Kovernber 1963
INHIBITION OF HOMOGENEOUS MOSOAMINE OXIDASES
859
In Vitro Inhibition Studies with Homogeneous Monoamine Oxidases XLFRED
BURGER^^^^
AND S.4K.4RI XIARAlard
Department of Biochemistry and Biophysics, ['niversily o j Hawaii, Honolulu, Hawaii Eeceived June 16, 11/66 The inhibition of the deamination of five substrates by highly purified beef liver mitochondrial monoamine oxidase was measured using several known M A 0 inhibitors. Inhibition values for the deamination of benzylamine by pure beef plasma LIB0 were also determined. The relative potencies of t,he inhibitors varied with the substrates used and did not always follow literature data obtained with less homogeneous M A 0 preparations. They did not run parallel for the two enzymes used. Harmine and harmaline were much less potent as inhibitors of the purified enzymes than observed previously with tissue homogenates.
The often conflicting mechanistic and quantitative literature data on inhibitors of monoamine oxidase (lL40) have been reviewed ably and critically by Zirkle and Kaiser and by Biel, et al., in a recent monograph.2 The difficulties of obtaining reasonably matching inhibition values encountered in various studies are illustrated for harmine, tranylcypromine (I), and S-benzyl-S-methyl-X-propargylamine (111)in Table I. The nature of the inhibition, the need for preincubation with the inhibitor, the presence of oxygen during l)ieincubation, aild especially the source of the enzyme, its state of purity, and the substrate to be deaminated all determine the relative potency of the inhibitor. I n turn, the nature of the inhibition (competitive us. iioiiconiyetitive, reversible us. iioiireversible) depends 011 the s ~ b s t r a t e011 , ~ a conibinatiori of this factor with bourcbe and purity of the e n ~ y m e , and ~ - ~on experimental co~iditions.~ -4s a result of diverse procedures, potency values varying from each other by factors as high as 1400 have bceri reported.2a The enzyme preparations used in all but one5of these studies were crude tissue homogenates, or, a t best, solubilized mit~chondria.~This, and simies specificity, may have accounted for much of the observed discrepancies. These differences should he minimized by use of homogeneous enzymes from the qame species. The purification of mitochondrial l I A 0 , the enzyme which is responsible for the deaniinatiori of important natural neuromodulatory substrates, has been attempted several times.* A 20-fold con(witration of mitochondrial N A O from beef liver has been d e ~ c r i b e d ,but ~ this preparation was insoluble aiid could be sedimented easily. Indeed, the pH activity curves of this preparation alone and of the enzyme plus iproniazid were parallel, but with another (1 I (a) On leave of absence from the University of Virginia, Charlottesville, Va., where inquiries should be sent. (b) Supported by a Special 1;elloaship from the Sational Institute of General Rledical Sciences, 1-F3G3I-21,405-01, Kational Institutes of Health, U. 8. Public Health Service. i r ) On leave of absence from Hokkaido Gakugei University. Hakodate, Japan. (d) Supported by a grant from the S-ational Institute of General .\Iedical Sciences, GhI-06i33-05, rational Institutes of Health, C. S. Public Health Service. (2) (a) C. L. Zirkle and C. Kaiser in "Psychopharmacological Agents," Yol. I , M. Gordon, Ed., Academic Press Inc., New York. S . Y., 1964. pp. . Drucker, ibid., pp. 359-443.
&I. J. Nimmo, S a t w e . 184, 547 (1959). (5'1 L. h,l. Barbato and L. G. Abood, Biochim. Biophya. Acta, 67, 531 ( 1sCi:o.
inhibitor (I) a second maximum appeared at pH 7.0, suggesting to Barbato and ilbood the presence of two monoamine oxidases.5 ,4 recent short announcement* of 3IAO prepared by sonication of rat liver mitochondria and by chromatography to an over-all 350fold purification has not been followed up further. We had available M A 0 from beef liver mitochondria which had been purified highly by a new fractionation procedure by S a r a and Y a s u n ~ b u . ~The crude enzyme preparation was extracted with Triton X-100 and fractionated twice with ammonium sulfate. This was followed by absorption on alumina C, and then by DEAE-cellulose column chromatography to an approximately over-all 50-fold concentration. The resulting enzyme had a specific activity of more than 3000. It appeared interesting to use this enzyme in a study of the inhibitory effect of several 2-phenylcyclopropylamines10 aiid some other structurally different 31.40 inhibitors on the deamination of several natural substrates: dopamine, norepinephrine, tyramine, tryptamine, 5-hydroxytryptamine (j-HT), as well as benzylamine. I t was hoped that this study would complement previous measurements with less pure catalysts and with a narrower spectrum of substrates. I n addition, significant differences in inhibition values may indicate diff ereiit mechanisms by which the inhibitors block the deamination of various substrates in their natural surroundings. -2 relative lack of stereospecificity of inhibitors of RIA0 has been observed by several investigators. We had at hand the (*)-, (+)-, and (-)-trans and ( f)-cis isomers of 2-phenylcyclopropylaniine and felt that a further examination of the inhibitory potencies of these stereoisomers, using homogeneous enzymes and the substrates listed above, would illustrate more clearly the unspecificity of these isomers at the same active enzyme site. Beef plasma 11.40 has been obtained pure.12 Since this enzyme does riot dehydrogenate amines of the pheiiethylainine and indolylethylamine type, we tested the effect of the same series of inhibitors 011 the deamination of benzylamine by this enzyme. Interest in this enzyme was augmented by the observation of Tainada,
(4) -4. R . Maass and
(9) S. Nara, and K. T. Yasunobu, Biochem. Biophys. Res. Commun., io be published (10) C. Kaiser, B. R1. Lester, C. L. Zirkle, A. Burger, C. S. Davis, T. J. Delia. and L. Zirngibl, J. ,%fed. Pharm. Chem., 5, 1243 (1962). (11) Fur a summary, see ref. 2a, pp. 523-624. (12) 11. Yamads. and Istrutt.
.5-H'I3 5-HI' 5-HT S-H'r 5-HT 5-HT Tyramine Tyramine Tyramine Tyramine ,5-H T 5-HT
301) 9Oli h7
44 100 )
J
250 100-1000 101I
5-HT
10-7 10-5
5-HT
'*
.i-HT
13i
IO-fi
.i-I-Il' ,5-Il'l-
I7 20 (reserpine
10-G
R u t liver ICst liver Rat liver Rat brain Mouse brain Iiat br:tiii Beef liver Mouse liver Rabbit liver l h t brain 1::tt liver (+riiiiea pig liver Itat liver Itat bruin Vouse brain (;linea pig liver
reversal i n mice in vivo) J . 1). Taylor, a Jlef. 19. S.Udenfriend, H . IVitkop, B. ti. liedfield, and 11. \Veishach, Biochenz. PhurtmcoL., 1, 160 (1958). 31. Ozaki, 11. Weissbach, A. Ozaki, B. Witkop, and S. A. A. Wykes, Y. C. Gladish, and IT. B. JIartiri, S a t u w , 187, 941 (1960). Udenfriend, J . M e d . Pharnz. Cheni., 2, 591 (1960). e IS. Green aiid R. 15'. Erickson, J . Phvrmacol. Ezptl. Therap., 129, 237 (1960). K. Stock and E. Westerniann, =1rc,h.. E q t l . Pathol. Pharmakol., 243, 44 (1962). 0 I). It. Illaxwell, W.R. Gray, and E. 11. Taylor, 11. ltef. 7. Brz't. J . I'harmacol., 17, 310 (1961). Ref. 4. i P. -4. Shore and V.H . Colin, Jr., Biochenz. PhnrmucoZ., 5, 91 (1960). Green, iuipublished results. W. Schuler and E. JVysu, Arch. intern. pharmacodyn., 128, 431, 439 (1960).
et al.,l:j that the crystalline I\IXO from Aspergillus n i g e r does not only oxidize the typical aliphatic aiiiiiies affected by serum h1A0, but also tyramine, tryptamine, 5-HT, mescaline, phenethylarnine, arid norepinephrine. The fungal enzyme, moreover, eont'ains Cu2+ and resembles beef plasma MA0 in other respects.
Experimental Section ~~aterials.-Substrates, listed a t final concentrations used in t h e experiments, iiicluded tyramine hydrochloride, 5 x 31; dopamilie hydrochloride, 10-2 -11; tr>-ptaminehydrochloride, 2 X 1(1-3 .TI; S-hydrosytryptamiiie hydrogen oxalnte,l4 2 X ( )-iiorepinephriiie hydrochloride, 1 0 - 3 31: and benzylamine sulfate, 1.6 X I C 3 J!. The inhibitors studied were ( & ) trans-, ( +)-trans-, ( - )-lrnns-, and ( f)-cis-'?-plieiiylcycloprop~l:mine (as hydruchlorides) (2-phenyl lopropylarnine = I ) , ( i)-tra,,z.s-N,N-tlimethyl-~-(2-phen~-lcyclopropyl)amine(11) hytlrochloride, N-beiizyl-S-i-rieth~-l-?;-i2-pri1~~yiiylja~iiiie (111) hytiroc~hloride, a-methylphenethylhydrazine ( I V ) hydrochloride. 7-methos~--l-niethyl-9H-pyrido[3.4-b]indole (harmiiie) hydroc:hloride, 4,O-dihydrc1-7-1netliosy-l-1iiethyl-~3H-p~rido [3,4-b]iridole (harmsliiie), and ~,F)-dihydro-i-hydros~--l-iiiethyI-:3H-pyrido[:3.4-b]indole (harrnalol).l5 Substrate Specificity.---The relative substrate specificitl- iif highly purified beef liver mitochondrial LIAC) was determined by the niethid of C o i i ~ i y . All ~ ~ substrates used were 5 x 10F3 .If. The results are shCJwl1ill Table 11.
*
-. .
(18) 11. Yarnaila, 0 . .idacIii, 11. liiiniaeai. ami r i . Ogata, .1Zent. R e s . I m t , Food So.,Xlioto L-niz,., NO.26, 2 1 ( 1 9 6 6 ) : 11. Y a m a d a , 0. .\daclii. a n d l i . Ogata, ~ 1 0 B~i d. . Chern. ( T o k y o ) , 2 9 , 117 (1965). h f t e r conclusion of o u r n o r k we learned of tlie studies of C. 31. AlcEwen. ,Jr,, J . Bi07. Chem., 240, 200:3, 2011 (11165), on AI.40 from h u m a n plasma. This enzyme is probably also a copper protein and deaminates primary amines, among them, benzylusnine. tryptamine, tyramine, dopamine, a n d kyniiramine, hilt not histamine ani1 5-lI'V. 114) Ikcnuse the' erIirrirrients, except thosr w i t 1 1 Iienz~laniine.dependell of tile titration , i f S I T 8 liberated with K C O s froin t h e enzymatically draininated substrates, a common complex of 5-HT with creatine sulfate could lIOf
111'
ll.(d.
[l,i) \\P are grateful to l l r . I3ernard \ i i t k o p of tile S s t i o n a l lnatitutr[ i f Ileulth for samples of harmaline and harmalol. k 10) I : . J . ('una-a?. "Microdiffusion Analq-sis a n d Volumetric E r r o r , ' 4 t h Ed., Croslbv, Lockwooil and J o n , L t d . , London. 1957.
TABLE TI sUBSTK.1TE
SPECIFICITY
OF
BEEF IAIVER ~ ~ I T O C H O N D R I AJLI A O
Julrrtrstt.
Ilelntive rate of deamination
Tyramine Benzylamine Dopamine Tryptamine .i-Hydroxytr>-ptamint: Sorepineplirine
100 -3,
i r)
60 53 46 17
Methods.--I.:nzyme activity was measured by the rnctliod of Tabor, et al.," using benzylamine as the substrate. Protein (.oncentration was determined colorimetrically by the biuret rvxtiori measuring absorbancy a t 540 nip. The potencies of the inhibitors were assayed spectr~scopically~~ on the deamination of benzylamine, but for all other substrates a simplified in vitro volumetric method \vas evolved. Two-chamber porcelaiii Conway dishes were charged with 1 .()(I 1111. of tJL;2 boric acid, t o which 1 drop of broin cresol green indicator mis added. 111the outer chamber was placed a n amount of 0.1 Jf potassium phosphate buffer ( p H 7.4), chosen t o make :L total of I .00 nil. after addition of enzyme, inhibitor, and substrate. Enzyme (0.05-0.:3 nil., depending on its activity arid oti t hr substrate) a r i d iihibit,or (0.1 nil.) were added, aiid the mixture \vas itllowed t o stand under cover for l;i niiri. Then 0.1 ml. of substrate solution was admixed, and the dish was covered with $1 polished glass plate fastened with High Vacuum Silicori vacuum grease. After 30 min. at 20-24', a saturated K2COa solution (1 nil.) \\'as added rapidly, and the tightly (.overed dish was placed in an oven at 33" for 1 hr. The borat,e solution was titrated with i . 1 5 X 1 0 - 3 S H&04. Each determination was run in duplicatr. A blank of enzyme and buffer alone, and a coni rol of enzyme, buffer, arid substrate were run similarly each day. Inhibitor conceritrations were chosen t o bracket 50% inhibition values, and [IIj0 was extrapolated from these points. None of the inhibitors evolved NHI or amines under the esperimental conditioiis. Of the substrates used, only norepinephrine and benzylamine gave appreciable amounts of NH3 which were tietlucted froni t h e re:rction product values.
INHIBITIOK OF HOMOGEKEOUY ~IOSOAMISE OXIDASES
November 1963
861
TABLE 111 I n Vitro INHIBITION OF HIGHLY PURIFIED BEEF LIVERMITOCHONDRIAL MA0
IIlao, '%I-
r
Inhibitor
Benaylaminea
Tyramineb
DopamineC
Tryptamined
&Hydroxytryptaminee
1.4 X 10-7 5.6 X 6.3 X 1 . 6 X 10-6 3 . 9 x 10-6 1.8 x 10-6 2 . 5 x 10-7 7 . 1 x 10-7 7 . 1 x 10-8 3 . 2 X 10-7 2 . 5 x 10-6 1 . 8 x 10-5 5 6 X 4 . 5 x 10-6 4 . 4 x 10-0 8 x 10-7 3 . 5 x 10-7 8 . 9 x 10-7 8 . 9 X 10-8 5 . 6 X lo-' I1 7 . 1 x 10-8 5 . 6 x 10-7 6 . 3 x 10-7 3 . 2 x 10-7 7 . 1 x 10-7 2.5 X 4 x 10-7 2 . 2 x 10-7 i.1 X I11 7 . 1 x 10-8 3 . 2 X 10-5 2 . 2 x 10-5 1.8 X 4 . 5 x 10-6 IV 5 . 0 x 10-7 Benzylamine sulfate, 1.6 X 11; determined spectrophotometrically at 250 mp by the method of Tabor, et al." Tyramine.HC1, 5 X M. Dopamine. HC1, 1 X X. Tryptamine.HC1, 2 X .If. e 5-Hydroxytryptamine hydrogen oxalate, 2 X M. ( =t)-trans I ( )-trans I ( - )-trans I ( It )-cis I
+
0
Results
TABLE IT'
In Vztro I ~ H I B I T IOFO HIGHLY N PURIFIED BEEF LITEK ~IITOCHOXDRI IL hIAO BY H IRV i~i ALE. ILOIDS
The in vitro inhibitory concentrations, [I]5o, of seven inhibitors of beef liver mitochondrial M A 0 are listed So inliib. Inhibitor Concn., .11 Substrate in Table 111. They emphasize the significance of the Harmine 1.6 X Benzylamine 0 substrate in such inhibition experiments. For ex1 x 10-3 11 Dopamine ample, (*)-cis I has been reported18 to be as potent 1 x 10-3 17 Tryptamine as the (*)-trans isomer for the inhibition of the de1 x 10-3 5-HT 46 Benzylamine 0 amination of tyramine by M A 0 in a beef liver niitoHarmaline 1 . 6 X 10-5 1 x 10-3 5-HT 69 chondrial preparation ; under other conditions (rat Harmalol 1 . 6 X 10-5 0 Benzylamine brain homogenate, 5-HT) the cis isomer was about 6 times more potent than (*)-trans I.19 Comparative more potent inhibitor by some factor in the tissue values in Table I11 range from equipotency of the two homogenates, by reduction or 0-deinethylation, was isomers for the deamination of tyramine, to approxidiscounted when harmaline and harmalol were found mately the double potency for the cis isomer with to exhibit equally low activity under our in vitro condiother substrates. In accord with the l i t e r a t ~ r e , ~ ~ ~ ~ ~ tions. The fact that these condensed-ring indole (+)-trans I was 10-20 times more potent than the (-) alkaloids show only even low-grade activity (in the isomer. These data confirm the relative nonspecificity M region) in inhibiting the deamination of 5-HT of purified niitochondrial M A 0 toward geometrical, by the beef liver enzyme points to a possible differbut not toward optical isomers. ence in mechanismz3between the action of these alkaThe tertiary amine, (*)-trans 11, is a powerful inloids and of those inhibitors which do not contain an hibitor of the mitochondrial enzyme, ranging in potency indole systein. with (*)-cis I. The effect of some of our inhibitors on pure beef ?IT-Benzyl-S-methyl-N-(2-propynyl) amine (111) was M A 0 was measured spectros~opically~~ using plasma found to be one of the most potent inhibitors of our benzylamine as a substrate. Sone of the other submitochondrial M A 0 for all substrates used, while strates used with the mitochondrial liver enzyme could a-methylphenethylhydrazine (IV) was from 7-55 be tested because they are not deaminated by plasma times less active than 111. This relation was reversed AIA0.12 A comparison of the inhibition values in with pure beef plasma RIA0 (vide infra). V with those in the benzylamine column in Table I n view of the low rate of deamination of norepinephrine (Table 11),only one inhibitor was tested with T I B L E 1this substrate; (*)-trans I inhibited the reaction of I n Vztro INHIBITION OF PCRE BEEFPLILM i X k O norepinephrine 60% at a concentration of 10-5 AI. Inhibitor 11150, .w The most unexpected finding was the significant ( f)-trans I 6 . 3 X 10-6 lack of potency of harmine and related alkaloids in our 3.2 X ( Mrans I experiments. Harmine and harmaline have been 1 . 4 x 10-4 ( - ,-trans I reported as being among the most potent in vitro in( f)-cis I 2 . 8 x 10-4 hibitors of bIAO in rat brain and rat liver homogenates, IV 1 . 6 X 10-7 using 5-HT as a substrate,z1or guinea pig liver homogeBenzylamine as the substrate. nate using tyramine.22 As an inhibitor of the deaniinaTable I11 reveals the uniformly higher inhibitor contion of four substrates by highly purified beef liver centrations needed to affect the reactions of the plasma mitochondrial -1IAO (Table IV), harmine was from enzyme. A notable exception was the hydrazine IV to tinies as potent as in the tests with the which was the most potent inhibitor in our series for above tissue homogenates from other species. The plasma N A O , while it did not occupy such a position chance that harmine may have been metabolized to a with the niitochondrial enzyme. Plasma M A 0 has (18) S harkar, Dissertation, Northwestern UniT e r s i t r , E r a n s t o n , I11 , been shown to be inhibited by c8arbonyl reagents of 1961. (19) C L. Zirkle, C Kaiser, 11. H. Tedesclii, K. E. Tedeschi. and .I,Burthe hydrazine class, apparently by reaction with the ger, J . U r d . Pliarm. Chem.. 6 , 1265 (1962). pyridoxal cofactor of the enzyme.z4 It is possible that (20) J IC. N o r a n , Dissertation. Uni\ersitv of O t t a w a , O t t a n a , C a n a d a , IT' inhibits the enzyme in this fashion. 1962.
+
Q
(21) See Table I, footnotes a-d. (22) A Pletscher, H. Besendorf, H. P. Baclitold, and K. F. Gez, H e l ~ . l'hyszol. Pharmacal. Acta. 17, 202 (1959).
(23) Cf. ref. 19; for a review of this situation, see ref. 2a p. 531. (24) H. Painada and Ii. T . T a s u n o b u , J . Bzol. Chem., 298. 2869 (lYti3).
Notes
f r o m t h e Lnitvl f I ) (a1 s c i p p < j r t e , i l , i . C o n t r a c t N o . I.).\-~U-lHX-~I1~-203% Stnti,s Arniy AIriliral Research a n d T)c~velopmentCiimmanil, Office of tile Siirzeon Oenrral. ( 1 ) ) Presented a t the 1 4 l s t National XIeetine of thr . \ ~ n ~ r i < ,('Iiemiral a~t Society, Washingtiin. I ) , C . . March 1962. f c j T l i i s I S 28111i n a w r i e s of lialirrs concerned w i t l i t h e preparation a n d properties uf Iirw inonomer's anrl polymers; for the previous paper in this series, see ( ~ G . . Ovrrlicrgt-r, 11. Ringsdorf, and 13. .\rchen, .I. O r g . Chem., 3 0 , 2:12 (196,5], (11) l l i i u articlr i~taken from the dissertation of R . .ivchen sui,tnitted in partial fiilfillment of the reqiiirenirnts for tlir degree of Doctor
