Biochem. J. (1977) 161, 167-174Printed in Great Britain

167

Purification and Immunochemical Characterization of Monoamine Oxidase fromRat and Human Liver

By REG. G. DENNICK and R. JOHN MAYERDepartment ofBiochemistry, University Hospital and Medical School,

Clifton Boulevard, Nottingham NG7 2UH, U.K.

(Received 30 July 1976)

1. Monoamine oxidase from rat and human liver was purified to homogeneity by thecriterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecylsulphate. 2. The enzyme activity was extracted from mitochondrial preparations byTriton X-100. The enzyme was purified by (NH4)2SO4 fractionation followed bychromato-graphy on DEAE-cellulose, Sepharose 6B, spheroidal hydroxyapatite, and finallychromatography on diazo-coupled tyramine-Sepharose. 3. Distinct differences occur inthe chromatographic behaviour of the two enzymes on both DEAE-cellulose andspheroidal hydroxyapatite. 4. It is unlikely that the purification of the enzymes ontyramine-Sepharose is due to affinity chromatography and reasons for this are discussed.5. The purified enzymes did not oxidize 5-hydroxytryptamine and the relative activities ofthe enzymes with benzylamine were increased approx. 1.25-fold compared with theenzyme activities of mitochondrial preparations. 6. Immunotitration ofenzyme activity inextracts of mitochondrial preparations from rat liver was carried out with 5-hydroxy-tryptamine, tyramine and benzylamine. The enzyme activities were completely immuno-precipitated by the same volume of antiserum. Similar results were obtained with theantiserum to the enzyme from human liver.

Monoamine oxidase [monoamine-02 reductase(deaminating), EC 1.4.3.4] has been isolated andpartially purified from many tissues ofvarious species(see Sandler & Youdim, 1972; Gorkin, 1973 forreviews). The work reported here describes thepurification of monoamine oxidase from rat andhuman liver and subsequent immunochemicalstudies on the enzymes.Monoamine oxidase has been purified from ox

liver (Nara et al., 1966; Gomes et al., 1969) andhuman liver (Norstrand & Glantz, 1973) but themethods used are extremely complicated and time-consuming. The procedure described here involvesmodification of the existing purification schemes(Nara et al., 1966; Gomes et al., 1969; Youdim &Collins, 1971; Houslay & Tipton, 1973; McCauley &Racker, 1973; Houslay & Tipton, 1975) with theaddition of a final chromatographic step on diazo-coupled tyramine-Sepharose. Monoamine oxidasecan be purified considerably from Triton X-100extracts of mitochondrial preparations by chromato-graphy on tyramine-Sepharose (Dennick & Mayer,1976). When used in combination with conventionalpurification procedures the method gives prepara-tions of monoamine oxidase from rat and humanliver that are homogeneous by the criterion of poly-acrylamide-gel electrophoresis in the presence ofsodium dodecyl sulphate.

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Antisera to the purified enzymes have been used toassess the relationship ofthe two forms ofmonoamineoxidase which are functionally defined by substratespecificity.

Materials and Methods

Animals

Adult Wistar rats and sheep were obtained fromthe Joint Animal Breeding Unit, University ofNottingham School of Agriculture, Sutton Boning-ton, Leics., U.K. Human liver was obtained within24h of death from a 70-year-old male who died as aresult of a myocardial infarct. The subject had nohistory of psychiatric illness. The liver was kindlysupplied byDr. G. Stirling, Department ofPathology,University of Nottingham, U.K.

Materials

NAD+ was purchased from Boehringer Corp.(London) Ltd., London W5 2TZ, U.K. Tyraminehydrochloride, benzylamine, 5-hydroxytryptamine(creatinine sulphate complex), diaminobenzylaminehydrochloride, 1,4-diaminobutane and CoomassieBrilliant Blue R were purchased from Sigma (Lon-don) Chemical Co., Kingston-upon-Thames, Surrey,

R. G. DENNICK AND R. J. MAYER

U.K. 2-Mercaptoethanol, sodium dodecyl sulphate,dimethylformamide, N-methylpyrollidone, NN'-methylenebisacrylamide, acrylamide, NNN'N'-tetra-methylenediamine, riboflavin and spheroidal hy-droxyapatite were purchased from BDH Chemicals,Poole, Dorset, U.K. CNBr was purchased fromHopkin and Williams Ltd., P.O. Box 1, RomfordRM1 1HA, Essex. p-Nitrobenzoyl azide was pur-chased from Eastman(Kodak Ltd), Kirkby, LiverpoolL33 7UF, U.K. Glycerol (B.P. grade) was purchasedfrom Hills Pharmaceuticals, Briercliffe, BurnleyBB1O 2JY, Lancs., U.K. Triton X-100 was purchasedfrom Lennig Chemicals Ltd., Croydon, LondonCB9 3NB, U.K. Sepharose 4B and 6B were pur-chased from Pharmacia (G.B.) Ltd., London W.5,U.K. DEAE-cellulose (Whatman grade DE-52) wasobtained fromW. and R. Balston(Modified Cellulose)Ltd., Maidstone, Kent, U.K. Bio-Beads SM-2 (20-50mesh) were purchased from Bio-Rad LaboratoriesLtd., 27 Homesdale Road, Bromley, Kent, U.K. TheAmicom Ultrafiltrator was obtained from Amicon,High Wycombe, Bucks., U.K. Conductivity measure-ments were made with a Radiometer Conductivitymeter type CDM-2d. The MSE 'Atomix' waspurchased from MSE Scientific Instruments, ManorRoyal, Crawley, West Sussex, U.K. Benzylaminehydrochloride was prepared by treating the base,dissolved in diethyl ether, with conc. HCI. Thefiltered precipitate was recrystallized twice fromacetone/water (10:1, v/v). Freund's adjuvant wasobtained from Difco Laboratories, Detroit, MI,U.S.A. All other chemicals were of A.R. grade.

Aldehyde dehydrogenase

Ox liver aldehyde dehydrogenase was prepared bythe method of Houslay & Tipton (1973).

Assay ofmonoamine oxidase

Monoamine oxidase was assayed by the coupledspectrophotometric method of Houslay & Tipton(1973) with ox liver aldehyde dehydrogenase. Theassays were carried out at 30°C in plastic cuvetteswith a 1cm light-path and contained, in a finalvolume of 1 ml, 20mM-potassium phosphate, pH7.2,aldehyde dehydrogenase (0.1,umol/min), 0.45mM-NAD+, 2.3mM-tyramine hydrochloride and enzyme(20-300,ug of protein). The reaction was started bythe addition of substrate and the change in A340was measured. Where indicated benzylamine hydro-chloride (final concn. 2.3mM) or 5-hydroxytrypt-amine (final concn. 1.1 mM) was used as substrate.

Fractions eluted from chromatographic columnswere more conveniently assayed by a modification ofthe method of Tabakoff & Alivisatos (1972) using p-dimethylaminobenzylamine as substrate. Assayscontained, in a final volume of 1.2ml, 100mM-

potassium phosphate, pH7.2, 2.5 mM-p-dimethyl-aminobenzylamine and enzyme (20-300.ug ofprotein). The samples were incubated at 30°C for 1 hand the A355 was determined. The reaction waslinear with time for at least 1 h.

Protein determination

Protein at high concentrations (5-30mg/ml) wasdetermined by the method of Gornall et al. (1949) andat lower concentrations by the method of Wang &Smith (1975).

Preparation ofbutyl-tyramine-SepharoseButyl-tyramine-Sepharose was prepared by a

modification of the method of Cuatrecasas (1970).Sepharose was activated with CNBr by the method ofPorath et al. (1973). Sepharose 4B (50ml) was washedextensively with water in a sintered-glass funnelby gentle suction. The Sepharose was suspended in50ml of SM-potassium phosphate buffer, pH 11.4 at5°C, prepared by dissolving 3.33 mol of K3PO4and 1.67mol of K2HPO4/litre. CNBr (4g) wasdissolved in 20ml of N-methylpyrollidone/water(1:4, v/v). This solution was added to the Sepharosesuspension and the mixture was gently stirred for10min at 5-10°C. The suspension was then exten-sively washed with ice-cold water on a sintered-glassfunnel. The Sepharose was added to lOOml of 2M-1,4-diaminobutane (previously adjusted to pH10with 6M-HCI) and gently shaken overnight at 4°C.The aminobutyl-Sepharose was suspended in 60ml of0.2M-sodium borate buffer, pH9.3, and 0.175M-p-nitrobenzoylazide in dimethylformamide (40ml) wasadded. The mixture was stirred for 1 h at roomtemperature (20°C). The p-nitrobenzamidobutyl-Sepharose was washed with I litre of dimethyl-formamide/water (1:1, v/v) and then suspended in60ml of 0.1 M-sodium dithionite in 0.5M-NaHCO3,pH 8.5, for 1 h at 40°C. Thep-aminobenzamidobutyl-Sepharose was washed thoroughly with water,cooled to 4°C, and added to 200ml of0.1 M-NaNO2 in0.5M-HCI at 4°C. The mixture was stirred for 7min at4°C and then washed with 500ml of ice-cold 1%(w/v) urea followed by rapid washing with 2 litresof ice-cold water. The diazonium-Sepharose wasthen immediately added to 50ml of lOmM-tyraminehydrochloride in 0.2M-sodium borate buffer, pH 10,and gently shaken at 4°C overnight. After coupling,the orange butyl-tyramine-Sepharose was washedextensively with water at room temperature andstored in water containing a few drops of toluene at4°C. Before use columns of butyl-tyramine-Sepha-rose were washed with lOOml of 1 M-KCI in 10mM-potassium phosphate buffer, pH7.2, containing 1.5%(w/v) Triton X-100 and 20% (v/v) glycerol and subse-quently equilibrated in the same buffer without KCI.

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LIVER MONOAMINE OXIDASE

Purification ofmonoamine oxidase

All operations were carried out at 4°C unlessotherwise stated.

Preparation ofTriton X-100 extractsofmitochondria.Mitochondrial preparations were obtained from lOOg(fresh wt.) of rat or human liver by the method ofHawkins (1952) and washed once in 0.25M-sucrose(previously adjusted to pH7.0 with 0.5M-NaHCO3).Rat liver was homogenized with a Potter-Elvehjemhomogenizer whereas the more fibrous human liverrequired homogenization for 1 min in a MSEAtomix homogenizer. The washed mitochondrialfractions were resuspended in 20mM-potassiumphosphate buffer, pH7.2, to give a final volume oflOOml and stored frozen at -15°C for at least 48h.The mitochondrial preparations were slowly thawedand then centrifuged for 60min at lOOOOOgav.. Thepellet was resuspended by gentle homogenization in20mM-potassium phosphate buffer, pH7.2, contain-ing 1.5% (w/v) Triton X-100 (100ml) and the mixturewas stirred for 1.5h. The suspension was thencentrifuged for 1.5h at lOOOOOgav. and the super-natant used as the source of the enzyme.Removal ofTriton X-100 and (NH4)2SO4 precipita-

tion. Triton X-100 was removed from the supernatantby means of Bio-Beads SM-2 previously equilibratedin 20mM-potassium phosphate buffer, pH7.2, by themethod of Holloway (1973). The Triton X-100extract (lOOmI) was stirred at 0°C and approx. lOOg(dry wt.) of Bio-Beads were added in lOg portionsover a period of 1-2h until the A280/A260 ratio of thesupernatant remained constant. The suspension wasfiltered by gentle suction on a sintered-glass funneland the Bio-Beads were carefully washed with lOOmlofice-cold 20mM-potassium phosphate buffer, pH7.2.The filtrate and washings were combined and solid(NHl4)2SO4 was added slowly, with stirring at 0°C, togive a concentration of 361 gAlitre of initial volume.The solution was maintained at pH7.2 by the additionof0.5 M-KOH. The solution was stirred for 30min andthe precipitate collected by centrifugation for 20minat 25000gav.. The supernatant was discarded and theprecipitate suspended in lOOml of 20mM-potassiumphosphate buffer, pH7.2, and dialysed overnightagainst 50vol. of the same buffer. The human liverpreparation remained insoluble at this stage ofpurification but its activity was not impaired.

Chromatography on DEAE-cellulose. Each di-alysed preparation was diluted to 200ml with 20mM-potassium phosphate buffer, pH7.2, and applied to acolumn (2.5cmx20cm) of DEAE-cellulose pre-viously equilibrated in the same buffer. The columnswere washed with 100ml of the buffer.

(a) Elution of monoamine oxidase of rat liver.Monoamine oxidase from rat liver was eluted withapprox. 200ml of the same buffer containing 0.2%(w/v) Triton X-100. Solid (NH4)2SO4 was added to

Vol. 161

the eluate slowly, with stirring at 0°C to give a con-centration of 361 g/litre of initial volume. The solu-tion was maintained at pH7.2 by the addition of0.5 M-KOH. The solution was stirred for 30min andthe precipitate collected by centrifugation for 20minat 25000ga.. The pale-yellow floating precipitate wascarefully removed and dissolved in 5ml of 20mM-potassium phosphate buffer, pH7.2, containing0.1 mM-mercaptoethanol and 1.5% (w/v) TritonX-100. The solution was dialysed overnight against50vol. of the same buffer.

(b) Elution of monoamine oxidase of human liver.Monoamine oxidase from human liver was eluted bymeans of a linear gradient (200ml) of Triton X-100(0-1%, w/v) in 20mM-potassium phosphate buffer,pH7.2, containing 0.2M-KCI. Fractions containingmonoamine oxidase activity were pooled and con-centrated with an Amicon Ultrafiltrator with aPM1O membrane. The concentrated solution (5ml)was subsequently dialysed overnight against 50vol.of 20mM-potassium phosphate buffer, pH7.2, con-taining O.lmM-mercaptoethanol and 1.5% (w/v)Triton X-100.

Chromatography on Sepharose 6B. Each dialysedpreparation was chromatographed on a column(3cmx 80cm) of Sepharose 6B equilibrated in 20mM-potassium phosphate buffer, pH7.2, containing0.1 mM-mercaptoethanol and 1.5% (w/v) TritonX-100. The fractions with the highest specific activitywere pooled and dialysed overnight against 50vol. of10mM-potassium phosphate buffer, pH7.2, contain-ing 0.1 mM-mercaptoethanol.

Chromatography on hydroxyapatite. Each dialysedpreparation was applied to a column (2cm x O0cm)of spheroidal hydroxyapatite equilibrated in 10mM-potassium phosphate buffer, pH7.2, containing0.1 mM-mercaptoethanol. The column was washedwith 50ml of the same buffer.

(a) Elution of monoamine oxidase of rat liver.Monoamine oxidase from rat liver was eluted in twophases: (i) by applying 50ml of 200mM-potassiumphosphate buffer, pH7.2, containing 0.1 mM-mer-captoethanol; (ii) by subsequently applying 50ml ofthe same buffer containing 0.2% (w/v) Triton X-100.The second fraction had the highest specific activityand was used in the next purification step.

(b) Elution of monoamine oxidase of human liver.Monoamine oxidase from human liver was elutedfrom the column by 50ml of 200mM-potassiumphosphate buffer, pH7.2, containing 0.1 mM-mer-captoethanol. No further activity could be eluted byadding Triton X-100 to the buffer.The preparations of monoamine oxidase from rat

or human liver were dialysed overnight against 50vol.of 10mM-potassium phosphate buffer, pH7.2 con-taining 1.5% (w/v) Triton X-100 and 20% (v/v)glycerol.

Chromatography on butyl-tyramine-Sepharose. The

169

R. G. DENNICK AND R. J. MAYER

dialysed preparations were applied to columns(2cmxIOcm) of butyl-tyramine-Sepharose equili-brated in 10mM-potassium phosphate buffer, pH7.2,containing 1.5% (w/v) Triton X-100 and 20% (v/v)glycerol. The columns were washed with 2-3 columnvolumes of the same buffer and monoamine oxidaseactivity was eluted by applying a linear gradient(40ml) of KCI (0-0.2M) in the same buffer.

Polyacrylamide-gel electrophoresis in the presenceofsodium dodeceyl sulphate. Polyacrylamide disc gelelectrophoresis of protein samples, and subsequentstaining for protein was performed by the method ofBetts & Mayer (1975) in 5% (w/v) or 10% (w/v)polyacrylamide gels.

Preparation of antisera to monoamine oxidase fromrat or human liver

Samples ofmonoamine oxidase were dialysed over-night against water, freeze-dried and dissolved inwater (12ml). Antisera were raised in sheep byiWecting 240pg of purified enzyme with Freund'scomplete adjuvant on four occasions. Antigen wasinjected at six sites (four subcutaneously and twointramuscularly) on each occasion at 14-day intervals.The volume injected at each site was I ml (0.Snml ofantigen solution and 0.5ml of adjuvant). At 1 weekafter the final injection the animal was bled byjugular cannulation. The blood was left to clot andcentrifuged at 1500g",,. for 30mi. An equal volumeof neutralized satd. (NH4)2SO4 was added to theserum at 0°C and after stirring for 30mm the pre-cipitate was collected by centrifugation at 25000gav.for 20min. The precipitate was washed twice with(NH4)2S04 solution (291 g/litre) at 0°C, dissolved in avolume of 1OmM-potassium phosphate buffer,pH 8.0, equal to one-half the initial serum volumeand dialysed overnight against 5 litres of the samebuffer. The suspension was subsequently centrifugedat 25000g8,. for 20min. The supernatant was dialysedovernight against 5 litres of20mM-sodium phosphate,pH 7.0, containing 0.15M-NaCI. The preparation was

subsequently heated at 70'C for 30min, centrifugedat 25000gav. for 20min, and the supernatant wasstored frozen at -15C.

Imm,unotitrations

Triton X-100 extracts of mitochondrial prepara-tions from rat and human liver were depleted ofmicellar Triton X-100 with Bio-Beads SM-2 asdescribed above. Immunotitrations were carried outwith antisera or control sera that had been processedas described above. Samples (0.2-0.3 ml) of TritonX-100-depleted extract were mixed with processedsera and adjusted toafinal volumeof 1 ml with 20mM-sodium phosphate, pH7.0, containing 0.15M-NaCI.The solutions were kept at 40C overnight and thencentrifuged at 14000gav. for 6min. Samples (0.1-0.25 ml) of the supernatants were assayed for mono-amine oxidase activity with 5-hydroxytryptamine,tyramine and benzylamine. Immunoprecipitates werewashed twice with 20mM-sodium phosphate, pH7.0,containing 0.15M-NaCl and protein was measured bythe method of Lowry et al. (1951) with bovine serumalbumin as standard.

Experimental and Results

Monoamine oxidase from rat and human liver waspurified to homogeneity by the criterion of poly-acrylamide-gel electrophoresis in the presence ofsodium dodecyl sulphate. Typical purificationschemes for the two enzymes are shown in Tables 1and 2. The purified monoamine oxidase from rat liverhad a specific activity of 16.1 nmol/min per mg ofprotein when assayed with tyramine although theeluate from the Sepharose 6B column had a higherspecific activity of 41.2nmol/min per mg of protein.The specific activity of monoamine oxidase from ratliver declined after chromatography on hydroxy-apatite and butyl-tyramine-Spharose. The prepara-tion of monoamine oxidase from human liver had afinal specific activity of 12.5 nmol/min per mg of

Table 1. Purification ofmonoamine oxidasefrom mitochondrialpreparations ofrat liverExcept where indicated all assays were carried out with tyramine as substrate.

Mitochondrial preparationTriton X-100 extractDEAE-cellulose eluateSepharose 6B eluateHydroxyapatite eluateButyl-tyramine-Sepharose

eluate

Vol.(ml)100100185601012

Protein(mg)

12844124510.55.62.3

Totalactivity

(nmol/min)113982864243312538

Specificactivity

(nmol/minper mg ofprotein)

0.882.014.241.222.316.1

Yield(100)72.656.338.010.93.3

Relativeenzyme activities

(tyramine/Purification 5-hydroxytryptamine/

(fold) benzylamine)1:0.23:0.47

2.3 1:0.20:0.5016.146.825.318.3 1:0:0.57

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LIVER MONOAMINE OXIDASE

Table 2. Purification ofmonoamine oxidasefrom mitochondrialpreparations ofhuman liverExcept where indicated all assays were carried out with tyramine as substrate.

Mitochondrial preparationTriton X-100 extractDEAE-cellulose eluateSepharose 6B eluateHydroxyapatite eluateButyl-tyramine-Sepharose

eluate

Vol.(ml)10010080481410

Protein(mg)84020075217.21.75

Totalactivity

(nmol/min)25728288494022

Specificactivity

(nmol/minper mg ofprotein)

0.31.41.22.35.5

12.5

Yield(100)10934.219.015.58.5

Relativeenzyme activities

(tyraminetPurification 5-hydroxytryptamine/

(fold) benzylamine)1:0.35:0.70

4.7 1:0.35:0.533.9 _7.7

18.341.6 1:0:0.90

2~~~~~~~~~~~~~~~~~~I o A

~0.t10

0 0. 1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 tAntiserum (ml)

Fig. 1. Immunotitration ofmonoamine oxidase activities inextracts ofmitoehondrialpreparationsfrom rat liverSamples (0.2ml) ofTiton X-100-depleted extracts ofmitochondrial preparations were incubated overnightat 4°C with antiserum. The mixtures were centrifugedat 14000g,. for 6niin and enzyme activities assayedin the supernatants. Enzyme activity was assayedwith tyramine (s), 5-hydroxytryptamine (s) andbenzylamine (O). *, Protein in the inmmunoprecipi-tates.

protein when assayed with tyramine and was purified41.6-fold with respect to the mitochondrial prepara-tion. Although the enzyme from human liver had alower final specific activity than the enzyme from ratliver it was recovered in higher yield. Both purifiedpreparations did not oxidize 5-hydroxytryptamineand slight increases (approx. 1.25-fold) in the relativeactivity of the purified enzymes towards benzylaminewere observed compared with the relative activity ofthe enzymes in mitochondrial preparations.

Polyacrylamide-gel electrophoresis of samples ofpurified monoamine oxidase (20-100.ug of protein)in the presence of sodium dodecyl sulphate showedone band which stained for protein. In some cases thisstained band was coincident with a faint yellow bandthat was observed in unstained gels (results notshown).

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The use of Bio-Beads SM-2 was very effective inremoving micellar Triton X-100. In preliminary ex-periments (NH4)2SO4 fractionation of Triton X-100extracts of mitochondrial preparations (0-30% satn.)gave a floating precipitate which contained approx.40% of the enzyme activity. Further (NH4)2S04fractionation (30-60% satn.) gave a sedimentingprecipitate that contained the remainder of the en-zyme activity. Further work showed that the first(NH4)2SO4 fractionation (0-30% satn.) precipitatedTriton X-100 from the extract which appeared in thefloating layer. Treatment of Triton X-100 extractswith Bio-Beads SM-2 removed micellar Triton X-100and subsequent (NHF)2SO4 fractionation (0-60%satn.) and centrifugation gave a sedimenting pre-cipitate. This treatment avoids the loss of enzymeactivity associated with the first (NH4)2SO4 fraction-ation (0-30% satn.) and facilitated the subsequentbinding ofthe enzyme to DEAE-cellulose. Themono-amine oxidase activity of human liver was renderedinsoluble by these procedures but this did not impairits enzyme activity.

Significant differences were noted in the behaviourof the two enzymes when chromatographed onDEAE-cellulose and hydroxyapatite. The mono-amine oxidase from human liver was bound morestrongly to DEAE-cellulose and therefore a highersalt and detergent concentration was required for theelution of the enzyme. However, monoamine oxidasefrom human liver bound less strongly to hydroxy-apatite than the enzyme from rat liver. It was there-fore eluted by 200mM-potassium phosphate buffer,pH7.2, without the addition of detergent. The twoenzymes behaved identically on butyl-tyramine-Sepharose.The results in Fig. 1 show that the enzyme activities

with 5-hydroxytryptamine, tyramine and benzyl-amine are completely immunoprecipitated fromTriton X-100-depleted extracts of mitochondrialpreparations of rat liver by the same volume of pro-cessed antiserum. Similar results were obtained withmitochondrial extracts from human liver (Fig. 2).

171

- togA

- 50

~~~~~lo ~~~~~~-s

0.1 0.2 0.3 0.4 0.5 0.6 0.7Antiserum (ml)

R. G. DENNICK AND R. J. MAYER

0~0-

Cd

0

Fig. 2. Immunotitration ofmonoamine oxidase activities inextracts ofmitochondrialpreparations from human liverSamples (0.3 ml) of Triton X-100-depleted extracts ofmitochondrial preparations were incubated overnightat 4°C with antiserum. The mixtures were centrifugedat 14000gav. for 6min and enzyme activities assayedin the supematants. Enzyme activity was assayedwith tyramine (e), 5-hydroxytryptamine (U) andbenzylamine (o). *, Protein in the immunoprecipi-tates.

Incomplete precipitation of monoamine oxidaseactivity occurred when extracts of mitochondrialpreparations in 1.5% (w/v) Triton X-100 were treatedwith antiserum. This observation agrees with that ofHartman et al. (1971). The removal of micellarTriton X-100 with Bio-Beads SM-2 resulted in thecomplete immunoprecipitation of monoamine oxi-dase activity. Monoamine oxidase was not precipi-tated with control sera.

Discussion

The purification scheme for monoamine oxidasedescribed here combines the classical purificationprocedures with a novel 'affinity' chromatographicstep using diazo-coupled tyramine-Sepharose. Baket& Hemsworth (1975a,b) have used inhibitors otmonoamine oxidase as affinity ligands and haveachieved a fourfold purification of the enzyme with ahydrazine-Sepharose column and a 40-fold (1 %yield) purification with a 1-m-aminophenyl-2-cyclopropylaminoethanol-Sepharose column fromextracts prepared by sonicating mitochondrial pre-parations. More recently Toraya et al. (1976) purifiedamine oxidase (ninefold, 32% yield) from extracts ofAspergillus niger by chromatography on a column ofaminohexyl-Sepharose, a matrix that failed to purifymonoamine oxidase from rat liver (Dennick & Mayer,1976).

Previous work (Dennick & Mayer, 1976) hasshown that monoamine oxidase can be purified20-fold and in 50% yield from Triton X-100 extractsof mitochondrial preparations from rat liver bychromatography on butyl-tyramine-Sepharose. The

purification achieved with this system was criticallydependent on the chain length of the spacer arm andthe ionic strength of the equilibrating buffer. A butylspacer arm, 10mM-potassium phosphate buffer,pH7.2, and the presence of 20% (v/v) glycerol werenecessary for binding and subsequent elution fromthe colum with KCI. It is noteworthy that Aunis etal. (1973) prepared highly purified dopamine 46-hydroxylase (EC 1.14.17.1) using ethyl-tyramine-Sepharose as an affinity matrix. These workersfound that the enzyme was simply retarded on theaffinity column and that washing with equilibratingbuffer was sufficient to achieve considerable puri-fication.

It is unlikely that the purification of monoamineoxidase from rat and human liver on butyl-tyramine-Sepharose is due to affinity chromatography.Substrate fails to elute the enzyme and proteinremains bound to the column even after washing with0.5M-KCI. This suggests that the purification ispossibly due to non-specific hydrophobic and/orionic interactions. The terms 'detergent' (Wilchek,1974) or 'apolar' (Phan & Mahler, 1976) chromato-graphy have been applied to such systems.The use of Bio-Beads SM-2 to remove micellar

Triton X-100 from enzyme preparations had severaladvantages. The binding of monoamine oxidase toDEAE-cellulose is critically dependent on the con-centration of Triton X-100. As little as 0.075% (w/v)Triton X-100 can prevent binding to DEAE-cellulose(Houslay & Tipton, 1975). Other workers haveattempted to remove Triton X-100 by fractionationwith (NH4)2SO4, which at a concentration of 27%satn. precipitates micellar Triton X-100 (R. G.Dennick & R. J. Mayer, unpublished observation).Thus Youdim & Collins (1971), Houslay & Tipton(1973) and Youdim (1976) have used an initial(NH4)2SO4 fractionation step (0-30% satn.). Thisprocedure was not used in the work described heresince it involves discarding a considerable fraction(40%) ofmonoamine oxidase activity. After removingmicellar Triton X-100 with Bio-Beads SM-2 followedby (NH4)2SO4 fractionation (0-60% satn.) anddialysis against detergent-free buffer, monoamineoxidase was recovered in high yield and subsequentlybound easily to DEAE-cellulose.The differences in behaviour of the enzymes from

rat and human liver during chromatography onDEAE-cellulose and hydroxyapatite are difficult toexplain. The human liver enzyme preparation wasobtained by more vigorous homogenization than theenzyme preparation from rat liver and contained aconsiderable quantity of fatty material. After removalof Triton X-100 and (NH4)2SO4 fractionation theenzyme preparation from human liver was insolubleand was applied to the DEAE-cellulose column as afine suspension. This may account for the conditionsthat were needed to elute the enzyme from theDEAE-

1977

172

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LIVER MONOAMINE OXIDASE 173

cellulose column and the concomitant slight decreasein specific activity. The behaviour of monoamineoxidase from rat liver during chromatography onhydroxyapatite is similar to that reported for theenzyme from ox liver (Yasunobu et al., 1968; Gomeset al., 1969). These workers obtained two fractionswith monoamine oxidase activity. Fraction Cl wasobtained by eluting the column of hydroxyapatitewith 0.2M-potassium phosphate buffer, pH7.4, andfraction C2 was obtained by elution with 0.2M-potassium phosphate buffer, pH7.4, containing0.15% (w/v) sodium deoxycholate. Fraction C2 hadthe highest specific activity and was shown to have amol.wt. of 1200000 whereas fraction Cl had amol.wt. of 400000. However, Hartman et al. (1971)showed that fractions Cl and C2 were immuno-logically identical and suggested that fraction C2 wasa trimer of fraction Cl representing a larger fragmentfrom the outer membrane of the mitochondrion. Inthe work reported here monoamine oxidase from ratliver was eluted from hydroxyapatite by 0.2M-potassium phosphate buffer, pH7.2. Subsequently afraction of higher specific activity could be obtainedby elution of the column with 0.2M-potassiumphosphate buffer, pH7.2, containing 0.2% (w/v)Triton X-100.

Several groups of workers have raised antisera tomonoamine oxidase from bovine (Hidaka et al.,1971; Hartman et al., 1971; McCauley & Racker,1973) and rat (Youdim & Collins, 1975) liver. Theantisera have been used in the qualitative (Youdim &Collins, 1975) and quantitative (Hidaka et al., 1971;Hartman et al., 1971) assessment of the nature of livermonoamine oxidase. The data have been interpretedto mean that liver monoamine oxidase is assembledfrom the same protein subunit into forms of differentmolecular weight (Hartman et al., 1971) or that twodistinct forms of liver monoamine oxidase exist(Youdim & Collins, 1975).The results in Figs. 1 and 2 show that monoamine

oxidase is quantitatively precipitated from TritonX-100-depleted extracts of mitochondrial prepara-tions from rat and human liver. The volume of anti-serum required to completely precipitate enzymeactivity is the same when 5-hydroxytryptamine,benzylamine or tyramine is used as substrate. Theresults indicate that these enzyme activities areassociated with a single immunogenic macromolecu-lar species and consequently are completely pre-cipitated from the mitochondrial extracts by anidentical volume of antiserum.The results show that the functional forms of

monoamine oxidase which may be resolved bypharmacological (Johnston, 1968) or kinetic(Houslay& Tipton, 1974) means are associated with a singlemacromolecular species. The data would support thecontention of Houslay & Tipton (1973) that themultiple forms of monoamine oxidase activity are

associated with a single protein species which ismodified by differential binding of phospholipid.The purification procedure for the enzymes des-

cribed in the present paper gives homogeneouspreparations of monoamine oxidase by the criterionof polyacrylamide-gel electrophoresis in the presenceofsodium dodecyl sulphate. The enzyme preparationsfrom both rat and human liver are not active agai-nst5-hydroxytryptamine but the antisera against theseantigens immunoprecipitate enzyme activity of theso-called A and B forms of the enzyme (5-hydroxy-tryptamine and benzylamine respectively) andactivity characteristic of both forms (tyramine). Theresults show that a specific B form of the enzyme wasnot purified and that the different enzyme activitiesreside with a common antigenic species.

It is now possible to study immunochemically thesynthesis and degradation of the enzyme in rat liverin different physiological states.

We are grateful to Miss A. J. Cook for skilled technicalassistance. R. G. D. is supported by a grant from theWellcome Foundation.

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