Human Liver Aldehyde Oxidase: Differential Inhibition of Oxidation

The Journal of Clinical InvestigationVol. 46, No. 9, 1967

Human Liver Aldehyde Oxidase: Differential Inhibition ofOxidation of Charged and Uncharged Substrates *

DAVID G. JOHNS(From the Department of Pharmacology, Yale University School of Medicine,

New Haven, Connecticut)

Summary. Human liver aldehyde oxidase (aldehyde: 02 oxidoreductase,EC 1.2.3.1) has been purified 60-fold and some of its properties studied.Like aldehyde oxidase from other mammalian species, human liver aldehydeoxidase is an enzyme with dual substrate specificity, possessing the ability tocatalyze not only the oxidation of aldehydes to the corresponding carboxylicacids, but also the hydroxylation of a number of nonaldehydic heterocycliccompounds; its relative activity towards the latter group of substrates is low,however, when compared with that of liver aldehyde oxidase from rabbit andguinea pig. When the aromatic aldehyde benzaldehyde is used as substrate,human liver aldehyde oxidase, like the rabbit enzyme, is strongly inhibitedby menadione, estradiol-17,8, antimycin A, Triton X-100, and N-alkylpheno-thiazines; the human enzyme differs from the rabbit enzyme, however, in be-ing relatively insensitive to oligomycin and Amytal. Like the rabbit enzyme,the human enzyme can catalyze the 3-hydroxylation of phenazine methosul-fate (PMS) and the 6-hydroxylation of N-methylnicotinamide (NMN).With the rabbit enzyme, however, the aerobic hydroxylation of these sub-strates proceeds by a conventional mechanism, while 'with the human enzyme,the aerobic hydroxylation of PMS and NMN is anomalous in that the reactionis inhibited only by agents with affinity for the substrate-binding site, such ascyanide and N-alkylphenothiazines, and not by agents which inhibit the"internal electron transport chain" of the enzyme, such as menadione anddiethylstilbestrol. This mode of oxidation appears to be unique to sub-strates with a positively charged quaternary nitrogen; the hydroxylation ofother nonaldehydic heterocyclic substrates for the human enzyme is sensi-tive to conventional aldehyde oxidase inhibitors.

Introduction

The enzyme hepatic aldehyde oxidase (alde-hyde: 02 oxidoreductase, EC 1.2.3.1) was firstdescribed by Gordon, Green, and Subrahmanyan(1). They demonstrated that the enzyme was aflavoprotein distinct from the rather similar en-zyme xanthine oxidase (xanthine: 02 oxidoreduc-

* Submitted for publication 18 April 1967; accepted25 May 1967.

This work was supported by grants CA-02817 andCA-08010 from the U. S. Public Health Service.Address requests for reprints to Dr. David G. Johns,

Department of Pharmacology, Yale University School ofMedicine, 333 Cedar Street, New Haven, Conn. 06510.

tase, EC 1.2.3.2); like xanthine oxidase, however,it could catalyze the oxidation of a variety of ali-phatic and aromatic aldehydes. The source ofenzyme for these early studies was hog liver. In1946, Knox (2) demonstrated that aldehyde oxi-dase from rabbit liver possessed the ability tocatalyze not only the oxidation of aldehydes, butalso the hydroxylation of a number of nonalde-hydic heterocyclic compounds, e.g., N-methylnico-tinamide and quinine. He noted also that theability of the rabbit liver enzyme to catalyze theoxidation of the latter group of substrates wasmuch greater than that of aldehyde oxidase fromother mammalian species. Mahler, Mackler,

1492

HUMAN LIVER ALDEHYDE OXIDASE

Green, and Bock (3) further purified the hog liverenzyme, and demonstrated the presence of molyb-denum and iron. Hurwitz (4) demonstrated theability of rabbit liver aldehyde oxidase to catalyzethe oxidation of pyridoxal, and described a num-ber of properties of the enzyme, in particular, itsinhibition by cyanide and p-hydroxymercuribenzo-ate. Rajagopalan, Fridovich, and Handler (5),and Rajagopalan and Handler (6, 7) have pub-lished a detailed and comprehensive study of therabbit liver enzyme; they prepared rabbit liveraldehyde oxidase of greater than 90% purity anddemonstrated the presence of coenzyme Q10 inthe purified enzyme.Our attention was directed to this enzyme by

our finding that, in several species, it was thepreviously unidentified catalytic agent responsiblefor the 7-hydroxylation (8) and consequent bio-logical inactivation (9) of the anti-tumor agent3',5'-dichloromethotrexate, a 6-substituted 2,4-di-aminopteridine (10). We subsequently noted thatthe rabbit liver enzyme could catalyze also theoxidation of several pyrimidines (11) and purines(12) of pharmacologic interest. Since the rabbitliver enzyme has much greater catalytic activitytowards nonaldehydic heterocyclic compoundsthan does aldehyde oxidase from other species, itappeared that information on the properties ofhuman liver aldehyde oxidase was necessary be-fore these findings could be extended to man. Itwas found, however, that the human enzyme hadnot been described, despite the possible signifi-cance of this enzyme both in the oxidation of alde-hydes arising in the course of intermediary metab-olism (particularly in the metabolism of ethanoland its congeners) and in the hydroxylation ofpharmacologically active heterocyclic compounds.Studies concerned particularly with the sensitivityto inhibitors and with the substrate specificity ofhuman liver aldehyde oxidase were therefore car-ried out, and are described below.

Methods

Gifts of the following compounds are gratefully ac-knowledged: trifluoperazine, trimeprazine, chlorpromazine(CPZ), and N-methylphenothiazine from Dr. E. J. VanLoon, Smith, Kline & French Laboratories, Philadelphia,Pa.; promethazine and promazine from Dr. F. E. Ham-erslag, Wyeth Laboratories, Philadelphia, Pa.; 3',5'-dichloromethotrexate (DCM) from Dr. J. M. Rueg-

segger, Lederle Laboratories Div., American CyanamidCo., Pearl River, N. Y.; 2-hydroxy-5-fluoropyrimidinefrom Dr. Z. Budesinsky of the Research Institute ofPharmacy and Biochemistry, Prague; 4-hydroxypyrazolo[3,4-d]pyrimidine from Dr. G. B. Elion, Burroughs Well-come & Co., Tuckahoe, N. Y.; and disulfiram fromAyerst Laboratories, New York, N. Y. 2,6-Dichloro-phenolindophenol (DCIP), a,a-dipyridyl, and DEAE-cellulose were obtained from the Eastman Kodak Co.,Rochester, N. Y.; phenazine methosulfate (PMS), N-methylnicotinamide (NMN), diethylstilbestrol, coenzymeQio, progesterone, and p-chloromercuribenzoate from Cal-biochem, Los Angeles, Calif.; oligomycin, flavin adeninedinucleotide (FAD), flavin adenine mononucleotide(FMN), quinacrine, and ammonium sulfate (enzymegrade) from Mann Research Laboratories, Inc., NewYork, N. Y.; estradiol-17,6 from California Foundationfor Biochemical Research, Los Angeles, Calif.; benzalde-hyde, acetaldehyde, propionaldehyde, methanol, potassiumcyanide, ammonium chloride, and Versene Fe-3 fromFisher Scientific Company, Pittsburgh, Pa.; Amytal anddiethyldithiocarbamic acid from K & K Laboratories, Inc.,Plainview, N. Y.; o-chlorobenzaldehyde from ParagonTesting Laboratories, Orange, N. J.; o-nitrobenzaldehydefrom Matheson, Coleman, & Bell, E. Rutherford, N. J.;menadione and pyridoxal hydrochloride from NutritionalBiochemicals Corporation, Cleveland, Ohio; antimycin A,nicotinamide adenine dinucleotide (NAD), nicotinamideadenine dinucleotide reduced (NADH), nicotinamide ade-nine dinucleotide phosphate (NADP), nicotinamide ade-nine dinucleotide phosphate reduced (NADPH), fromSigma Chemical Co., St. Louis, Mo.; Triton X-100 fromRohm & Haas Co., Philadelphia, Pa.; and frozen maturerabbit liver from Pel-Freez Biologicals, Inc., Rogers,Arkansas.Enzyme assayvs. Spectrophotometric assay methods

were employed throughout. For assays at a single wave-length, a Gilford multiple sample absorbance recorderwith a Beckman DU monochromator was used; a Cary15 recording spectrophotometer equipped with a 0-0.1OD and 0-1.0 OD slide wire was used for serial assaysrequiring complete ultraviolet absorption spectra.

(a) Benzaldehyde as substrate. Examination of theultraviolet absorption spectra of benzaldehyde and ofbenzoate at equimolar concentrations at pH 7.8 revealedthat the latter compound lacked the strong absorptionmaximum of benzaldehyde at 249 mgu (2 max = 17,540)(Fig. 1). It therefore appeared possible that aldehydeoxidase activity could be assayed by monitoring the de-crease in absorption at 249 mt consequent upon the oxi-dation of benzaldehyde to benzoate. Serial spectra ofthe enzymic conversion of benzaldehyde as catalyzed bythe rabbit enzyme are shown in Fig. 2; details of theassay are given in the legend for the figure. In adaptingthe assay to the Gilford multiple sample absorbance re-corder, the 0-0.250 OD scale was used, and a cuvettewith all constituents of the reaction except benzaldehydeused as a blank. Sample cuvettes contained, in addition,5.6 X 10' M benzaldehyde. Because of the high proteinlevels required in the blank and sample cuvettes for

1493

DAVID G. JOHNS

0.6

0

0

0.4

0.2

BENZALDEHYDE

5 -5MAT pH 7.8

260

WAVELENGTH (mML)

FIG. 1. ULTRAVIOLET ABSORPTION SPECTRA OF BENZAL-

DEHYDE AND BENZOIC ACID IN SODIUM PHOSPHATE BUFFER

AT PH 7.8.

measurable activity at early stages' of the purification ofthe human enzyme, proper adjustment of the deuteriumlamp mirror for maximum light intensity at 249 m/Awas found to be essential. The assay could be performedsuccessfully with two randomly selected monochromatorsrequiring slit widths of 0.35 and 0.42 mm, respectively,when blanked against distilled water at 249 mi/h; theassay was not practicable with a third monochromatorrequiring a slit width of 1.02 mm when blanked underthe same conditions. Rates were measured over a periodof 2 min; detailed assay conditions are described in latersections.

(b) PMS as substrate. The spectra of PMS and itsoxidation product, methylpbenazine-3-one, have beenpublished by Rajagopalan and Handler (13). In theroutine assay used here, the sample cuvettes containedPMS, 0.5 Amole, sodium phosphate buffer, pH 7.8, 100/Amoles, Versene Fe-3, 15 gg, ammonium chloride, 100,umoles, and enzyme solution, containing varying amounts

of protein, 0.1 ml; total volume was 1 ml. The increasein absorption at 520 miu consequent on the oxidation ofPMS was monitored at 230C over a period of 4 minwith the Gilford multiple sample absorbance recorder;controls without substrate and without enzyme were in-cluded in all assays. A molar extinction coefficient of11,700 for methylphenazine-3-one at 520 miu was as-

sumed (13). Fresh PMS solutions were preparedhourly, as accumulation of enzyme-inhibitory PMS oxida-tion products was noted in PMS stock solutions exposedto air.

(c) Dichlorophenolindophenol (DCIP) as electron ac-

ceptor. The enzyme assays described above were per-formed aerobically, with molecular oxygen as electronacceptor; reaction rates were determined from disap-pearance of substrate, in the case of benzaldehyde, or

appearance of product, in the case of PMS. With ali-phatic aldehyde substrates, however, conversion of sub-strate to product was not accompanied by measurablespectral changes. In these instances, DCIP, rather thanoxygen, was used as electron acceptor, and the rate ofreaction followed by measuring the rate of reduction ofthis compound at 600 mA. In these assays, 0.25 ml of a

DCIP solution of 10 mg/100 ml was added to the cuvette;sodium phosphate, Versene Fe-3, and ammonium chlorideconcentrations were the same as in the PMS assay.Rates were measured over a period of 4 min at 230C'and a- molar extinction coefficient of 19,100 was assumedfor DCIP (14).

(d) Slowly oxidized substrates. The enzymic conver-sions of 2-hydroxy-5-fluoropyrimidine to 5-fluorouracil(11), DCM to 7-hydroxyDCM, and NMN to NMN-6-pyridone were followed by serial ultraviolet absorption

I.0

0.8

5

0.6

0

3

0.4

4

0.2

210 230 250 270 290 310

WAVELENGTH (mj )

FIG. 2. ENZYMIC CONVERSION OF BENZALDEHYDE TOBENZOATE BY RABBIT LIVER ALDEHYDE OXIDASE. The refer-ence cuvette contained sodium phosphate buffer, pH 7.8,500 gmoles, Versene Fe-3, 20 jsg, and rabbit liver alde-hyde oxidase, 45 Iug, in a total volume of 3 ml. Thesample cuvette contained the same constituents, plus benz-aldehyde, 0.17 Smole. Spectra were recorded at 4-minintervals.

1494


spectra, with the Cary 15 recording spectrophotometer.Reference cuvettes contained sodium phosphate buffer, pH7.8, 500 /moles (for DCM, buffer of pH 6.9 was used),Versene Fe-3, 20 ltg, ammonium chloride, 600 /moles, andenzyme, in a total volume of 3 ml. The sample cuvettecontained the same constituents plus substrate. Since therate of hydroxylation of these three substrates was slowwhen the human enzyme was used, maximum possibleamounts of enzyme protein were added to reference andsample cuvettes, compatible with accurate recording ofspectra of substrate and product.

Partial purification of rabbit liver aldehyde oxidase.Aldehyde oxidase was purified approximately 90-foldfrom rabbit liver by ammonium sulfate fractionation andchromatography on Sephadex G-75 and DEAE-cellulose,as previously described (10). Enzyme activity was as-

sayed with DCM as substrate, and protein was deter-mined by the method of Waddell (15).

Partial purification of human liver aldehyde oxidase.It early became apparent that fresh human liver biopsymaterial would yield insufficient enzyme for comprehen-sive studies. Previous studies with the rabbit, mouse,

guinea pig, and monkey enzymes indicated, however, thataldehyde oxidase was recoverable from the livers ofthese species with no detectable alteration in yield or inproperties, as long as 24 hr after death, provided thatthe livers were stored cold (but not frozen) during thisinterval. For partial purification of the human enzyme,

therefore, postmortem human livers were obtained at timeof autopsy, within 4 hr after death, with the cooperationof the Department of Pathology, Yale University Schoolof Medicine. Livers from patients with a history ofhepatic disease, and livers with gross pathologicalchanges, were excluded; otherwise no selection was made.A total of nine livers was used in this study, fromthree female and six male subjects ranging in age from4 to 71 yr. No marked variation in enzyme activity was

noted among livers from which enzyme was preparedon the day of collection; two livers which were frozenafter collection and used for enzyme preparation some

weeks later were found to have much lower enzyme ac-

tivity. A similar observation of low yield of aldehydeoxidase from horse liver after freezing and thawingwas reported by Carpenter (16).Some studies were repeated with enzyme from fresh

wedge biopsy material obtained at laparotomy; no dif-ferences were noted from enzyme obtained from nec-

ropsy liver.Livers were homogenized for 1 min in 2.5 volumes of

0.05 M sodium phosphate buffer, pH 7.8, containing Ver-sene Fe-3, 0.005%, the homogenate centrifuged at 14,600g at 40C for 30 min, and an aliquot of the supernatantfraction removed for determination of enzyme activity.The supernatant was then heated with continuous stirringfor 10 min at 60'C in a water bath. Heat-precipitatedprotein was removed by centrifuging at 14,600 g for30 min, the supernatant fraction collected, and sufficientsaturated ammoniacal ammonium sulfate solution addedto give 45%o saturation; saturated ammoniacal ammonium

sulfate solution was prepared by adding 60 ml of concen-trated (29%o) ammonium hydroxide solution to 940 ml ofsaturated ammonium sulfate solution. The supernatantsolution was allowed to stir slowly at room temperaturefor 30 min, and the precipitate recovered by centrifugingas above. The precipitate was extracted with sufficientammoniacal ammonium sulfate solution of 30%o saturationto give a protein concentration in the supernatant of ap-proximately 25 mg/ml (in a typical preparation, this re-quired 120 ml of ammonium sulfate solution when liver ofstarting weight 1300 g was used). The supernatantwas recovered by centrifugation, and the precipitate sub-jected to successive reextractions with ammoniacal am-monium sulfate of 15%o saturation, and with distilled wa-ter. Typically, most of the aldehyde oxidase activitywas present in the 15-30% ammonium sulfate fraction,although activity was usually detectable in the 0-15%ammonium sulfate fraction; if the specific activity of thelatter approached that of the 15-30% fraction, the twofractions were combined before the next step, whichotherwise was performed on the 15-30%o ammonium sul-fate fraction alone.

In earlier runs, salts and diffusible low molecularweight compounds were removed at this stage of the puri-fication by overnight dialysis of the 15-30%o ammoniumsulfate fraction against distilled water at 40C; it was laterfound, however, that an alternate procedure, desalting bygel filtration through Sephadex G-75, resulted in less lossof enzyme activity then did overnight dialysis, possiblybecause gel filtration could be carried out much morerapidly. In a typical run, enzyme solution containing320 mg of protein was applied to a Sephadex G-75 col-umn, 2 X 18 cm, previously washed for several hourswith distilled water. The protein solution obtainedfrom the Sephadex column was then applied to a col-umn of DEAE-cellulose, which had been preequilibratedwith distilled water, and eluted stepwise with Tris-chloride buffers, pH 7.5, of concentrations 0.05, 0.15, 0.25,and 0.50 mole/liter. In a representative run, 280 mg ofprotein was applied to a DEAE-cellulose column of 2 X18 cm; the enzyme activity typically appeared in the0.25 M Tris effluent, although activity was often detectablein the 0.15 M Tris effluent also.The 0.25 M Tris effluent from several such columns

was combined and concentrated osmotically to 0.1 vol-ume by applying powdered sucrose to the external sur-face of a dialysis bag containing the protein solution.Protein was precipitated again from the concentratedTris effluent by adding saturated ammoniacal ammoniumsulfate solution until 45%o saturation was reached. Theprecipitated protein was redissolved in distilled water togive a solution of approximately 5 mg/ml. The purifi-cation procedure for the human enzyme is summarized inTable I.For determination of the specific activity of the frac-

tions obtained during purification, the benzaldehyde as-say described above was used. Before assay, aliquotsfrom the early fractions were subjected to recentrifuga-tion at 105,000 g for 1 hr in a Spinco model L centrifuge;

1495

DAVID G. JOHNS

TABLE I

Partial purification of human liver aldehyde oxidase

Total Total Total SpecificFraction volume protein activity activity Yield Purification

ml mg units* units/mg % -foldSupernatant from crude homogenate 4000 260,000 3900 0.015Supernatant from crude homogenate 3550 124,000 3100 0.025 79 1.7

after heat treatment (60'C for10 min)

Ammonium sulfate (15-30%) 140 5,379 1824 0.339 47 23DEAE-cellulose (precipitated with 262 1,362 1248 0.915 32 61ammonium sulfate and redissolved)

* One unit = amount of enzyme producing A249 mMu of 1 optical density unit per 10 min at 230C with benzaldehyde,5.6 X 10-5 mole per liter, as substrate. In all assays, activity was determined from initial rates.

assays were carried out on the supernatant.' The otherassay methods described above, although of value withthe partially purified enzyme, were not useful for deter-mining over-all fold purification, since they were notapplicable to the determination of aldehyde oxidase ac-

tivity in supernatants from crude homogenates: the DCIPassay gave a high blank rate in the absence of sub-strate,2 while the PMS assay gave a spuriously high ratewith crude homogenates, due possibly to the presence ofone or more enzymes, in addition to aldehyde oxidase,with PMS oxidase activity. This additional PMS oxi-dase activity could not be inhibited by cyanide, quinacrine,or methanol, and therefore was not attributable to alde-

1 Rajagopalan, Fridovich, and Handler (5) have shownrabbit liver aldehyde oxidase to be a supernatant en-

zyme; the hog liver enzyme appears to be mitochondrial,but is readily released into the supernatant fractionduring homogenization (17).2A similar observation was reported by Palmer, with

the hog liver enzyme (18).

hyde oxidase (5). The benzaldehyde oxidase activity ofthe crude fractions, on the other hand, was completelyinhibited by cyanide (1 X 10' mole per liter), diethyl-stilbestrol (4 X 10' mole per liter), and menadione (2.5 X10' mole per liter), and is therefore tentatively attribu-table to aldehyde oxidase; the validity of these assump-

tions is discussed in a later section.The rapid assay methods used in the purification of

the rabbit liver enzyme by Rajagopalan, Fridovich, andHandler (5) (the conversion of NMN to its 6-pyridone)and by the present author (10) (the conversion of DCMto 7-hydroxyDCM) were of no value in determining foldpurification of the human enzyme: the rates of oxidationwere too low to be measured in crude fractions, and theconversion of these substrates could be demonstratedspectrophotometrically only with partially purified hu-man liver aldehyde oxidase.Most of the studies described below were carried out

both with human liver aldehyde oxidase purified 20- to30-fold (the "15-30% ammonium sulfate enzyme") and

TABLE II

Effect of inhibitors on the conversion of benzaldehyde to benzoate by human liver aldehyde oxidase*

Inhibitor concentration Inhibition

a,a-Dipyridyl 1 X 10-4 mole per liter 53Chlorpromazine 5 X 10-1 mole per liter 51Diethylstilbestrol 4 X 10-v mole per liter 431Progesterone 6 X 10-s mole per liter 71§p-Hydroxymercuribenzoate 2 X 10-5 mole per liter 88Oligomycin 60 /g/ml 28Quinacrine 5 X 10-5 mole per liter 51Estradiol-17t7 8 X 10-8 mole per liter 50$Methanol 0.3 mole per liter 67Potassium cyanide 1 X 10-4 mole per liter 66Antimycin A 8 ,ug/ml 46$Triton X-100 8 X 10-4 %o 65Menadione 1.5 X 10-5 mole per liter 55Testosterone 6 X 10-5 mole per liter 44

* Sample cuvettes contained benzaldehyde, 0.057jumole, sodium phosphate buffer, pH 7.8, 100,gmoles, Versene Fe-3,15 ,ug, ammonium chloride, 100 Amoles, and enzyme solution, 0.03 ml, in a total volume of 1 ml. Initial rates weremeasured, except where otherwise noted.

T At the end of 5 min.§ At the end of 3 min.

1496


40- to 60-fold (the "DEAE-cellulose enzyme"). No sig-nificant differences were noted in the human enzyme atthese two stages of purification.

Results

Inhibitors of benzaldehyde oxidationBenzaldehyde (5.6 X 10-5 mole per liter) was

used as substrate, and enzyme activity monitoredat 230C by measuring the rate of conversion tobenzoate at 249 m/ (see above).

(a) Steroids as inhibitors. As shown in TableII, the oxidation of benzaldehyde to benzoate byhuman liver aldehyde oxidase was inhibited by avariety of agents: the inhibition by the steroidestradiol- 17,f and the nonsteroidal estrogen di-ethylstilbestrol was particularly marked (Fig. 3).Estradiol-17,8 is the most potent inhibitor of hu-man liver aldehyde oxidase found in the presentstudies; 50%o inhibition of the rate of oxidationwas noted at an estradiol concentration of 8 X 10-8mole per liter, when control and inhibited rateswere measured 5 min after the start of the reac-tion. The slow development of maximal inhibitionat low estradiol concentrations is apparently dueto a slow rate of binding of the steroid to theenzyme: when estradiol (8 x 10-8 mole per liter)

* CONTROLo DIETHYLSTILBESTROL 4 x 10- 7 M

0.100 -A -ESTRADIOL 2xlO7 Mx PROGESTERONE

6x10 M,

E

~j.0.050

0

O0 1 2 3 -4 5MINUTES

FIG. 3. INHIBITION OF HUMAN LIVER ALDEHYDE OXI-DASE BY ESTROGENS AND PROGESTERONE. The rate of oxi-dation of benzaldehyde (5.6 X 10' mole per liter) wasfollowed by the change in optical density at 249 m,;reaction conditions were identical to those given inTable VII.

> 7CH3

N - METHYLPHENAZINE (PMS)S

NC

(C H2)3

CH3 -N - CH3

CHLORPROMAZINE (CPZ)

FIG. 4. STRUCTURAL FORMULAS OF N-METHYLPHENA-ZINE AND CHLORPROMAZINE.

and enzyme were preincubated for 5 min beforethe reaction was started by adding substrate,maximal inhibition was noted at zero time. Thesituation thus differs from that seen with antimycinA, where preincubation did not shorten the pe-riod required for maximal inhibition to develop(see below).The nonphenolic steroids progesterone and tes-

tosterone were much weaker inhibitors of theenzyme.

(b) N-alkylphenothiazines as inhibitors. Thepossible activity of N-alkylphenothiazines as in-hibitors of human liver aldehyde oxidase was sug-gested by the resemblance of the phenothiazinestructure to that of the known aldehyde oxidasesubstrate PMS (N-methylphenazine) (Fig. 4).The N-alkylphenothiazines studied here are in-active as substrates for the enzyme; this inactivityappears to be due, not to the substitution of sulfurfor one of the ring nitrogens, but to the bulkyN-alkyl side-chains of the phenothiazines tested:N-methylphenothiazine, unlike the other membersof this group, but like N-methylphenazine (PMS),possesses substrate activity for the rabbit liverenzyme.3The N-alkylphenothiazines are more potent in-

hibitors of the human enzyme than of the rabbitenzyme, irrespective of whether benzaldehyde orPMS is used as a substrate (Tables III and IV):with benzaldehyde (5.7 x 10-5 mole per liter) as

3 G. S. Farnham and D. G. Johns. Unpublished data.

1497

DAVID G. JOHNS

CPZ (moles per liter x 105)FIG. 5. COMPETITIVE INHIBITION BY CHLORPROMAZINE OF OXIDATION OF

PMS (5 X 10' MOLE PER LITER) BY ALDEHYDE OXIDASE FROM HUMAN LIVER

AND RABBIT LIVER (DIXON PLOT). Reaction conditions were identical to thosegiven in Table IV. AO, aldehyde oxidase; CPZ, chlorpromazine.

substrate, the level of chlorpromazine (CPZ) re-quired for 50%to inhibition was 2 x 10-5 mole perliter in the case of the rabbit enzyme, but 5 x 10-6mole per liter in the case of the human enzyme.Both with the rabbit and with the human enzyme,CPZ is formally competitive with substrate (Fig.5).

(c) Inhibitors of electron transport. AntimycinA, a compound active in mitochondrial systems asan inhibitor at a site subsequent to the reduction

TABLE III

N-Alkylphenothiazines as inhibitors of the conversion ofbenzaldehyde to benzoate by aldehyde oxidasefrom human

liver and rabbit liver*

Inhibition

Human RabbitInhibitor* enzyme enzyme

Promethazine 32 53Promazine 41 63Trimeprazine 49 85Chlorpromazine 51 50Trifluoperazine 72 63

* Control cuvettes contained benzaldehyde, 0.057;mole, sodium phosphate buffer, pH 7.8, 100 pmoles,Versene Fe-3, 15 pg, ammonium chloride, 100 ;smoles, andenzyme solution, 0.03 ml, in a total volume of 1 ml.

T Inhibitor concentrations were 5 X 10-6 mole per literfor assays with human liver aldehyde oxidase, and 2 X 10-6mole per liter for assays with rabbit liver aldehyde oxidase.

of coenzyme Q (19), was effective as an inhibitorof benzaldehyde oxidation by human liver alde-hyde oxidase (Fig. 6; Table V); the level re-quired was slightly higher than that required togive equivalent inhibition of the rabbit liver en-zyme. At low levels of antimycin A, maximal in-hibition was not seen until several minutes afterthe start of the reaction; the rate of onset of inhibi-tion was not increased by preincubation. As withthe rabbit enzyme (6), antimycin A may, there-

TABLE IV

N-Alkylphenothiazines as inhibitors of the 3-hydroxylationof PMS by aldehyde oxidase from human liver

and rabbit liver*

Inhibition

Human RabbitInhibitor: enzyme enzyme

Trifluoperazine 30 70Promazine 43 79Promethazine 44 62Chlorpromazine 45 48Trimeprazine 60 88

* Control cuvettes contained PMS, 0.5 gmole, sodiumphosphate buffer, pH 7.8, 100 itmoles, Versene Fe-3,15 jig, ammonium chloride, 100 jmoles, and enzyme solu-tion, 0.1 ml, in a total volume of 1 ml.

t Inhibitor concentrations were 5 X 10-6 mole per literfor assays with human liver aldehyde oxidase, and 2 X 10-5mole per liter for assays with rabbit liver aldehyde oxidase.

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TABLE V

Electron transfer inhibitors: relative effectiveness as inhibitors of aldehyde oxidase fromhuman liver and rabbit liver

Concentration of inhibitor required to reduce reaction rateto 50% of control rate

Enzyme source Substrate Antimycin A Oligomycin Amytal

pg/mi Ag/mi moles/literRabbit liver Benzaldehyde* 2 0.4 3 X 10-4Human liver Benzaldehyde* 8 >60T >3 X 10-4§Rabbit liver PMSI' 2 0.4 5 X 10-4Human liver PMSII >40 >60 >1 X 10-3

* Control cuvettes contained benzaldehyde, 0.057 Mole, sodium phosphate buffer, pH 7.8, 100 Mmoles, Versene Fe-3,15 pg, ammonium chloride, 100 jsmoles, and enzyme solution, 0.03 ml, in a total volume of 1 ml.

$ Slight inhibition was noted at this concentration (see Table II).§ Higher Amytal concentrations could not be used because of the absorption of this compound at the wavelength

used for the assay (249 mM).11 Sample cuvettes contained PMS, 0.5 ;&mole, sodium phosphate buffer, pH 7.8, 100 Mumoles, Versene Fe-3, 15 Mg,

ammonium chloride, 100 ;moles, and enzyme solution, 0.1 ml, in a total volume of 1 ml.

fore, react with a reduced form of the humanenzyme.

By contrast, oligomycin and Amytal, both ef-fective inhibitors of the rabbit liver enzyme (6),were ineffective as inhibitors of human liver alde-hyde oxidase (Table V). Slight inhibition of thehuman enzyme was noted when the oligomycinconcentration was raised to 60 ,pg/ml; this concen-

tration, however, is of a different order of mag-

nitude from that required to inhibit the oxidationof benzaldehyde by the rabbit liver enzyme underidentical conditions (0.4 jug of oligomycin per ml)(Table V).

(d) Other inhibitors. Cyanide, quinacrine,methanol, p-hydroxymercuribenzoate, menadione,and Triton X-100 were effective inhibitors of hu-

* CONTROL

0 20jLG ANTIMYCIN A /ML

:1.

E0M 0.100

04

4 aO5O

00

0 1 2 3 4 5MINUTES

FIG. 6. INHIBITION OF HUMAN LIVER ALDEHYDE OXI-

DASE BY ANTIMYCIN A. Substrate: benzaldehyde, 5.6 X10' mole per liter.

man liver aldehyde oxidase at levels comparableto those reported for the rabbit liver enzyme (6)(Table II). The iron-chelating agent, a,a-di-pyridyl, was an effective inhibitor of the oxidationof benzaldehyde by the human enzyme at a con-

centration of 1 x 10-4 mole per liter. 4-Hydroxy-pyrazolo [3,4-d] pyrimidine (3 x 104 mole per

liter), a highly potent inhibitor of human liverxanthine oxidase (20), was ineffective as an in-hibitor of human liver aldehyde oxidase.

(e) Coenzymes. NAD, NADH, NADP, andNADPH had no effect on the rate of oxidation ofbenzaldehyde by the 40- to 60-fold purified hu-man enzyme. Some oxidation of NADH to NADwas noted in crude fractions; further investigationshowed this to be due to the presence of alcoholdehydrogenase, catalyzing the reduction of ben-zaldehyde to benzyl alcohol. DEAE-cellulose

TABLE VI

Aldehydes as substrates for human liveraldehyae oxidase*

Initial reaction velocity(Benzaldehyde = 100)

Benzaldehyde 100Propionaldehyde 97Acetaldehyde 73Pyridoxal 25o-Chlorobenzaldehyde 18o-Nitrobenzaldehyde <5Formaldehyde <5

* Sample cuvettes contained substrate, 2 Mmoles, DCIP,25 Mg, sodium phosphate buffer, pH 7.8, 100 Amoles,Versene Fe-3, 15 yg, ammonium chloride, 100 Mmoles, andenzyme solution, 0.1 ml, in a total volume of 1 ml. Reac-tion rates were determined by monitoring the rate ofreduction of DCIP, at 600 myA.

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DAVID G. JOHNS

3

2

Km = I.x IO 5moles per liter

I_________ _ x 10 4

[BENZALDE HYDE]FIG. 7. OXIDATION OF BENZALDEHYDE BY HUMAN

LIVER ALDEHYDE OXIDASE (LINEWEAVER-BURK PLOT).Sample cuvettes contained sodium phosphate buffer, pH7.8, 100 /Amoles, Versene Fe-3, 15 lig, ammonium chloride,100 /Amoles, enzyme solution, 0.02 ml, and substrate asindicated, in a total volume of 1 ml. The rate of oxida-tion of benzaldehyde was followed by the change inoptical density at 249 mu.

chromatography separated all alcohol dehydroge-nase activity from the aldehyde oxidase prepara-tion.Added FAD, FMN, and coenzyme Q10 did not

affect the rate of oxidation of benzaldehyde by thehuman enzymye.

Other aldehyde substrates

A number of aliphatic and aromatic aldehydesserved as substrates for human liver aldehyde oxi-dase; in no instance, however, did the maximalreaction velocity exceed that of benzaldehyde(Table VI). The apparent Km of benzaldehydefor the human enzyme, as determined by the Line-weaver-Burk method, was extremely low for thisenzyme (1.1 x 10-5 mole per liter) (Fig. 7); forother substrates, Michaelis constants ranged from5 X 10-5 to 5 X 103 mole per liter. Formalde-hyde, unlike the other aliphatic aldehydes tested,was an extremely poor substrate for human liveraldehyde oxidase; a similar observation was re-ported by Palmer (18) for the hog liver enzyme.Early workers (1), using the hog liver enzyme,

suggested that the low substrate activity withformaldehyde might be due to denaturation of theenzyme by this compound; this view would notappear to be tenable with human liver aldehydeoxidase, since the oxidation of the substrate PMSby the human enzyme was not significantly in-hibited by formaldehyde (6 x 10-3 mole per liter),although marked inhibition, as a consequence ofcompetition for the substrate-binding site, wasnoted in the presence of other aliphatic aldehydes(see below).

Inhibitors of PMS oxidation by human liver al-dehyde oxidase

It early became apparent from inhibition stud-ies that the mode of PMS oxidation by humanliver aldehyde oxidase differed in some importantrespects from the mode of oxidation of benzalde-hyde. Parallel control studies with the rabbitliver enzyme, on the other hand, showed benzalde-hyde and PMS to be handled similarly by thelatter enzyme.

(a) Inhibitors equally effective against PMSand benzaldehyde oxidation by human liver alde-hyde oxidase. Cyanide, quinacrine, N-alkylpheno-thiazines (Tables III and IV), methanol, and

ZLE0

4

00

0.100

0.050

.Ax

0A00

CONTROL -3FORMALDEHYDE 6x10 MACETALDEHYDE 6x 10- M2-OH-5-F-PYRIMIDINE lxi-3 MBENZALDEHYDE 4 x 10 -4MDICHLOROMETHOTREXATE2xlO-4M /l

2 3 4MINUTES

5

FIG. 8. INHIBITION BY OTHER SUBSTRATES OF OXIDA-TION OF PMS (5 X 10' MOLE PER LITER) BY HUMANLIVER ALDEHYDE OXIDASE. The rate of conversion of PMSto N-methylphenazine-3-one was followed by the change inoptical density at 520 my; reaction conditions were iden-tical to those given in Table IV.

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TABLE VII

Estrogens and progesterone as inhibitors of human liver aldehyde oxidase

Concentration of inhibitor required to reduce reaction rate to 50% of control

Substrate Estradiol-17,6 Progesterone Diethylstilbestrol

moles/literBenzaldehyde* 8 X 10-8 4 X 10-5 5 X 10-vPMSj 1 X 10-4 >4 X 10-5 (8 X 10-6)§

* Sample cuvettes contained benzaldehyde, 0.056 jsmole, sodium phosphate buffer, pH 7.8, 100 gmoles, VerseneFe-3, 15 jug, ammonium chloride, 100 /umoles, and enzyme solution, 0.03 ml, in a total volume of 1 ml. Rates weremeasured 5 min after the start of the reaction.

t Sample cuvettes contained PMS, 0.5 Mmole, sodium phosphate buffer, pH 7.8, 100 Mmoles, Versene Fe-3, 15 Mg,ammonium chloride, 100 Mmoles, and enzyme solution, 0.1 ml, in a total volume of 1 ml. Rates were measured 5 minafter the start of the reaction.

§ Stimulation, 50% maximal.

p-hydroxymercuribenzoate were equally effectiveas inhibitors of the oxidation of both PMS andbenzaldehyde.

Other substrates for the enzymes, both alde-hydes and nonaldehydic heterocyclic substrates,were effective inhibitors of PMS oxidation (Fig.8), a result compatible with utilization of the samesubstrate-binding site for both aldehyde and non-aldehydic heterocyclic substrates. Benzaldehyde(4 x 10-4 mole per liter) was particularly effectiveas an inhibitor of PMS oxidation. Formaldehyde,as noted above, apparently has low affinity forthe substrate-binding site of human liver aldehydeoxidase, as indicated by its very slight ability toinhibit PMS oxidation, shown here, and its lowsubstrate activity (Table VI).

(b) Compounds effective as inhibitors of ben-zaldehyde oxidation, and ineffective as inhibitorsof PMS oxidation by human liver aldehyde oxi-dase. Menadione, a strikingly effective inhibitorof the oxidation of both benzaldehyde and of PMSby the rabbit liver enzyme, and of benzaldehydeby the human liver enzyme, is ineffective in in-hibiting the oxidation of PMS by human liveraldehyde oxidase (Fig. 9). Similarly, the ster-oid inhibitors (Table VII), Triton X-100 (5 x10-4%), and antimycin A (Table V), highly po-tent inhibitors of benzaldehyde oxidation, are in-effective as inhibitors of PMS oxidation by thehuman enzyme.

(c) Compounds effective as inhibitors of benz-aldehyde oxidation which stimulate the rate ofPMS oxidation by human liver aldehyde oxidase.Diethylstilbestrol, a potent inhibitor of benzalde-hyde oxidation by the human enzyme (Table II),stimulates significantly the rate of PMS oxidation

(Fig. 10); the maximal rate reached was 165%that of the control. Half-maximal stimulationwas seen at a diethylstilbestrol concentration of8 x 10-6 mole per liter. Stimulation of PMS oxi-dation was also noted with the sulfhydryl com-pound diethyldithiocarbamic acid, a weak inhibitorof benzaldehyde oxidation. The mode of stimula-tion of PMS oxidation by the two compoundswould appear to differ, since the effects were ad-ditive when both were added at levels sufficientto give a maximal effect with either compoundalone (Fig. 11).No examples were found of compounds effective

as inhibitors of PMS oxidation and ineffectiveas inhibitors of benzaldehyde oxidation by humanliver aldehyde oxidase.

0.6

E 0.4-0

O /~~~~~~~~~~

< 0.2 2.

MINUTESFIG. 9. DIFFERENTIAL INHIBITION BY MENADIONE (2.5

X 10-5 MOLE PER LITER) OF OXIDATION OF PMS (5 X 10'MOLE PER LITER) BY ALDEHYDE OXIDASE FROM RABBIT LIVERAND HUMAN LIVER. Experimental conditions were identi-cal to those given in Table IV.

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DAVID G. JOHNS

Z 1500

0

0I~

z

01004I-

ax0

cn

7 50a.UL0

w

I-

a:- n

I0 6

DIETHYLSTILBESTROL (moles per liter)

FIG. 10. STIMULATION BY DIETHYLSTILBESTROL OF OXIDATION OF PMS(5 X 10-' MOLE PER LITER) BY HUMAN LIVER ALDEHYDE OXIDASE. Experi-mental conditions were identical to those given in Table IV. Diethylstilbes-trol was added in ethanol solution (0.01-0.02 ml); corresponding volumesof ethanol alone were added to control cuvettes.

Other nonaldehydic heterocyclic substrates

The oxidation of N-methylnicotinamide, which,like PMS, has a positively charged quaternary ni-trogen, followed the same pattern as that of PMS,

O CONTROLo DDCA 8x10 5M

0.150 DES 6x 105MX DDCA DES

E

0.100

N X

I-

0

0.050

1 2 3 4

MINUTES

FIG. 11. ADDITIVE STIMULATION BY DIETHYLSTILBES-

TROL, DES, (2 X 10' MOLE PER LITER) AND DIETHYLDI-

THIOCARBAMIC ACID, DDCA, (8 X 10' MOLE PER LITER)OF OXIDATION OF PMS (5 X 10' MOLE PER LITER) BY

HUMAN LIVER ALDEHYDE OXIDASE. Diethylstilbestrol anddiethyldithiocarbamic acid were added in ethanol solu-tion; corresponding volumes of ethanol alone were addedto control cuvettes.

i.e., the reaction was sensitive to inhibition by cy-anide, quinacrine, and N-alkylphenothiazines, butnot to inhibition by menadione or diethylstilbestrol.In contrast, the oxidation of the uncharged non-aldehydic substrate 2-hydroxy-5-fluoropyrimidinewas sensitive to both the latter agents (TableVIII); thus, this compound, like benzaldehyde,apparently utilizes the complete "internal electrontransport chain" of aldehyde oxidase.

TABLE VIII

Effect of inhibitors on the conversion of 2-hydroxy-5-fluoro-pyrimidine to 5-fluorouracil by human liver

aldehyde oxidase*

Inhibitorconcentration Inhibition

moles/liter %Potassium cyanide 1 X 10-4 71Menadione 2.5 X 10-5 46Diethylstilbestrol 3.3 X 10-5 93

* Reference cuvettes contained sodium phosphate buffer,pH 7.8, 500 ,umoles, Versene Fe-3, 20 jsg, ammoniumchloride, 600,gmoles, and enzyme, 0.1 ml, in a total volumeof 3 ml. Sample cuvettes contained the same constituents,plus 2-hydroxy-5-fluoropyrimidine, 0.54 pmole. Ultra-violet absorption spectra were recorded at 20-min intervals,using a Cary 15 recording spectrophotometer with a0-1.0 OD slide wire. Reaction rates were determinedfrom the rate of decrease in the absorption maximum of thesubstrate at 310 mMA (11).

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0

lo-410- 5


Discussion

The careful delineation of the "internal elec-tron transport chain" of rabbit liver aldehyde oxi-dase by Rajagopalan and Handler (6) permits aready explanation of the results which have beenobtained in inhibition studies with the human en-zyme. As these authors have demonstrated, cy-anide, p-hydroxymercuribenzoate, methanol, andquinacrine attack the enzyme at or near the sub-strate-binding site. The present studies suggestthat N-alkylphenothiazines, being formally com-petitive with substrate, also bind at the substratesite, irrespective of whether the rabbit or the hu-man enzyme is used. All these agents are ef-fective in inhibiting the oxidation of either chargedor uncharged substrates by the human enzyme.However, a second class of inhibitors, menadione,Triton X-100, antimycin A, and the steroids, ap-parently influence the reduction and reoxidation ofthe components of the "internal electron transportchain" of the rabbit enzyme (molybdenum, FAD,coenzyme Q1,O and iron) and do not bind at thesubstrate site [Rajagopalan and Handler (6)].These agents do not affect the oxidation ofcharged substrates such as PMS and NMN byhuman liver aldehyde oxidase, but are highly ef-fective inhibitors of the oxidation of unchargedsubstrates such as benzaldehyde and 2-hydroxy-5-fluoropyrimidine, when oxygen is used as electronacceptor. It would appear therefore that, underthe conditions used in the present studies, the un-charged substrates for the human enzyme utilizethe entire "internal electron transport chain" ofthe enzyme, and are sensitive to inhibitors whichact at any site of the chain, while positively chargedsubstrates can utilize only the substrate site, andare sensitive solely to inhibitors binding at thelatter. Thus, whereas with the rabbit enzyme,under aerobic conditions, the electronic structureof the substrate appears to be of little importancein determining the route of electron transport(7), with the human enzyme, the electronic struc-ture of the substrate is of much greater sig-nificance.The present studies do not establish whether

molecular oxygen acts as electron acceptor in theoxidation of the charged substrates by the humanenzyme, or whether the substrate itself serves thisfunction: Rajagopalan and Handler (13) have

shown that, with the rabbit liver enzyme, underanaerobic conditions, PMS can act as an electronacceptor for aldehyde oxidase; and thus, whenPMS is used anaerobically as a substrate for therabbit enzyme, equimolar amounts of N-methyl-phenazine-3-one and leuko PMS are generated.Such a situation may apply with the human en-zyme even under aerobic conditions: since thesite of electron egress is early in the chain whenPMS is used as electron acceptor (6), the in-sensitivity of PMS oxidation by the human en-zyme to electron chain inhibitors such as mena-dione, which act late in the electron transfer path-way, would thus be accounted for.The unusual situation of a compound such as

diethylstilbestrol acting as either an inhibitor orstimulator of the catalytic activity of an enzyme,depending on which type of substrate is used, iscompatible with the two routes of oxidation pos-tulated for human liver aldehyde oxidase. Sincediethylstilbestrol and diethyldithiocarbamic acidappear to bind at sites of the internal electrontransport chain remote from the substrate-bindingsite, such compounds could inhibit the oxidation ofa compound requiring the complete transport chain(e.g., benzaldehyde) while fortuitously causing afavorable alteration in conformation at the sub-strate site. Such a favorable conformationalchange would only be demonstrable when a sub-strate not requiring the entire electron transportchain, such as PMS, was used.

These studies throw little light on the longstanding problem of the metabolic role of alde-hyde oxidase. Several other enzymes in additionto aldehyde oxidase are known to possess theability to catalyze the oxidation of aldehydes,among them xanthine oxidase (EC 1.2.3.2); thesteroid-insensitive NAD-linked aldehyde' dehy-drogenase studied by Racker (21), Deitrich,Hellerman, and Wein (22), and others (EC1.2.1.3); and the steroid-sensitive NAD-requiringaldehyde dehydrogenase of Maxwell and Topper(23). The studies of Richert and Westerfeld(24) suggest that NAD-linked aldehyde dehy-drogenases, rather than metalloflavoproteins suchas aldehyde oxidase and xanthine oxidase, are ofprimary importance in the metabolism of acetalde-hyde arising from the oxidation of ethanol. Itwould appear likely, therefore, that the primaryphysiologic role of hepatic aldehyde oxidase may

1503

DAVID G. JOHNS

not be the oxidation of aliphatic aldehydes. Fromthe present studies with the human enzyme, andfrom earlier studies by other authors with liveraldehyde oxidase from other mammalian species,three other possible functions for this enzyme maybe postulated. The first of these is that aldehydeoxidase may be serving a protective function as anonspecific drug-metabolizing enzyme. It hasbeen shown, for example, that the 7-hydroxylation(and consequent biologic inactivation) of theanti-neoplastic agent methotrexate by this enzymeproceeds so rapidly in the rabbit that, for allpractical purposes, a toxic level of this compoundfor this species cannot be obtained (25, 26). Ap-propriately substituted nonphysiologic purines(12) and pyrimidines (11) can likewise serve asexcellent substrates for rabbit liver aldehyde oxi-dase. The present studies with the human en-zyme would tend to suggest, however, that thedrug-metabolizing function of aldehyde oxidase isnot of importance in all species. In man, the rangeof nonphysiologic heterocyclic substrates that canbe hydroxylated by the enzyme is much narrowerthan in the rabbit or guinea pig, and the rate ofhydroxylation is very much slower.A second possibility is that these nonphysio-

logic heterocyclic compounds have the capacityto act as substrates as a consequence of their re-semblance to an as yet unidentified physiologicnonaldehydic heterocyclic substrate for the en-zyme. The use of aldehyde oxidase inhibitorssuch as those described in the present papershould permit the testing of this hypothesis: theadministration of such an inhibitor at an adequatedosage level should result in an increased urinaryexcretion of the hypothetical substrate, in the sameway that the xanthine oxidase inhibitor 4-hydroxy-pyrazolo[3,4-d]pyrimidine results in an increasedexcretion of the xanthine oxidase substrates xan-thine and hypoxanthine (27).The third, and perhaps most likely possibility,

is that the physiologic role of human liver aldehydeoxidase is the oxidation of aromatic aldehydesarising from dietary sources, or occurring in thecourse of intermediary metabolism. As notedabove, the aromatic aldehyde benzaldehyde has thehighest affinity for the human enzyme of all thesubstrates examined to date; in addition, the maxi-mnal reaction velocity for this substrate exceededthat for all other substrates tested. Similar re-stilts were obtained with the mouse and rabbit liver

enzymes. Whatever the physiologic role of theenzyme may be, however, the effectiveness as al-dehyde oxidase inhibitors of relatively nontoxicpharmacologic agents such as quinacrine and theN-alkylphenothiazines, and physiologic compoundssuch as estradiol-17,8, would suggest either thataldehyde oxidase, like xanthine oxidase, is not es-sential to life, or that it is present in great excessover the amount required in intermediarymetabolism.The partial structural analogy between the

phenothiazine nucleus and the isoalloxazine com-ponent of flavin adenine dinucleotide (FAD) haspermitted the hypothesis that N-alkylphenothiazinesmay inhibit flavoproteins in vivo by virtue of anaffinity for FAD-binding sites; this suggestionhas received experimental support from the ob-servation that N-alkylphenothiazines competewith FAD for the apoenzyme of D-amino acid oxi-dase (28, 29). This interpretation, however, can-not be extended to the inhibition by phenothia-zines of aldehyde oxidase; in this instance, as thepresent studies show, the inhibitors are formallycompetitive with substrate. Similarly, Rajago-palan and Handler (7) have shown that quina-crine inhibits rabbit liver aldehyde oxidase byformal competition with substrate. These resultscan most easily be interpreted by assuming anaffinity of the phenothiazine nucleus (or, in thecase of quinacrine, of the rather similar acridinenucleus) for the substrate-binding site of aldehydeoxidase. It would appear, in fact, that only sterichindrance from the long N-alkyl side-chains ofthe phenothiazines examined here prevents thesecompounds from acting as substrates; as notedabove, N-methylphenothiazine, like N-methyl-phenazine (PMS), is rapidly oxidized by the rab-bit liver enzyme.3 Thus, while these studies re-veal nothing about the mode of action of theN-alkylphenothiazines at the molecular level aspsychopharmacologic agents, they permit the al-ternative suggestion that, assuming these com-pounds act in vivo as enzyme inhibitors, the siteof action at the hypothetical target enzyme orenzymes of the central nervous system may be asubstrate-binding site, as with aldehyde oxidase,rather than an FAD-binding site, as with D-aminoacid oxidase. It would follow that the hypotheti-cal target enzyme need not be a protein with areadily dissociable flavin cofactor, and that theresemblance between the phenothiazine nucleus

1504


and the isoalloxazine component of FAD may befortuitous, and not of pharmacologic significance.

Acknowledgments

The author would like to thank Mrs. Lucilla Yangfor able te-hnical assistance, Dr. John Mooney for helpin securing fresh normal human liver tissue, and Doc-tors Joseph Bertino and Brian Hillcoat for helpful dis-cussions. He is indebted to Dr. David B. Ludlum forpointing out the difference in modes of enzymic oxida-tion of charged and uncharged substrates.

References

1. Gordon, A. H., D. E. Green, and V. Subrahmanyan.1940. Liver aldehyde oxidase. Biochem. J. 34:764.

2. Knox, W. E. 1946. The quinine-oxidizing enzymeand liver aldehyde oxidase. J. Biol. Chem. 163:699.

3. Mahler, H. R., B. Ma-kler, D. E. Green, and R. M.Bock. 1954. Studies on metalloflavoproteins.III. Aldehyde oxidase: a molybdoflavoprotein. J.Biol. Chem. 210: 465.

4. Hurwitz, J. 1955. Inhibition studies on aldehydeoxidase. J. Biol. Chem. 212: 757.

5. Rajagopalan, K. V., I. Fridovich, and P. Handler.1962. Hepatic aldehyde oxidase. I. Purificationand properties. J. Biol. Chem. 237: 922.

6. Rajagopalan, K. V., and P. Handler. 1964. He-patic aldehyde oxidase. II. Differential inhibitionof electron transfer to various electron acceptors.J. Biol. Chem. 239: 2022.

7. Rajagopalan, K. V., and P. Handler. 1964. Hepaticaldehyde oxidase. III. The substrate-binding site.J. Biol. Chem. 239: 2027.

8. Loo, T. L., and R. H. Adamson. 1965. Themetabolite of 3',5'-dichloro-4-amino-4-deoxy-N10-methylpteroylglutamic acid (dichloromethotrexate).J. Med. Chem. 8: 513.

9. Misra, D. K., R. H. Adamson, T. L. Loo, and V. T.Oliverio. 1963. Inhibition of dihydrofolate re-ductase by dichloromethotrexate and its metabo-lite. Life Sci. 2: 407.

10. Johns, D. G., A. T. lannotti, A. C. Sartorelli, B. A.Booth, and J. R. Bertino. 1965. The identity ofrabbit-liver methotrexate oxidase. Biochim. Bio-phys. Acta. 105: 380.

11. Johns, D. G., A. C. Sartorelli, J. R. Bertino, A. T.Iannotti, B. A. Booth, and A. D. Welch. 1966.Enzymic hydroxylation of 5-fluoropyrimidines byaldehyde oxidase and xanthine oxidase. Biochem.Pharmacol. 15: 400.

12. Loo, T. L., C. Lim, and D. G. Johns. 1967. Enzymichydroxylation of 6-methylthiopurine by hepaticaldehyde oxidase. Biochim. Biophys. Acta. 134:467.

13. Rajagopalan, K. V., and P. Handler. 1962. Oxida-tion of phenazine methosulfate by hepatic aldehyde

oxidase. Biochein. Biophys. Res. Communn. 8:43.

14. Singer, T. P., and E. B. Kearney. 1957. Deter-mination of succinic dehydrogenase activity.Methods Biochem. Analy. 4: 307.

15. Waddell, W. J. 1956. A simple ultraviolet spectro-photometric method for the determination of pro-ten. J. Lab. Clin. Med. 48: 311.

16. Carpenter, F. H. 1951. Studies on the purificationand properties of aldehyde oxidase from horse liver.Acta Chem. Scand. 5: 406.

17. Igo, R. P., B. Mackler, and H. Duncan. 1961.Liver aldehyde oxidase: the nature of hematincomponent. Arch. Biochem. Biophys. 93: 435.

18. Palmer, G. 1962. The purification and properties ofaldehyde oxidase. Biochinm. Biophys. Acta. 56:444.

19. Pumphrey, A. M., and E. R. Redfearn. 1959. Therate of reduction of the endogenous ubiquinone ina heart-muscle preparation. Biochenz. J. 72: 2P.

20. Watts, R. W. E., J. E. M. Watts, and J. E. Seeg-miller. 1965. Xanthine oxidase activity in humantissues and its inhibition by allopurinol (4-hydrox-ypyrazolo(3,4-d) pyrimidine). J. Lab. Clin. Mcd.66: 688.

21. Racker, E. 1949. Aldehyde dehydrogenase, a di-phosphopyridine nucleotide-linked enzyme. J. Biol.Chem. 177: 883.

22. Deitrich, R. A., L. Hellerman, and J. Wein. 1962.Diphosphopyridine nucleotide-linked aldehyde de-hydrogenase. I. Specificity and sigma-rho func-tion. J. Biol. Chem. 237: 560.

23. Maxwell, E. S., and Y. J. Topper. 1961. Steroid-sensitive aldehyde dehydrogenase from rabbitliver. J. Biol. Chem. 236: 1032.

24. Richert, D. A., and W. W. Westerfeld. 1957. Ace-taldehyde oxidation in molybdenum deficiency. J.Biol. Chem. 227: 533.

25. Redetzki, H. M., J. E. Redetzki, and A. L. Elias.1966. Resistance of the rabbit to methotrexate:isolation of a drug metabolite with decreasedcytotoxicity. Biochem. Pharmacol. 15: 425.

26. Johns, D. G., and T. L. Loo. 1967. Metabolite of4-amino-4-deoxy-N10-methylpteroylglutamic acid(methotrexate). J. Pharm. Sci. 56: 356.

27. Rundles, R. W., J. B. Wyngaarden, G. H. Hitchings,G. B. Elion, and H. R. Silberman. 1963. Effectsof a xanthine oxidase inhibitor on thiopurinemetabolism, hyperuricemia, and gout. Trans. As-soc. Am. Physicians. 76: 126.

28. Gabay, S., and S. R. Harris. 1965. Studies offlavin adenine dinucleotide-requiring enzymes andphenothiazines. I. Interactions of chlorpromazineand D-amino acid oxidase. Biochem. Pharmacol.14: 17.

29. Gabay, S., and S. R. Harris. 1966. Studies offlavin-adenine dinucleotide-requiring enzymes andphenothiazines. II. Structural requirements forD-amino acid oxidase inhibition. Biochem. Phar-macol. 15: 317.

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Human Liver Aldehyde Oxidase: Differential Inhibition of Oxidation