× ioS M -t cm. -1 at 600 nm. for DCIP (Steyn-Parv6 and Beinert, 1958); I 'o2 × lO -8 M-1 cm.- 1 at 42o nm. for K 3Fe(CN)6 (Schellenberg and Hellerman, 1958); 6'26 × io -s M -1 cm. -~ at 34o nm. for NADPH (Kornberg and Horecker, 1953); i8"o× lO 3 M -~ cm. -1 at 26o nm. for NADP (Pabst Laboratories Biochemicals, 1967); and I"69× xo -4 M -t cm. -1 at 234 rim. for PCMB (Boyer, I954).

NADPH-cytochrome c reductase was kept at o-5 ° C. during all operations except where noted.

RE SULTS

ENZYME STABILITY

N A D P H - c y t o c h r o m e c reductase purified on DEAE-ce l lu lose loses about 3 o per cent of its activity after I m o n t h at - -15 ° C. in the eluting buffer. T h e enzyme purified in hydroxylapat i te is half as stable unde r these conditions, p robab ly due to the lower protein concentrat ion. Repeated thawing and freezing of purified enzyme greatly accelerate loss in activity dur ing storage at - -15 ° C. At room temperature , half of thc activity is lost within 12 hours and virtually all within 48 hours. At 55 ° C. all activity is lost within 2 minutes . T h e housefly reductase is unstable to dialysis. Dialysis against o.o75 M phosphate buffer, p H 7.8, for 16 hours inactivates nearly half of the enzyme; addi t ion o f IO -5 M 2-mercaptoe thanol to the dialysing buffer decreases the loss to 15-2o per cent, even t hough the dialysis tub ing was rout inely prct reated by boil ing for 30 minutes with E D T A . A buffer concentra t ion of o.o8 M phosphate , p H 7.8, was found to be the o p t i m u m dialysing buffer concentrat ion.

SUBSTRATE SPECIFICITY Housefly N A D P H - c y t o c h r o m e c reductase will reduce D C I P and ferricyanide in

addit ion to cy tochrome c. O the r electron acccptors have no t becn tested. Equivalent amounts of enzyme will reduce 5"z × lO -9 moles of cy tochrome c and 3"7 × lO-9 moles o f D C I P in I minute . Therefore , a l though cy tochrome c is reduced faster, since D C I P is a 2-electron acceptor, electron transfer to D C I P is more efficient. T h e enzyme is

i97z, I PURIFIED NADPH-CYTOCHROME c REDUCTASE I73

specific for NADPH, the rate of reduction of cytochrome c with NADH as substrate is less than i per cent of that with NADPH.

pH OPTIMUM Fig. t shows the pH optimum for cytochrome c reduction by NADPH-cytochrome c

reductase. KC1 was added to the various buffers in amounts needed to bring the ionic strength of each buffer to o.x2.

0.05 I

~" 0.03

F o.oz[ l:i d 0.011

O / I I I 6 .0 7.0 8 .0 9 .0

pH FIG. z . - -pH-act ivi ty curve for NADPH-cytochrome c reductase. I" 5 lag. of enzyme

was used (specific activity 3z units per mg.) O, Tris-HC1; r'l, Phosphate.

IONIC STRENGTH

Phosphate buffers at pH 7.8 were prepared at different ionic strengths, and their effect on the reduction of cytochrome c and DCIP by housefly reductase was studied. Cyto- chrome c reduction was studied using several different concentrations of NADPH for comparison with the results of Phillips and Langdon (i96z) on rat liver NADPH- cytochrome c reductase. Since initial studies with housefly reductase at assay concentra- tion of NADPH gave curves which resembled the curve obtained by Phillips and Langdon (I962) at rate-limiting NADPH concentrations, the effect of ionic strength at several concentrations of NADPH was studied and the results are shown in Fig. z. The initial peak at 4 times the NADPH assay concentration (z.2 ~tmoles NADPH) is appar- ently due to the contribution of NADPH itself to the ionic strength of the assay medium. The small peak at o'o 9 ionic strength cannot always be reproduced; however, the results at 0.05 ionic strength have been repeated in all instances.

TEMPERATURE

The effect of temperature on the enzymatic reduction of cytochrome c and DCIP is shown in F/g. 3. At about 40 ° C. the reaction rates were not linear during the first 1.5 minutes of the reaction; this is due to heat denaturation of the enzyme. Therefore, all

I74 WILSOt~ AND HODGSON Insect Biochem.

rates are expressed as the absorbance change during the first minute of the reaction. Using reaction rates extrapolated to the first to seconds resulted in a zo per cent increase

0.08

~0.06

004[/" r

6= 0.02[ o

IONIC STRENGTH FIG. 2.--Effect of ionic strength on NADPH-cytochrome c reductase. Ionic strength

refers to buffer only; 1"2 lag. of enzyme used (specific activity 6o units per mg.). n , Cytochrome c, 30o mlamoles NADPH; A, Cytochrome c, 12oo mlamoles NADPH; O, Cytochrome c, 3o mla-moles NADPH; A, DCIP, 3oo mlJmoles NADPH.

e -

• O . 1 6 -

E 0.12-

0.08 o ~)

,~ 0 . 0 4

d 0 0 = ,,, I ~ ~ ~ J ,,=:1 I 0 2 0 3 0 4 0 5 0 6 0

T E M P E R A T U R E , *C FIG. 3.--Effect of temperature on NADPH-cytochrome c reductase. 2"5 gg. of enzyme

used (specific activity 33 units per rag.) for cytochrome c and z'5 lag. (specific activity 27 units per mg.) for DCIP and D C I P + B S A . A, Cytochrome c; O, DCIP ; n , D C I P + B S A .

in peak height but no change in the shape of the curves. The concentration of BSA added to the cuvette for the DCIP plus BSA reactions was I. 5 rag., which produces a protein concentration equivalent to cytochrome c.

I97I, I P U R I F I E D N A D P H - C Y T O C H R O M E C REDUCTASE I75

KINETICS

Since the reduction of cytochrome c or DCIP by NADPH-cytochrome c reductase is a binary reaction, Michaelis constants for the substrates involved can be obtained by measuring the enzymatic rates when the concentrations of both substrates are varied and

O -!>° ~,

E == E

0.25 -

0.20 a

0 . 1 5 ~

0.10

0.05

O 0 , I I I ,, I , I I I 0.005 0DI0 0.015 0.020 0.0?.5 0.030 0.035 I (mpm01es)_ I NADPH

FZG. 4.reDouble reciprocal plot for reduction of cytochrome c with NADPH concen- tration as abscissa. A, z15 mpmoles cytochrome c; O, 8o mpmoles cytochrome c; n, 58 mgmoles cyto-chrome c; O, 34 mpmoles cytochrome c.

0.35 m

o. o

" o (I)

g 0.25

o.2o! t.1

~ 0.15 o E

0.10

-1:~o.o5

J 15/~M

° o 0 . ~ ' ' o.o,o QOI5 6.0'~ ' ' 0.025 I

Cyt ¢ ,(m/J m°les) ' !

Fxo. 5.--Relationship between the reciprocal of apparent maximum velocity and the reciprocal of cytochrome c concentration for NADPH-cytochrome c reductase.

then plotting the results as a double reciprocal plot. The concentration of each substrate is then extrapolated to infinity and the K , value obtained from this plot (Alberty, z956 ). Fig. 4 shows a double reciprocal plot for cytochrome c reduction. The parallel lines

z76 WILSON AND HODGSON Insect Biochera.

obtained indicate a ping-pong reaction mechanism (Alberty, z956 ). When the reciprocals of the cytochrome c concentrations were plotted against the reciprocals of the apparent maximum velocities (Fig. 5), a K , of z 7 IxM was obtained from cytochrome c. A Line- weaver-Burk (z934) plot (Fig. 6) for cytochrome c with fixed NADPH concentration

T --- 0 . 2 0 C

E

® 0 . 1 5

q)

° 0.10

o

ID

-~ 0.05 E ~L E

°o

yt c 17/JM

I I I , , ! I I 0.005 0.010 0.015 0.020 0.025 0.030

I .,(rap moles)_ I cit 0

FIG. 6.--Lineweaver-Burk plot for cytochrome c. 0.8 gg. of enzyme used (specific activity 24 units per rag.).

6

Ki NADP 15pM !

0 l I I I I 0 4 8 12 16 20 24 28

I M - i x104

FIO. 7.--Competitive inhibition of NADPH-cytochrome c reductase by NADP. z'7 lag. of enzyme used (specific activity 3t units per rag.), m, 25 pM NADP; O, z2/JM NADP; D, 6 pM NADP; O, 4 pM NAPD; A, o pM NADP.

(assay concentration) yielded a Km of z5!PM. This lower value was expected since the assay concentration of NADPH was less than saturating; however, Lineweaver-Burk plots provided a check on the double reciprocal plots since the Lineweaver-Burk plots were more accurate than the latter.

i97i , i PURIFIED NADPH-CYTOCHROME C REDUCTASE 177

Calculations of the Michaelis constants in an analogous manner for NADPH and DCIP yielded i2 laM and 52 laM respectively by the double reciprocal plot method and 9.i ~tM and 42 oM respectively by Lineweaver-Burk plots.

INHIBITORS

As shown in Fig. 7, NADP is a competitive inhibitor of the enzymatic reduction of cytochrome c. The Michaelis constant for inhibition was calculated to be 13 ~M.

At io -3 M, N A D H in the presence of enzyme inhibits cytochrome c reduction by N A D P H by approximately 4 per cent.

Housefly reductase can be inhibited by PCMB and N E M as shown in Fig. 8. The enzyme was preincubated with inhibitor for 5 minutes prior to assay with cytochrome c.

I00

z 80 0 I '-- 2, 7. 60 Z

I"-

z 4 0 h i

W

a. 20

PCMB~ "~:~3 o NEM

0 o I I 10-8 10-7 10-6 10-5 10-4 10-3 10-2

INHIBITOR, MOLARITY Fie.. 8.--Effect of p-chloromercuribenzoate and N-ethyl maleimide on NADPH-cyto-

chrome c reductase.

NADPH-cytochrome c reductase can be partially protected against inhibition by PCMB and N E M by preincubation with NADPH, and inhibition can be partially reversed by incubation of the inhibited enzyme with - - S H compounds. As shown in Table I, both cysteine and e-mercaptoethanol can partially reverse inhibition by PCMB ; e-mercaptoethanol is more effective. N A D P H will protect the enzyme from N E M inhibition more effectively than from PCMB inhibition. N A D H offers no protection from N E M inhibition.

In some experiments PCMB at concentrations of i o - 9 - i o -8 M was noted to cause a stimulation of activity of up to io per cent. This stimulation effect could not be repeated in all instances, and no stimulation was noted with NEM.

DISCUSSION Housefly NADPH-cytochrome c reductase is a moderately stable enzyme when stored

at - - i5 ° C. A temperature of about 35 ° C. rapidly inactivates the enzyme, and salt concentrations higher or lower than o.o8 M, p H 7.8, reduce the stability. This opt imum

17 8 WILSON AND HODGSON Insect Biochem.

salt concentration approximates the concentration (0-07 M) which elutes the enzyme from a DEAE-cellulose column during purification (Wilson and Hodgson, I97I ). Dialysis reduces the enzyme activity, in part due to an apparently heavy metal inactiva- tion since 2-mercaptoethanol partially reverses dialysis inactivation.

The activity of housefly reductase is sensitive to changes in the ionic strength, but is less sensitive than rat liver reductase (Phillips and Langdon, 1962 ) or rabbit liver reductase (Nishibayashi Omura and Sato, 1963) and exhibits a different behaviour towards changes in ionic strength from the mammalian enzymes. Ionic strength changes do not appear to influence N A D P H binding by the enzyme, contrary to the findings of Phillips and Langdon (i962) for rat liver reductase. Moreover, at ionic strengths greater than o.o4, the behaviour of the housefly reductase is very different

Table -/.--PROTECTION AGAINST AND REVERSAL OF INHIBITION OF NADPH-CYTOCHROME c REDUCTASE BY #-CHLOROMERCURIBENZOATE AND N-ETHYL I~{ALEIMIDE

INHIBITOR (molarity)

I X I0 -g PCMB I x IO " PCMB I x Io-" PCMB I × Io -~ PCMB 8 5: io -3 NEM 8 × Io -:~ NEM 8× Io -'~ NEM

ADDITIONS

None IO -5 M cysteine IO -s M 2-mercaptoethanol NADPH preincubation None NADPH preincubation NADPH preincubation (assay with NADPH)

PERCENTAGE INHIBITION

IOO

90 3z 9o 93 35 92

Enzyme used for PCMB studies was o'8 lag. of 74 units per mg. and for the NEM studies was I'5 lag. of 65 units per rag. 0-8 rag. of enzyme (specific activity 74 units per mg.) used for PCMB experiments and I'5 lag. (specific activity 65 units per mg.) for NEM experiments.

Five-minute incubation with inhibitor or sulphur reagents; z-minute preincubation with NADPH or NADH.

towards electron acceptor substrates; Phillips and Langdon (i962), varying ionic strength, found the behaviour of the rat liver enzyme to be independent of the choice of electron acceptors. It appears that increasing ionic strengths produces little effect on cytochrome c binding to housefly reductase but enhances D CIP binding, either by ionic charge interactions by the buffer with enzyme or substrate, or conformational changes in the enzyme molecule, induced by high buffer concentration, which are more favour- able to DCIP binding.

The temperature effects on reductase activity reflect both the activity and denaturation of the enzyme. At about 4 °o C. the initial velocity during the first minute of the reaction is not linear, indicating enzyme denaturation. Below 4 °0 C., the rate of D CIP reduction was reduced by BSA whereas at about 4 °0 C. the rate was increased; BSA may block the enzyme-substrate interaction or bind DCIP since it is known to bind organic molecules. Above 4 °o C. BSA apparently protects the enzyme from heat denaturation.

The kinetic analysis of NADPH-cytochrome c reductase indicates that the enzyme has a lower affinity for substrates than the mammalian enzyme. The K, , for DCIP is nearly thirtyfold greater than that obtained by Williams and Kamin (1962) for pig liver reduc- tase.

1971, I PURIFIED NADPH-CYTOCHROME C REDUCTASE 179

The importance of the 2'-phosphate group on N A D P H for activity of the enzyme is clearly demonstrated by the almost negligible cytochrome c reductase activity with N A D H and the inability of N A D H to protect reductase from N E M inhibition. Since N A D H at Io -s M inhibited cytochrome c reduction by N A D P H by 4 per cent, N A D H apparently does interact to some extent with the enzyme, but this binding is weak, as judged by the lack of protection by N A D H from N E M inhibition.

Housefly NADPH-cytochrome c reductase is more sensitive to sulphydryl reagents than the mammalian enzyme. 0.33 gM PCMB will cause 9 o per cent inhibition, whereas Williams and Kamin (i962) reported 2.o laM PCMB inhibited pig liver reductase by 87 per cent, and Phillips and Langdon (1962) reported i. 7 m M PCMB only inhibited rat liver reductase by 2o per cent. Furthermore, Masters, Kamin, Gibson, and Williams (~965) found that high concentrations (concentrations not specified) of N E M caused no inhibition of pig liver reductase. The results of the present work strongly implicate - - S H groups on the housefly enzyme to be catalytically active.

Housefly NADPH-cytochrome c reductase is similar to its mammalian counterpart in many respects. Some differences were found, such as kinetic rates, sulphydryl group inhibition, and behaviour with changing ionic strength of the surrounding media. These differences may be due to different preparative techniques, but the possibility of inherent dissimilarities in the enzyme from organisms as different as houseflies and mammals cannot be excluded.

ACKNOWLEDGEMENT

This work was supported in part by grants from the U.S. Public Health Services numbers ES-ooo44 and ES-ooo83. One of the authors (T. G. W.) gratefully acknowledges the support of an NDEA fellowship.

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c', J. biol. Chem., x36 , 747-774. KENNEDY, J. R., jun., and SOYKA, L. F. (x97o), ' Isolation and characterization of reduced nicotin-

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LINEWEAVER, H., and BURK, D. (1934), 'The determination of enzyme dissociation constants', J. Am. chem. Sot., 56, 658-666.

MASTERS, B. S. S., KAMIN, H., GIBSON, Q. H., and WILLIAMS, C. H., jun. (I965) , ' Studies on the mechanism of microsomal triphosphopyridine nucleotide-cytochrome c reductase', J. biol. Chem., 240, 921-931.

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PHILLIPS, A. H., and LANGDON, R. G. (I 96z), 'Hepatic triphosphopyridine nucleotide-cytochrome c reductase: Isolation, characterization, and kinetic studies', J. biol. Chem., 237 , 2652-266o.

SCHELLENBERG, K. A., and HELLERMAN, L. (I958), 'Oxidation of reduced diphosphopyridine nucleotide', J. biol. Chem., 231 , 547-556.

I80 WILSON AND HODGSON

ST~ZYN-PARv~, E. P., and BEINERT, H. (I958), ' On the mechanism of dedrogenation of fatty acyl derivatives of coenzyme A' , J . biol. Chem., 233, 843-852.

TaNO, T., and IMAI, K. (I968), 'Physiological studies on thiobacilli. Part V. Extraction of NADPH-cytochrome c oxidoreductase from Thiobacillus thiooxidans', Agric. Biol. Chem., 32, 284-286.

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WILSON, T. G., and HODGSON, E. (I97I), ' Microsomal NADPH-cytochrome c reductase from the housefly, Musca domestic, L. : Solubiliation and purification', Insect Biochem., I, 19-26.

Key Word Index: NADPH-cytochrome c reductase; microsomes, Musca domestica.