EXPERIMENTAL PARASITOLOGY 41, 186197

Entamoeba

(1977)

histolytica:

Diaphorase

Activities

EUGENE C. WEINBACH, DAN R. HARLOW, C. ELWOOD CLAGGETT, AND LOUIS S. DIAMOND Laboratory of Parasitic Diseases, National Institute of Allergy and infectious Diseases, National Institutes of Health, Bethesda, Marylund 20014, U.S.A. (Accepted for publication 1 April 1976) WEINBACH, E. C., HARLOW, D. R., CLAGGEIT, C. E., AND DIAMOND, L. S. 1977. Entamoeba histolytica: Diaphorase activities. Experimental Parasitology 41, 186-197. Diaphorase (EC 1.6.99.2), which catalyzes the oxidation of reduced nicotinamide adenine dinucleotides, was observed in the soluble fraction of axenically cultivated ,trophozoites of Entamoeba histolytica. At least two enzyme proteins are responsible for this activity; one with a high affinity for NADPH, the other with a low affinity for NADH and possibly also for NADPH. Activity was observed both polarographically with oxygen as the final acceptor, and spectrophotometrically with artificial electron acceptors. As determined polarographitally, oxidation of ‘the reduced nucleotides was greatly enhanced by vitamin K3. A fraction enriched in diaphorase was prepared from the soluble fraction of sonicated amoebae by acetone and ammonimn sulfate precipitations. The diaphorase was not inhibited by cyanide or antimycin, but was inhibited by p-chloromercuribenzoate, n-methylmaleimide, and antimalarial drugs. Compounds which chelate metals, e.g., substituted butanediones, were more potent inhibitors. Only partial inhibition was obtained with dicoumarol. The inhibitor studies, together with data reported elsewhere, indicate that the amoeba1 diaphorase is an iron-sulfur flavoprotein. Substituted benzoquinones and dichlorophenolindophenol were effective as electron acceptors; cytochrome c was ineffective. Amoeba1 diaphorase is similar to the analogous mammalian enzyme DT-diaphorase in its stimulation by vitamin K, and its reactions with artificial electron acceptors; however, it differs markedly in being composed of at least two enzyme proteins, and in its lesser sensitivity to the inhibitor dicoumarol. Bacterial diaphorase is severalfold more active with NADH than with NADPH, the reverse of results obtained with the amoeba1 enzyme. It is postulated that in E. hktolytica, diaphorase functions sequentially with transhydrogenase to catalyze the oxidation of the reduced nicotinamide nucleotides aerobically. This may account for a major portion of the oxygen uptake observed with the amoeba. INDEX DESCRIPTORS: Entamoeba hbtolytica; Protozoa; Diaphorase (EC 1.6.99.2); NADH; NADPH; Metabolic inhibitors; Metal chelators; Vitamin K,; Electron acceptors; Respiration. INTRODUCTION

Previous studies in our laboratory have provided conclusive evidence that axenitally cultivated trophozoites of Entamoeba histolytica, Strain HK-9, readily consume oxygen when this gas is provided (Weinbath and Diamond 1974). During the course of subsequent studies to elucidate the respiratory pathway, we observed that

Col~yright Q 1977 by Acackmic Press, Inc. All rights of reproduction in any form reserved.

this parasite has the enzymatic capacity to oxidize either of the reduced nicotinamide adenine dinucIeotides ( NAD ( I?) H ) . Because nicotinamide nucleotides have an essential role as coenzymes in both aerobic and anaerobic cellular metabolism, it was of interest to investigate the properties of the oxidations in more detail. The term “diaphorase” has been applied to any enzyme capable of catalyzing the

ISSN 0014 4x94

j%&UWf?bU

hi&k@XX

oxidation of NAD( P)H by artificial electron acceptors such as redox dyes, ferricyanide, or quinones. Although this term is neither an accurate description of these enzymes, nor the recommended systematic designation, ( NAD( P) H: (quinone-acceptor) oxidoreductase, EC 1.6.99.2), it is used in this communication for the sake of convenience. Ernster, Ljunggren, and Danielson (1960) isolated from rat liver a dicoumarol-sensitive flavoprotein, nr-diaphorase, that catalyzes the oxidation of NADH and NADPH by various redox dyes and quinones. Subsequent studies by these authors disclosed that the enzymatic activity was associated with a single protein (for review, see Hall, Lind, Golvano, Rase, and Ernster 1972). In contrast, our studies with E. histolytica, which are summarized here, revealed that the amoebae contain at least two distinct proteins that catalyze the oxidation of the reduced nicotinamide nucleotides. The present communication also describes a partial purification and some properties of the amoeba1 enzymes. MATERIALS

AND METHODS

Parasites The technique for axenic cultivation of E. histolytica (Diamond 1968) and method of harvest (Weinbach and Diamond 1974) used in this study have been described. Intact Amoebae Freshly harvested and twice-washed trophozoites were suspended in chilled buffered saline (0.11 M NaCI, 0.016 M KPHPOI, and 0.003 M KHaPO1; pH 7.45) so that each milliliter contained 6 mg of protein (approximately 6 x 10G organisms). These suspensions were used without further modification in studies with intact amoebae. Sonicate Sonication of the above suspensions, usually 20 ml, was accomplished with the

DIAI'HORASE

ACTIVITIES

Branson sonifer, the microprobe. were exposed to amps for lfj set

157

Model S75, equipped with The amoeba1 suspensions power setting No. 4 at 3.6 at 4 C.

The souicated suspensions were centrifuged at 110,OOOgfor 120 min. The clear, pale yellow supernatant fraction was decanted and used without further modification in experiments with the soluble fraction. Diaphorase activities of the pellet were estimated after it was resuspended in 10 ml of 0.01 hf phosphate buffer, pH 7.0. Acetone POW&Y Extracts The clear supematant fraction was added slowly (e.g., 17.5 ml over a 5min period) to 9 vol of acetone at -12 C in a beaker. The beaker was covered, and after 30 min the excess acetone was decanted, and the sticky, red precipitate was dried in uacuo over silica gel at 4 C. The dry, tan residue was weighed and suspended in 0.01 RI phosphate buffer, pH 7.0, in the ratio of 1 g of residue per 25 ml of buffer. The SLISpension was placed in a refrigerator at 4 C overnight to complete the extraction, and then centrifuged at 12,OOOg for 10 min. The clear, pale yellow supernatant fluid was used without further modification. Diaphorase activities in the pellet were estimated by suspending the residue in 5 ml of 0.01 M phosphate buffer, pH 7.0. Ammonium

Sulfate Fractions

For these preparations, 5 to 10 ml of packed cells (400 to 1000 mg of protein) was the starting material. The cells were suspended in approximately 15 to 30 ml of 0.25 111sucrose buffered with 50 mM Tris, pH 7.4, and sonicated, and acetone powders were prepared as described above. The acetone powder suspensions were dialyzed against running tap water at 4 C overnight to remove sucrose. The dialyzed suspension was cleared by centrifugation at 17,OOOg

1YS

WEINBACH ET AL.

for I5 min. The cIeared solution was fractionated with neutralized ammonium suIfate in three stages: O-40%, 40-55%, and !%65% saturation as described by Ernster, Danielson, and Ljunggren ( 1962). Diaphowe

Assays

Diaphorase activities were determined either polarographically, utilizing the Clark oxygen electrode ( Weinbach and Diamond 1974)) or spectrophotometrically, employing the Cary Model 15 recording spectrophotometer (Cary Instruments, Monrovia, California). All assays were done at least in duplicate; the values reported are avcrages.

(Lowry, Rosebrough, Farr, and Randall 1951). Crystalline bovine serum was used as standard. Materials Glass-distilled, deionized water was used to prepare solutions of all reagents, which were of the highest purity available commercially. NADH and NADPH were obtained from the Sigma Chemical Co., (St. Louis, Missouri). Enzymes used in the assays for nicotinamide nucleotides were purchased from the Boehringer Mamlheim Corp., New York, New York. RESULTS

Other Assays

Diuphorase Activities of Intact and Sonicated Amoebae

The oxidized and reduced nicotinamide dinucleotide contents of E. histolytica were determined enzymatically, after appropriate extraction procedures, by the methods described by Klingenberg ( 1963). Protein was determined by a modification of the biuret procedure (Szarkowska and Klingenberg 1963) after treatment of the amoeba1 suspensions with 0.5% sodium deoxycholate, or by the Folin phenol reagent

Suspensions of freshly harvested, intact Entamoeba histolytica exhibit active endogenous respiration. Addition of NADH or NADPH to the intact trophozoites had little effect on the respiratory rate (Figs. 1A and B). Sonication of the amoebae resulted in a marked reduced rate of endogenous respiration (Fig. 2). Addition of either NADH (Trace A) or NADPH (Trace B) to the sonicated preparations led to an

INTACT AMEBAE I

INTACT AMEBAE A.

I

El

FIG. 1. Effect of NAD(P)H on the oxygen uptake of freshly harvested trophozoites of Entameba hkitolytica. The cuvette contained approximately 6 x 10’ organisms (6 mg of protein) suspended in 1 ml of buffered saline (0.11 M NaCl, 0.016 M EHPO,, and 0.003 M KHPO,, pH 7.45) at 35 C. The arrows indicate additions of 1.0 pmole of NAD( P)H. The numbers below the tracings express the oxygen consumption in nano-atoms per minute.

Entamoeba

histolytica:

SONICATE

1s9

DIAPHOHASEACIYYITIES SONICATE

820

FIG. 2. Effect of NAD(P)H and vitamin K, on the oxygen uptake of sonicated trophozoites of Entamoeba histolytica. The cuvette contained 6 mg of amoeba1 protein in experiment A, and 1.5 mg of protein in experiment B, suspended in 1.0 ml each of buffered saline (see legend to Fig. 1). Arrows indicate additions of 1 pmole of NAD( P)H and 0.5 *mole of vitamin K,. The numbers below the tracing express the oxygen consumption in nano-atoms per minute.

increase in the respiratory rate, which was particularly pronounced with NADPH. It also may be seen in Fig. 2 that addition of vitamin KS, which had little effect on endogenous respiration of the sonicated preparations, (Trace B ), greatly enhanced the oxidation of the added nicotinamide nucleotides. The enhanced oxidation of either nucleotide was seen regardless of the order in which vitamin K3 was added. Considering that one-fourth as much protein was used in experiment B, it is clearly evident that NADPH was much more rapidly oxidized than was NADH under these conditions. Sonication, apparently, releases a natural carrier( s ) which is replaced or supplemented by vitamin K3. Similar results were obtained with stored preparations, which results in progressive lysis of the amoebae (Weinbach and Diamond 1974). Cellular Localization and Partial Purification of ‘the Diaphormes Using a procedure patterned after that described by Ernster, Ljunggren, and

Danielson (1960) for the purification of mammalian liver diaphorase, it was possible to considerably enhance the amoeba1 TABLE

I

Intracellular Localization and Partial Purification of Entamoeba histolytica Diaphorasen - ..-- __ Specific activity Fraction (natoms oxygen/ min/mg of protein) NADH Intact amoebae Sonicated amoebae 100,OOOgsupernata,rit 100,OOOgpellet Acetone powder of supernatant Extract Residue

NADPH __-

72

1416

95

1631

137

1818 66 2600 4619 311

I5 22 27 7

a OxidaGon was determined polarographically at 33 C in t,he presence of vitamin Ka. The cuvette contained 1 mM vitamin K, 1 mM NAD(P)H, and portions of the amoeba1 fractions suspended in 1 ml of buffered saline described in the legend to Fig. 1. The amounts of protein used in these assays were those found to give maximal rat.es of oxidation (see text).

190

WEINBACH

ET

TABLb:

AL.

II

Ammonium Sulfate Fractionation of Soluble Diaphorases from Entawboeba histolytica” -

Fraction

Total proteiu (lnd

Supernatant of sonicate Extract of acetone powder Ammonium Sulfate precipitates O-40% 40-55'//; X-65" /O'

Soluble at 65% Total recovery from ammonium sulfate precipitates

28s 84

NADH --_____~ Specific Specific activity activity (natoms Xproteiu WWw (w) of protein) 62 25

17,856 2,100

NADPH Specific activity (nat,oms O/min/mg of protein)

369,216 185,976 1,426 30,682 118,032 2,332

1

99

99

1,426

29 67 22

tic,7 1,072

1,334 7,377 44

1,166 3,004

_____-

1,282 2,214

23

16 53

Specific activity Xprotein (wd

152,472

0 The assay conditions were the same as described in the legend to Table I.

diaphorase activities. The data summarized in Table I disclosed that most of the diaphorase activities of E. histolytica were in the supernatant “soluble” fraction of the parasite. Activity obtained with NADPH greatly exceeded that found with NADH. Indeed, further purification of the amoeba1 enzymes by precipitation with acetone led to loss of activity with NADH and concomitant enrichment of the NADPH diaphorase activity. From these results, it appears that there are at least two diaphorases in E. histolytica; one which reacts with NADH and one which reacts with NADPH. The high activities shown in Table I for the intact amoebae are largely due to the presence of vitamin KS in these experiments, and not to oxidation of the added nucleotides (Weinbach and Diamond 1974). Assays of the various fractions for NADPH diaphorase activity disclosed an interesting phenomenon; viz., that increasing amounts of protein per test resulted in decreasing specific activities. Although the phenomenon was not studied further, its basis may be the presence of an inhibitor

or competing enzymes which are carried along in the fractionation procedures. Further purification of the soluble diaphorases is shown in Table II. The highest specific activities for both nucleotides were found in the fraction precipitating at 5565% ammonium sulfate and the fraction soluble at 65%. These data are further evidence that the amoebae contain a separate diaphorase for each of the reduced nicotinamide nucleotides. Gel Electrophoresis Figure 3 illustrates results obtained with polyacrylamide disc gel electrophoresis of acetone powder extracts. Gels A and D show the large number of proteins present in this crude extract. In Fig. 3, gel B, the NADPH diaphorase reduction of nitro-blue tetrazolium, may be seen as a diffuse band at the top of the gel and a distinct second band (arrow). In contrast, when NADH was used as a substrate, this second band was absent (gel C), and only the diffusely stained region at the top of the gel may be seen. The diffuse bands present in both gels may represent nonspecific diaphorase activity. Thus, it is clear that the specific

E&amoeba protein which catalyzes NADPII does not react with NADH.

histolytica:

-T * =7-n- 1n- s-7-,--n

oxidation

phosphate and glucose 6-phosphate dehydrogenase to regenerate the reduced nucleotide. The data depicted in Fig. 6 illustrate that the end product of diaphorase activity (NADP’) is inhibitory. The extent of the inhibition, also seen with the acetylpyridine analog ( AcPyADP+ ), is determined by the ratio of reduced to oxidized nicotinamide nucleotides. This feedback inhibition may be an important mechanism for regulating the redox state of the endogenous nicotinamide nucleotides. In this regard, it is of interest that the nucleotide pool of intact E. histolytica is maintained largely in the oxidized state (Table III). Although the concentration and ratio of NAD+ to NADH are within the range of the values reported for rat liver mitochondria, these values differ for NADP’ and

Properties of the Amoeba1 Diaphorases Reaction products. These were determined in order to establish that the observed oxygen uptake resulted from oxidation of the exogenous reduced nicotinamide dinucleotides. The oxidation of NADH to NAD+ was confirmed by the restoration in absorption at 340 nm upon addition of ethanol and alcohol dehydrogenase to the reaction mixture. Figure 4 shows this restoration after NADH had been oxidized to NAD’ in the presence of the diaphorase. Similarly, Fig. 5 shows the restoration after the rapid oxidation of NADPH to NADP+. Because of the high activity of the diaphorase with NADPH, it was necessary to inactivate it before adding glucose 6-

Frc. 3. Polyacrylamide disc gel electrophoretogram of acetone powder extracts of Entamoeba The technique of Neville (1971) was used, with the substitution of 0.1% deoxycholate for 0.1% sodium dodecyl sulfate in the upper reservoir buffer to preserve enzyme activity. After electrophoresis at 4 C, each gel was split lengthwise and one half (gels A and D) was stained for protein with Coomassie blue by the method of Weber and Osborn (1969); however, the electrophoretic destaining procedure used by these authors was deleted. Gel halves to be used for determination of the diaphorase activity (gels B and C) were stained with nitro-blue tetrazolium in the presence of (B) 1 mM NADPH or (C) 1 mM NADH. histolytica.

192

WEINBACH

ET AL.

07 NADH 0.6

OXIDATION

-

/ -0

3

6

9

12 MINUTES

15

I8

ADH 21

FIG. 4. Reaction product of NADH oxidation catalyzed by acetone powder extracts of Entumoeba histolytica. The reaction medium contained 7.2 mg of protein of acetone powder extract, 1 mM vitamin K, and 50 mM Tris, pH 7.5 in a final volume of 3 ml. The instrument was adjusted to read zero absorbance, and at the time indicated, approximately 0.3 *mole of NADH added. When the reaction was completed, the pH was adjusted to 11.4 with NaOH and the folIowing were added: 0.1 ml of 95% ethanol and approximately 600 units of crystaIline yeast alcohol dehydrogenase ( ADH) , Temperature = 24 C.

0.7

NADPH

,

3

FIG. 5. Reaction

6

0

OXIDATION

3 MINUTES

6

3

Ii:

product of NADPH oxidation cataIyzed by acetone powder extracts of The reaction medium contained 2.4 mg of protein of acetone powder extract, 1 mM vitamin Ks and 50 mM Tris, pH 7.5 in a final volume of 3 ml. The instrument was adjusted to read zero absorbance and at the time indicated, approximately 0.3 pmole of NADPH was added. When the reaction was completed, the diaphorase was inactivated by boiling for 5 min, the reaction mixture was centrifuged, the pH was adjusted to 7.6, and the following was added: 3 pmo1e.s of glucose B-phosphate (C-6-P) and approximately 2 units of crystalline glucose 6-phosphate dehydrogenase. Temperature = 24 C. Entamoeba

histolytica.

Entamoeha

histolytica:

~APHORASE A~I\‘ITIES TABLE

1”

193 II I

Xicotinamide Nucleotide Colltenl-of_Il,tacl Entamoeba histolyticaa

ntnoles/mg of protein

Nicotinamide nucleotide NADf NADH NAD+ + NADH XADP+ NtlDPH N.4I)P+ + NSDPH

1.5 0.7 2.2

1.0 0.3 1.::

a I~::tch nnmber is the average of at least i hree illdependent. drtePmilratiorls.

FIG. 6. End product inhibition of Entamoeba diaphorase. Numbers between curves are the ratios of NADPH: NADP’ or AcPyADP+ ( acetylpyridine adenine dinucleotide phosphate). Initial reaction rates were determined spectrophotometrically at 24 C. The reaction medium contained 50 mM Tris, pH 7.5; 20 mM dichlomrophenolindophenol; 330 PM NADPH; and 28 pg of protein of acetone powder extract in a final volume of 3 ml.

Izistolytica

malian respiratory chain, weakly inhibited the oxidation of NADPH only. In contrast, compounds known to chelate metals, particularly the transition metals (Keller and Parry 1956), were highly inhibitory to the amoeba1 diaphorase. This was observed in both spectrophotometric (Table V) and polarographic (Table VI) experiments. Table V illustrates that the inhibitors were equally effective with either TABLE

Inhibitor

NADPH ( Slater, Bailie, and Bouman 1961). In mitochondria, the concentration of NADP+ and NADPH is greater than that found in amoebae, and the nucleotide is largely in the reduced state. Effect of inhibitors. Oxidation of the reduced nicotinamide nucleotides was not affected by cyanide or antimycin, but was inhibited by p-chloromercuribenzoate, nand various antimalarial ethylmaleimide, drugs (Table IV). Only partial inhibition was obtained with dicoumarol, a potent inhibitor of mammalian nT-diaphorase (Ernster, Ljunggren, and Danielson 1960). With the exception of dicoumarol, the inhibitors were equally effective with either nucleotide as respiratory substrate. Amytal and rotenone, inhibitors of site 1 in the mam-

IV

Effect of Inhibitors on Edxnoeba histolytica Diaphorase* Concentration

Inhibition

(111M)

(79

NADH

-__ Cyanide Antimycin Rotenone Amyt’al Dicoumarol p-Chloromercuribenzoate iV-Ethylmaleimide Atabrine Chloroquine Primaquine Amodiaquin

1.0 10 pg/ml 0.05 2.0

-

0 0 0 0

NADPH 0 0 10

0.2

22

22 4x

0.3 6.0 5.0 5.0 3.0 5.0

88 85 53 83 81 70

x7 76 67 87 80 86 -

a Oxidation determined polarographically at 35 C. The cuvet,te contained 0.5 mill vitamin Ka, 1 mM NAD (P)H, and the supernatant fraction of sonicated amoebae (3.7 mg of protein) in a final volume of 1 ml of buffered saline. Other details as in the legend to Fig. 1.

194

WEINBACH

TABLE

V

Inhibition of Entamoeba histolytica Diaphorase as Determined Spectrophotometricallya Compound

Concentration required for 507o inhibition NADH Mf)

4,4,4-Trifluoro-l-(2-naphthyl)l,&butanedione 4,4,4-Trifluoro-l-(Bthienyl)1,8butanedione 4,4,4-Trifluoro-l-phenyl1,3-butanedione 4,4,4-Trifiuoro-l-(2-furyl)1,3-butanedione l,l,l-Trifluoro-2,4-hexanedione l,l,l-Trifluoro-2,4-pentanedione

NADPH Wf)

1.s

0.9

3.2

1.1

2.6

1.2

3.9 4.4 3.3

1.6 2.7 2.8

a Oxidation of NAD(P)H determined spectrophotometrically at 340 nm. The cuvette contained 0.5 mM vitamin K, 1 mM NAD(P)H, and the supernatant fraction of sonicated amoebae (0.12 mg of protein) in a final volume of 1 ml of buffered saline. Temperature = 24 C.

of the nicotinamide nucleotides. It also may be seen in these tables that similar data were obtained with two different types of amoeba1 diaphorase preparations and electron acceptors. The close correspondence of the data obtained by the two types of experiments is evidence that the inhibitors are reacting with sites on or in close proximity to the primary dehydrogenase proteins. Electron acceptors. The amoeba1 diaphorase transfers electrons to a variety of acceptors Table VII). The relative activity of these compounds was similar to that found by Ernster, Danielson, and Liunggren (1962) for the mammalian DT-diaphorase. Substituted benzoquinones were more effective than the napthoquinones. Cytochrome c was ineffective with either the mammalian or the amoeba1 diaphorases. Additional obsermtions. The amoeba1 diaphorase (NADPH) is remarkably stable. Enzymatic activity of partially purified preparations (e.g., 5.565% ammonium sul-

ET AL.

fate precipitate) retained complete activity for 2 weeks at 4 C. In order to prevent bacterial contamination, however, preparations were stored frozen at -20 C. Frozen preparations were fully active for at least 6 months. In this respect, the amoeba1 enzyme resembles the mammalian nr-diaphorase (Ernster, Danielson, and Ljunggren 1962) more closely than it does the bacterial enzyme ( Wosilait and Nason 1954). Polarographic determination of the pH optimum of the purified diaphorasc revealed essentially similar activity over a broad range in the region of pH 7.0-8.5. Enzymatic activity was negligible below pH 5.5 and above 9.5. TABLE Inhibition

VI

of E&amoeba histolgtica Diaphorase as Determined Polarographicallya Compound

Concentration required for 5O’Y ,* inhibition (Inn/)

Substit,uted but,anediones 4,4,4-Trifluoro-l- (2-naphthyl)1,3-butanedione 4,4,4-Trifluoro-l-phenyll&butanedione 4,4,4-Trifluoro-l-(2-thienyl)1,3-butanedione 4,4,4-Trifluoro-l-(2-furyl)1,3-butanedione I,l,l-Trifluoro-2,4-hexanedione l,l,l-Trifluoro-2,4-pentanedione 4,4,4-Trifluoro-I-(3-pyridyI)I ,3-butanedione

0.1 1.6 2.6 2.0 3.2 3.8 4.8

Iron reagents Bathophenanthroline Salicylaldoxime I,lO-Phenanthrolirre 01,or’-Dipyridyl 8-Hydroxyquinoline 2,2’,2”-Tripyridine

0.1 1.5 3.0 3.8 7.5 17.5

= Oxidation determined polarographically at 35 C. The cuvette contained 1 mM vitamin KS, 1 rnhf NADPH, and 9.4 rg of protein of the 55-6570 ammonium sulfate precipitate in a final volume of 1 ml of 50 m&l Tris-250 mM sucrose, pH 7.4.

Entamoeba

histolytica:

DISCUSSION

Evidence has been accumulated during this study showing that diaphorase activities may be recovered from the soluble fraction of axenically cultivated trophozoites of Entamoeba histolytica. Abundant data (Figs. 2, 3, 5, 6, Tables I and II ) have indicated that the amoeba1 diaphorase, unlike the mammalian nT-diaphorase discovered and studied extensively by Ernster and co-workers (for review, see Hall, Lind, Golvano, Rase, and Ernster 1972), is not associated with a single protein which catalyzes the oxidation of both NADH and NADPH. Catalytic activity is associated with at least two proteins of E. histolytica. One appears to react specifically with NADPH, whereas other proteins (seen as diffuse bands in Fig. 3, gels B and C) react with both nicotinamide nucleotides. A distinguishing characteristic of the DTdiaphorase is its extreme sensitivity to dicoumarol (complete inhibition at 1 PM; Ernster, Ljunggren, and Danielson 1960) ; in contrast, the amoeba1 enzyme is only feebly affected by this compound (Table IV). In some respects, however, the amoebal enzyme ( s) resembles the nT-diaphorase of mammalian tissues: enrichment of specific activities by acetone (Table I) and ammonium sulfate (Table II) precipitations, sensitivity to sulfhydryl reactants and flavoantagonists (Table IV), and relative effectiveness of artificial electron acceptors ( Table VII). The amoeba1 diaphorase does not appear to be analogous to the pyridine nucleotidemenadione reductase from Escherichia coli ( Wosilait and Nason 1954). The bacterial enzyme was only one-third as active in transferring electrons to vitamin K3 from NADPH as it was with NADH. In contrast, the most active amoeba1 enzyme was manyfold more effective with NADPH than it was with NADH (Table I). Other properties of the amoeba1 diaphorase which distinguish it front the bacterial enzyn~e

195

DIAPHORASE ACIWITIES TABLE

VII

ISlectron Acceptorsof Entamoeba histolytica

Diaphorase”

Acceptor 2,B-Dichlorophenolindophenol (DCPIP) 1,4-Naphthoquinone 2-Methyl-1,4-naphthoquinone (Vitamin Kg) Methyl-p-benzoquinone Cptochrome c

K,

Relative

(fiM)

activity6

7.3 11.5

100 17x

13.0 11.0 -

178 224 0.014

RThe reaction mixture consisted of 50 mM Tris, pH 7.5, 1 m111 NADPH, and 28 pg of protein of acetone powder extract in a total volume of 3 ml. For 1,4-naphthoquinone, methyl-p-benzoquinone, and vilamin K,, oxidation of NADPH was measured using Em#” = 6.22. For reduction of DCPIP, E ,,,,P” = 21.0; for reduction of cytochrome c, E m.~~6w = 18.3. All values were calculated from initial rates at 24 C. b Based on the rate with DCPIP taken arbitrarily as 100.

are its pH optimum, metal ion, and flavin requirements. Results of this study provide additional insight into the chemical composition of the prosthetic groups of the amoeba1 diaphorase. The inhibition observed with the antimalarial drugs, Atabrine, Chloroquine, Primaquine and Amodiaquin (Table IV) is presumed to involve flavin binding (Ernster, Danielson, and Ljunggren 1962). Recently, we have demonstrated by difference spectrophotometry the presence of flavoproteins in E. histolytica ( Weinbach, Harlow, Takeuchi, Diamond, Claggett, and Kon 1976). Although no accurate determination of the flavin content of the enzyme has been made, there is little doubt that the amoeba1 diaphorase( s) is a flavoprotein. We conclude from the data summarized in Tables V and VI that metals, particularly the transition metals, are essential for diaphorase activity. It is probable that the inhibition, observed both with the iron reagents and with the substituted butancdiones (Table VI), is due to chelation of

196

WEINBACH

the iron necessary for catalysis. This conclusion is supported by data presented elsewhere, disclosing the presence of ironsulfur centers in diaphorase preparations of E. histolytica (Weinbach, Diamond, Claggett, and Kon 1976; Weinbach, Harlow, Takeuchi, Diamond, Claggett, and Kon 1976). Our present concept of the physiological role of the amoeba1 diaphorase is that it provides a mechanism for the oxidation of NADH generated during glycolysis. Direct oxidation of NADH may occur in &o, but based on the in vitro experiments reported here, this reaction appears to be of little significance quantitatively. The recent discovery of transhydrogenase in E. histolytica (Harlow, Weinbach, and Diamond 1976) makes the following sequence plausible: NADH is oxidized by reducing NADP’. This reaction is mediated by transhydrogenase. The diaphorase, in the presence of oxygen, catalyzes the oxidation of NADPH, thereby restoring NADP’. Thus, transhydrogenase and diaphorase, functioning sequentially, maintain the NAD+ which is required for glycolysis to proceed. Although other mechanisms for oxidizing NADPH may be present, e.g., reduction of acetaldehyde to ethanol via alcohol dehydrogenase ‘(Reeves, Montalvo, and Lushbaugh 1971), the diaphorase-transhydrogenase system provides an aerobic mechanism for regulating the redox state of nicotinamide nucleotides. Indeed, this system may account for a major portion of the respiration observed with intact E. histolytica. ACKNOWLEDGMENTS We gratefully acknowledge the Richard W. Hendler in performing electrophoresis experiments, and Dr. for determining metal ion and flavin of the amoeba1 diaphorase.

help of Dr. the disc gel T. Takeuchi requirements

REFERENCES DIA~IONU, L. S. 1968. Techniques cuhi_ _ _ of axenic -_ _.

vation

of

Entamoeba

histolytica

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