Properties of a pyridine nucleotide-menadione oxidoreductase from mung bean seedlings

Phytochemistry.

1962, Vol. I, pp. 131 to 136.

Perpaman Press Ltd.

Priicd

in England

PROPERTIES OF A PYRIDINE NUCLEOTIDEMENADIONE OXIDOREDUCTASE FROM MUNG BEAN SEEDLINGS H. SHICHIand D. P.

HACKETT

Department of Biochemistry, University of California, Berkeley, California. (Received 14 February 1962)

Abstmct-A soluble NAD(P)Hs : 2methyl-l&na hth uinone oxidomductase (menadione reductase) has been partially puritied from etiolated munt bean &furZ us oureus)seedlings. The ewyme is equally active with NADHs and NADPH*, and the & or NADH, is 2.14x 11Y4M. It shows diaphorase activity and can be assayed conveniently with ferricyanide (& = 2.27x lo-’ M). A number of l&naphth uinone derivatives were tested as electron acceptors, and of these menadione was the most effective; oYli? y the related amino-naphthol, Vitamin KS, was more active. The enzyme also reduces 1,~benzoquinone, but not Vitamin K1 or ubtquinone UQia. The menadione reductase is completely inhibited by 1F M dicumarol, but it is not a%cted by lo” M DNP. INTRODUCTION RBCENTwork

on both animals and plants has focused attention on the possible roles of the Vitamin K- and ubiquinone-type quinones in electron transport1 A number of different enzymatic systems which catalyse the reduction of quinones by reduced pyridine nucleotides have been isolated. These vary in their electron acceptor spec&ities, and some of the enzymes have even been descri&ed as diaphorases. Wosilait and Nason* fist obtained an enzyme from pea seeds that reduces benxoquinone. They then found that pyridine nucleotide-2-methyl-1,4-naphthoquinone oxidoreductase (menadione reductase) activity is widely distributed in higher plar~ts.~ This activity has since been measured using soluble extracts from potato t.ube~%,~ wheat germ,” and tobacco roots? and the enzyme has been partially purified from the last source. The present paper describes some properties of a soluble mung bean enxyme which catalyses the reduction of a variety of quinones and is particularly active with menadione (Vitamin KJ. EXPERIMENTAL

AND RESULTS

Enzyme Purification

Fifty lb of 3- to 5day-old etiolated mung bean (PheoZr~ aureus Roxb.) seedlings were macerated in 14 lb batches with water in a Waring blender for 15 sec. The homogenate was squeezed through two layers of cheesecloth to give the crude extract (approximately 18 l.), which was adjusted to pH 5 with 1 N acetic acid and allowed to stand overnight at 5”. The pH 5 supernatant fraction (PH 5-super) was siphoned off from the precipitated material, adjusted to pH 7 with NH,OH, and enough ammonium sulphate (AS) added to it to make a 60 per cent saturated solution. After standing overnight, the sediment was collected by filtration on a Buchner funnel, dissolved in 150 ml of O-1M phosphate buffer, pH 7.5, and dialysed against distilled water overnight at 5”. The dialysed solution (AS O-60 per cent ‘G.E.W.Ww

LMEand C. M. O’ER,

Qninoaes in Electron llunsm,

.&YW O~ILAFI and A. Nm, J. Biol. C&m. 296,255 (1954). ’ W: D: Woan&~ and A. NA~ON,J. B&l. Churn.2OS,785 (1954). ’ D. P. HACKEIT, PlrmtPhysiol. 33,8 (1958). 6 H. H. CL.UM and A. Nm, Plant Physbl. 33,354 1958). ’ B. C. %L.ERand H. J. EVANS,TothwxoScf. 2,132 ( 1958). 131

Little, Brown, Boston

H. SHICHIand D. P. HA~~EIT

132

sat,) was refractionated with ammonium sulphate and the fraction precipitating between 45 and 55 per cent saturation at pH 7 was collected. This was dissolved in buffer, dialysed against distilled water overnight at 9, and centrifuged to remove any insoluble residue. To the dialysed solution (As 45-55 per cent sat.), 30 per cent by volume of cold (-30’) acetone was added with constant stirring at 0”. Ten minutes after the addition of acetone, the precipitate was collected by centrifugation and discarded. The acetone concentration of the supernatant fraction was increased to 60 per cent by adding more cold acetone; the resultant precipitate was then collected by centifugation and dissolved in 5 ml of 0.1 M phosphate buffer, pH 7.5 (acetone 30-60 per cent). This was used as the final preparation. For the routine enzyme assay ferricyanide was used as the electron acceptor. TABLB 1.

%JMMARY OF ENzyMa

PURIFICA’TION’

Specific activity (units/mg protern)

Electron donor

Fraction

(A)NADH,

(B)NADPH,

I

B/A

Preliminary studies showed that the major fraction of the activity was precipitated when the pH 5 supematant was 60 per cent saturated with ammonium sulphate; none was precipitated by 40 per cent saturation. With acetone, all of the activity was precipitated when the concentration (by volume) was between 30 and 60 per cent. The results of the fractionation procedure described above are given in Table 1: the enzyme was purified either g-fold or l&fold, depending on whether NADH,, or NADPH, was the electron donor. Only 1.4 per cent of the total activity in the pH 5 super&ant was recovered in the final fraction.

05----

t - -. .. _-I

I

-..._.._. f-

-.-.

5 0.4 zi s.

-

L_

f @3 t P i 02 ET 1; 0.1.

:; .'

I 0

02

1 04

Protein

I 06

I_ IO

0.6

concentration,

pg

OF NADHI OXIDATION. OneunitofenqrmeactivitydafinedacracbangeinaxtinctionoPWlinalcmocllat340~ during the initial 20 sec. Away mixtum as described in text. FIQ.

l.lEFFEm

OF ENZYME

cmxmmum

ON

RAZZ

Assay Conditions

For the standard assay, the reaction mixture contained 1-Oml of 0.2 mM NADH, or NADPH* dissolved in 0.12 M phosphate buffer (pH 7~9,0-01 ml of enzyme solution, and 0.01 ml of O-01M potassium ferricyanide or some other acceptor. The rate of decrease in extinction at 340 nqu was followed at room temperature and was corrected for any nonenzymatic oxidation of the coenzyme by the acceptor. The rate of the reaotion was directly proportional to the enzyme concentration (Fig. 1); the pH optimum is 7.8 (Fig. 2); and the Michaelis constant (Kj,,)for ferricyanide in this reaction is 2.27 x lo-* M (Fig. 3).

A series of 0.12 M

activity

As shown in Table 1, the purifkd enzyme is almost equally active, on a protein basis, with NADHl and NADPH,. This is chamcteristic of a number of menadione reductases and diaphorases. The dependence of the reaction rate on the concentration of NADH, was determined and the K, for this donor was calculated to be 2-14x lO-*M (Fig. 4). This

I /farrvzyanide,

Fm. 3.

~MINATION

I/M

OF- ENZYME AFFINITY FOR THE EU!CTRON ACCEPTOU (FFJUUCYANtDE).

H. SHICI-II and D. P. HA-

134

value is close to the comparable Michaelis constants for pea seed quinone reductase* and tobacco root diaphorase.’ Electron Acceptors The abilities of a number of compounds to act as electron acceptors for the oxidation of NADHs by this enzyme are compared in Table 2. Both unsubstituted benzoquinone and a-naphthoquinone are active, whereas anthroquinone is not. The effect of substitution in the 2- and 3-positions of naphthoquinone was examined. Menadione, the 2-methyl derivative, is particularly active, and it was assigned an arbitrary value of 100. Activity is completely abolished by substitution in the 2-position with the electron-repelling methylamino

I

I 0

4

I 8 I/NADH,

Fro. 4.

DErBRumATloN

I

I

I I2

16

20

I, 24

28

I /mg per ml

OF ENZYME AFRNITY FOR THE

FJ..ECTRON

DONOR

(NADH*).

or hydroxy groups. Of the several 2,3disubstituted 1,4naphthoquinones, only the dichlorocompound showed sign&ant activity. This indicates that the 3-position does not necessarily have to be free in order for the quinone to react. There was no reaction with phthiocol (2-hydroxy-Imethyl-1,4naphthoquinone), a compound which is very effective in catalysing photosynthetic phosphorylation.7 Vitamin Kg, 4amino-2-methyl-1-naphthol, was even more active than menadione; K, must be very rapidly converted to the quinonimine form. It is of interest that this nitrogen analogue is more effective than the natural vitamins in the promotion of prothrombin formation. Since the naturally-occurring quinones UQ,, and Vitamin K1 are extremely insoluble, special methods must be used to solubilixe or emulsify them for use in aqueous systems. Solubilization by Tween 80 (polyoxyethylene sorbitan mono&ate) and BRLI-35 (polyoxyethylene lauryl alcohol) did not make possible the reduction of these quinones by the mung bean enzyme. As shown in Table 2, ferricyanide is a considerably less effective acceptor than several of the quinones. At equal concentrations, 2,6dichloro-phenol-indophenol is even less effective than ferricyanide. The low diaphorase activity of the mung bean enzyme distinguishes it from some other menadione reductases. Molecular oxygen, cytochrome c, o-lipoic acid, and acetyl-pyridine NAD did not serve as electron acceptors (Table 2). This indicates that the enzyme shows no oxidase, cytochrome c reductase, lipoate dehydrogenase, or transhydrogenase activity. Inhibitors One of the properties which has been most useful for the characterization of the diaphorases and quinone reductases is their response to certain inhibitors. 2,4Dinitrophenol ’ D. I. AUNCW,Fe&rationProc. 20, 1012 (l%l).

135

Menadionereductasefrommungbeansecdl@s TABLE 2. RELATIVEAm

OF &Wl'RON

Accepro~s+

Solvents

Acceptort uinone ‘:~oquinone (NQ) 2&thyl-NQ (KJ

ethanol

t

z&s

2-sulphonyI-NQ 2-methylamino-NQ 2-aniline-NQ 2-hydroxy-3-methyl-NQ

E&form

2,3-dihydroxy-NQ 2-hydroxy-3-methoxy-NQ 2-hy@xy-3_(3’-p~~pyl)-NQ

g&d=P-sQ

2-methylJ-phytyl-NQ (K3 4-amine-2-methyl-l-naphthol ubiquinone UQ10 ferricyanide cytochrome E 9 Dr-a-li ic acid 11 3-acetyp” pyridine-NAD oxygen

(K3

Zi!ZZlorm ethanol chloroform ethanol chloroform ethanol BRIJ-35 ethanol BRIJ-35 water water water water

Relative activity

41 12 38 x 0 ;:; uo 8 122 2! x 0

+ Standard assay for NADH, oxidation as described in text. Final concentration of acceptors = 5X lO_’M. t NQ equals 1,4-naphthoquinone. $ Solvent in which acceptor was dissolved before addition to reaction mixture. K1 and UQ10 solubilized in BRIJ-35 according to method of Wosilak~ f R.eduction of cytochrome c (lO_’M) measured at 550 mp. 11Reaction mixture contained 001 pmole NADH8 and 3 holes lipoic acid in 1 ml pH 69 phosphate buffer. (DNP), at 1O-4M, did not inhibit the menadione reductase activity. The response to . dxumarol (1O-6M) depends on the nature of the electron acceptor: the oxidation of NADHa was inhibited 60 per cent when menadione was the acceptor, but only 8 per cent with ferkyanide. At lo-4 M, dicumarol inhibited the reaction 97 per cent. Several other inhibitors were tested, using ferricyanide as electron acceptor: activity was inhibited 26 per cent by lo-4 M PCMBA (parachloromercuribenibenzoicacid), 26 per cent by 10” M Atabrine and 15 per cent by 1O-4M Amytal. DISCUSSION

The properties of the mung bean menadkme reductase distinguish it from other enzymes isolated from higher plant tissues. The pea seed quinone reductase reduces benzoquinone but not menadione, and it is very sensitive to DNP.s The wheat germ diaphorase is active with memukme but not with 1,4_naphthoquinone. s Phylloquinone reductase from spinach leaves shows some activity with both Vitamin K1 and cytochrome c, and it is not inhibited by dicumar01.~ The tobacco root diaphorase is almost equally active with 1,4benzoquinone, l&raphthoquinone, menadione, and 2,6dichlorophenol-indophenol as electron acceptors.6 The pH optima of the tobacco and mung bean enzymes differ by one unit. More definitive comparisons of all these enzymes must await their further purikation. In some, but not all, respects the mung bean enzyme is similar to the mammakn enzymes BG. WOLF,F. KIEFFERand C. MAR-

Fdmtion

Pm.

Is.354 (1959).

136

H. &mm

and D. P. Hm

which can act as pyridine nucleotide-menadione oxidoreductases.*la All of them are inhibited by dicumarol, but they differ in their responses to DNP and PCMBA. A comparison of the inhibitor sensitivities and the electron acceptor speciScities suggests that the mung bean enzyme most closely resembles the Vitamin K-reductase of Mllrki and Martiu~~~ This enzyme has been extensively purikd and shown to be a flavoprotein. The physiological roles of the various menadione reductases have not been deSnitely established. It is signiscant that in both plant and animal homogenates most of the activity is found in the soluble fraction, and it was from a particle-free fraction that the mung bean enzyme was prepared. This intracellular localization suggests that the enzyme is not a part of the normal respiratcry chain and that it probably functions in hydrogen transport in the cytoplasm itself. The fact that naturally occurring quinones were not effective with this enzyme might be the result of our inability to duplicate the physiological state in which the electron acceptors may be bound in some intracellular membrane. Several of the mammalian enzymes are active with Vitamin K1 and/or ubiquinone (UQo),eJ1~’ but neither of these natural quinones was active with Martius’ highly puriikd enzyme.” The natural acceptor for the soluble mung bean enzyme might even be menadione itself, although this quinone has not yet been clearly identified in Nature. Acknowledgemen?~The the naphthoquinoms.

authors are indebted to Drs. M. W. Foote and R. What&y for samples of some of

’ W. D. Wosurr, J. Biol. Chem. us, 11% (NO). “‘F.Mi~~1andC.M~~~1~~,Biockun.2.333,111(1%0). l1 T. E. CONOVER and L. ENSTER, Biochm. B&p&s. Res. Corn. 5 26 (NO). I* A. GIUDIT~Aand H. J. STRXKER,Biochim. Biophys. Acfu 48, 10 (l%l).