SOME PARAMETERS AFFECTING THE ACTIVITY O F MONOAMINE OXIDASE IN PURIFIED BOVINE BRAIN MITOCHONDRIA' SABITGABAY,FRANCES M. ACHEEa n d GULRIZMEN?ES* Biochemical Research Laboratory, Veterans Administration Hospital, Brockton, MA 02401, U.S.A. and Harvard School of Dental Medicine, Boston, MA U.S.A. (Received 29 October 1975. Accepted 20 January 1976) Abstracl~ Some parameters affecting the activity of monoamine oxidase (MAO) in purified beef brain

mitochondria were investigated. and diversities in enzyme properties were found as a function of sub>(rate.The deamination of the biogenic amines: serotonin. dopamine, tyramine, tryptamine, phenylethylilminc and two non-physiological amines, kynuramine and rn-iodobenzylamine, was studied. Anions in high concentrations inhibited enzyme activity with kynuramine being the substrate most affected. Among the biogenic amines, the activity with the indolalkylamines showed greater sensitivity to monovdent anions such as chloride than to polyvalent ions such as phosphate whereas the opposite was true with the phenylalkylamines. However, pyrophosphate ion had little or no effect on M A 0 activity, regardless of substrate. The inhibition of kynuramine and serotonin deamination was non-competitive but mixed competitive inhibition was found with tyramine and phenylethylamine. The activity of M A 0 was markedly affected by pH, and it had been previously reported that the substrates showed different pH optima in their oxidation. The effect of pH on activity has been attributed in part to changes in the ionization of the substrate and the hypothesis that the true substrate is the non-protonated amine. This was reflected in kinetic studies showing high substrate inhibition with increased pH. It was calculated that phenylethylamine would have the highest percentage of un-ionized amine at pH 8.2 and 9.1. At these pHs, there was more pronounced inhibition with high substrate concentrations of phenylethylamine than with the other substrates. In contrast. there was little inhibition with high substrate concentrations of tyramine which was the most ionizable of the substrates tested. When K, values obtained at pH 7.4, 8.2 and 9.1 were corrected for ionization of the substrate, the corrected K , was lowest at p H 7.4 for all substrates. Less than 50'" of M A 0 activity was lost when beef brain mitochondria was heated at 5 0 C for 20 min. However. there was only a slight variation with substrate in the thermal inactivation experiments. It is concluded that the mitochondria1 membrane environment surrounding the enzyme imposes certain restrictions on the enzymatic activity with respect to the different substrates which, in turn, are also affected by such parameters as pH and ions. The results are discussed in terms of the relationship of these factors to the question of enzyme multiplicity.

DESPITF numerous investigations, there continues t o be much uncertainty about the basic characteristics of monoamine oxidase (EC 1.4.3.4) (see COSTA& SANDLER, 1972; USDIN,1974). In our previous publication (ACHEE er ol., 1974), t h e preparation of brain mitochondria and some properties of MAO, as found in this purified brain mitochondrial fraction, were described. As more information about solubilized M A 0 becomes available, it is of critical importance t o know

how much of this information reflects t h e in cico state. Enzymes embedded in, or tightly b o u n d to membranes represent heterogeneous systems in which the microenvironment plays a n important role. Thus, for such enzymes, it is important t o understand the significance of the membrane attachment, h o w the characteristic microenvironment might affect the m o d e of action (GOLDMAN, 1973). To this end we have sought t o characterize M A 0 in the intact mitochondria. This paper furthers our observations on the properties of ' This article constitutes the third paper in a series enti- M A 0 in brain mitochondria and. in particular. tled 'Studies of monoamine oxidases'. For the first two reports on factors which affect the enzyme activity & VALCOGRT,1968; ACHEE such as pH. anions a n d heat. A preliminary communipapers, see references, GABAY ('1 d., 1974. cation of some of these findings h a s been presented ' Present Address: Ege University. Department of Bio- (GABAY er al., 1973). chemistry, Faculty of Medicine, Izmir. Turkey. Financial means were met by a fellowship grant from the American Association of University Women. Washington, D C and MATERIALS AND METHODS a travel fellowship from the Senior Fulbright-Hays ProReoymtA All radiochemicals [ l-"C] dopamine hydroPam, Washington. DC. U.S.A. Ahbruciution used: MAO. monoamine oxidase (EC bromide (6 28 mCi mmol), [ I-"C] 8-phenylethylamine hydrochloride (7 mCi/mmol). [2-'"C) tryptarnine bisuccinate 1.4.3.4).

416

FRANCES M. ACHEEand GULRIZMENTES SABITGABAY.

(47 3 mCi mmol). [I-''C] tyramine hydrobromide (10.73 mCi mmol) and [?-'"C] serotonin binoxalate (17.7 mCi mmoll. and scintillation chemicals were obtained from Yen England Nuclear. Boston, MA. The cation resins. AG 5OW-X8. 2 W o O mesh and Amberlite CG-50, 100-200 mesh, were obtained from Bio-Rad Corporation, Richmond. CA and A. H. Thomas Company. Philadelphia, PA. respectively. Horseradish peroxidase (EC 1.1 1.1.7) was a product of Boehringer Mannheim Corporation. New York. NY. Other chemicals were purchased as follows: kynuramine hydrobromide. Research Plus Labs., Inc., Denbille, NJ : 4-hydroxyquinoline. Aldrich Chemical Company. Inc.. Milwaukee. WI: homovanillic acid. Grade A. Calbiochem. La Jolla. CA m-lodobenzylamine-HCI was of Wyeth Labs.. Inc.. Philadela gift from Dr. W. F. BRYCE phia. PA, who synthesized it beginning with m-iodotoluene. following essentially the steps of ZELLERet u/. (1965) and the references cited therein. The melting point was 191-192-C: a 3.3 x lo-' M solution in 0.067 M-phosphate buffer, pH 7.4 had an absorbance of 0.260 at 253 nm. These properties compared favorably with the melting point of 193.C for m-iodobenzylamine-HCI reported in the literature (SHAPIROer al.. 1962) and the molar-extinction coefficient of 780 (ZELLERet a/., 1965). Preparation of mirochondrral frucrion. The washed mitochondrial fraction, P,, was prepared from bovine cerebral cortex as described previously (ACHEEet a/.. 1974) using differential centrifugation in an 0.4 M sucrose medium containing 0.001 M-EDTA and 0.0204 polyethylene sulfonate. pH 7.0. The mitochondrial fraction was routinely washed twice with the same medium containing 8% Ficoll to remove the lipid and fluffy white material. The quality of the preparation was assessed through the use of enzyme marker studies and electron microscopy as previously described (ACHEEer al., 1974). The mitochondria1 fraction was deemed to be reasonably free of contamination by other subcellular organelles and appeared to have a fair degree of morphological integrity. The ratio of M A 0 activity in this fraction compared to that of the homogenate was found to be approx 4:5 for the substrates tested: kynuramine, tyramine, tryptamine, dopamine, serotonin, phenylethylamine and m-iodobenzylamine. Monuamine oriduse assays 1. Specrrofluoromerric ussay of kynuramine oxidation. The method of KRAML(1965) was modified as described previously (ACHEEer a/., 1974) and used for the routine assay of kynuramine activity. 2. Radiometric assays. MA0 activity with the biogenic amines, dopamine, phenylethylamine, tryptamine, tyramine and serotonin was routinely determined using i4C-labelled substrates. Briefly, in a standard assay procedure, 50-100 pg of mitochondrial protein, 50 mM-phosphate buffer, pH 7.4 and appropriate concentrations of substrate were incubated in a total volume of 0.20ml at 3 7 T for 20 min. At the end of the incubation period, the reaction was stopped by the addition of 0.2 ml of cold 1.2 N-perchloric acid, the reaction tubes were chilled immediately and centrifuged (1000g,,,) to remove the protein. 0.2ml of the supernatant was applied to an 0.5 x 3.5 cm ion exchange resin column and the reaction products washed from the column with distilled water to a final volume of 5.0ml. A sample of the eluate was counted in 15 rnl of the scintillation solution consisting of 0.0250//:,POPOP, and 0.825% PPO in toluene mixed with Triton X-100 in a 2:1 ratio (v/v). Counting was done in a Unilux I Liquid Scintillation

counter (Nuclear-Chicago. Chicago, IL) with a counting efficiency of approx 65qb. This represents a somewhat lower effictency (approx 57;) than had been achieved with the more hazardous and costly naphthalene-dioxane scintillation solution previously used. Efficiencies were determined and quenching of samples corrected for by the channel ratio method. The procedures used previously (ACHEE e f (if., 1974) for assay of serotonin and tryptamine M A 0 activity involved the use of solvent extraction by ether and toluene. respectively. While good results were obtained with these methods, the use of ion exchange resins was found to he more convenient and less time consuming. The use of the same method to separate the products from all the various radioactive substrates greatly simplified the comparison of these substrates. In addition, papers reporting on the extraction vs ion exchange separation procedures have seemed to favour the latter methods (SOUTHGAIT & COLLINS, 1969; JAIN er a/.. 1973). Comparable results were found in this laboratory with the two methods, so that it was felt that the observed activities would not be affected by the change in separation methods. From earlier studies, it was found that optimal substrate concentrations for the given assay conditions were as folM ; dopamine. phenylethylamine lows: tyramine. I x and serotonin, 5 x W 4 M and tryptamine. 2 x 10.'~. The specific radioactivity of each substrate was diluted to 3-5 mCi/mmol with appropriate unlabelled substrate just prior to use. AG 50W-X8 resin. washed with distilled water, was used for the separation of tyramine and dopamine metabolites; Amberlite CG-50 resin, washed by the method of PISANO(1960), was used for the other substrates. In all of the assays, appropriate blanks were run to correct for the small amount of substrate not adsorbed to the column. The blanks were prepared by adding the substrate after perchloric acidification of the reaction mixture. followed by chilling and separation as in the test. 3. Specrrophorumerric assay of m-iodobenry/nminr m i dation. M A 0 activity with m-iodobenzylamine as substrate was determined by a slight modification of the method of ZELLERer al. (1965). The standard reaction mixture consisted of 3.3 x W 4M-m-iodohenzylamine, 50 mM-phosphate buffer. pH 7.4 and 15@200 pg of mitochondrial protein in a final volume of 1.0ml. Increase in absorbance at 253 nm with time was measured at 37 C using a Gilford Model 2000 recording spectrophotometer. The test sample was measured against an enzyme blank containing no substrate. Activity was expressed in terms of nmol of ni-iodobenzyaldehyde produced using an extinction coefficient of 9200 for the aldehyde at 253nm in phosphate buffer (ZELLERet a/., 1965). This same extinction coefficient was used in the pH optimum studies, since it was found that the change of pH and buffers did not significantly aflect the adsorption of m-iodobenzaldehyde at 253 nm. 4. Amine osidution measured by hydrogen peroxide production. The hydrogen peroxide released during oxidative deamination was determined essentially by the method described by SNYDER& HENDLEY(1968). This method is based on the findings of GUILBACLT et n/. (1967) that homovanillic acid can form a fluorophore in the presencc of peroxidase and H1O,. In a typical assay. 50mM-phosphate buffer. pH 7.4 or 7.8, 0.04 mg horseradish peroxidase and 50-100 pg mitochondrial protein were preincubated at 37°C for 10 min in a DubnofT metabolic shaker bath. After preincubation 0.25 mg homovanillic acid and appropriate amounts of substrate were added and the reaction allowed ~~

~

417

Parameters affecting M A 0 activity in brain mitochondria to incubate for 1 h with shaking at 37°C. At the end of the incubation period, the reaction was stopped by placing the reaction vessels in a boiling water bath for at least 1 min. The fluorescence of the samples were read at an emission wavelength of 420nm. excited at 320nm. The amount of H Z 0 2produced was determined from a calibration curve using a freshly prepared H,O, standard solution M-stock concentration) which was run similarly to and concurrently with the enzyme samples. Blanks in which the enzyme was placed in the boiling water bath before the addition of substrate were also run. M A 0 activity was determined with the substrates tyramine. tryptamine and kynuramine using 2 x lo-’ M, 2 x 1 0 - 4 and ~ 2 x M final concentrations, respectively. Catecholamines and hydroxyindoles are substrates of peroxidase er al., 1964) and, therefore, interfere with the (SAUNDERS measurement of H,O, by any coupled peroxidase assay. Thus, the deamination of other M A 0 substrates of interest, as serotonin and dopamine, cannot be measured by this assay procedure. This method is superior to determinations of hydrogen peroxide production from amine oxidation using o-dianisidine in having a greater sensitivity. In addition, o-dianisidine itself has been found to act as a competitive inhibitor of M A 0 (MCEWENet a/., 1968). 5 . Amine oxidation measured b j ammonia production. The ammonia produced in the M A 0 reaction with tyramine, tryptamine and kynuramine as substrates was determined using the microdiffusion technique, modified from the procedure of COTZIAS& DOLE(1951). The enzyme reaction mixture, consisting of 50 mM-phosphate buffer, pH 7.4, 5 W 7 0 0 pg mitochondrial protein and substrate in a final volume of 3.0 ml, was incubated in a glass-stoppered Erlenmeyer flask for 1 h at 37°C in a Dubnoff metabolic shaker. At the end of the incubation time, the flasks were immediately placed in an ice bath and a 2 ml sample of the reaction mixture quickly transferred to the outer ring of a prepared Conway diffusion dish. The outer ring of the dish contained 1 ml of a saturated K , C 0 3 solution. The center well contained 1.0ml of a boric acid indicator solution. The top ring of the dish was coated with silicone stopcock grease and the dish was sealed tightly, using a glass plate. Diffusion was allowed to take place for 3-5 h at room temperature. A t the end of the diffusion period, the center well solution was titrated with 0.01 N-HCI using a syringe microburette (Micro-Metric Instrument Co., Cleveland, OH) capable of delivering quantities as small as 0.5 pl. Appropriate reagent and enzyme blanks were also run. Activity was expressed as nmol of ammonia produced per min per mg protein at 37’C. Eflect of hearing on M A 0 acticitj For studies or the thermostability of MAO, samples of the mitochondrial suspension were incubated in a water bath at the desired temperature for various time intervals. One vol of a stock mitochondria1 suspension in 0.27 M-sucrose was added at zero time to nine volumes of the same solvent which had been pre-heated to the desired temperature. The final protein concentration of the mitochondrial suspension being heated was approximately 1 mg/ml. At the end of the heating period, the samples were placed in an ice bath for 10 min before the remaining M A 0 activity was determined using the standard assay procedure for each substrate as described above. The control sample was a mitochondrial suspension which had not been subjected to heating prior to assay. In some studies. either 5 0 m ~ phosphate buffer, pH 7.4, or 10 mM-pyrophosphate buffer,

pH 8.2, was used as the solvent system in which heating of the mitochondrial suspension was carried out. All other operations remained the same as given. Kinetic studies

The kinetic constants. K , and V,,,, were determined using the standard assay procedures as described above. At least 5-7 substrate concentrations were used for each determination with the concentrations being varied over a 10- to 40-fold range. In several instances, the concentration was varied over a wider range, up to 50- to 100-fold or more in order to ascertain the effect of high substrate concentration on activity, but only the velocities obtained with the lower ranges were used for determination of the constants. The substrate concentration ranges used for these studies varied with substrate as follows: tyramine, 2 x 1 0 - 5 t 0 2 x IO-’~;serotonin,5 x 1 0 - 5 t 0 5 x I O - ’ M ; kynuramine, 2 x lo-’ t o 2 x I O - ’ M ; tryptamine, 1 x to 1 x ~ O - ’ M to 5 x 1 0 - 4 ~ dopamine. ; 4 x and phenylethylamine. 5 x to 1 x lo-’ M. The constants were derived from double reciprocal plots (l/v vs I/s) as described by LINEWEAVER & BURK(1934). Linear graphs were obtained and the line of best fit was determined by linear regression and correlation analysis. performed with the aid of a programmed Monroe 1785 calculator. The correlation coefficients ( r ) for all the kinetic data plots were 0.98 or greater. Two to seven kinetic determinations were performed for each constant. Ionization constants

The pK, values for the amino group of phenylethylamine, serotonin, tyramine, tryptamine and kynuramine in aqueous media at 25°C are 9.79, 10.0, 10.52, 10.2 and 9.94, respectively. These values were taken from MCEWENet al. (1969) and the references cited therein. The pK, values at 37°C were estimated to be 9.43, 9.60, 10.13, 9.83 and (1964) 9.58, respectively, using the equation of PERRIN governing the temperature variation of ionization of bases. From these ionization constants, it is possible to estimate the percentage ionized form using a modified form of the Henderson-Hasselbalch equation (ALBERT & SERGEANT, 1971):

“A Ionized

=

100 1

+ antilog (pH-pK,)

Protein determination

Protein concentrations were determined by the method of LOWRYer a/. (1951) using bovine serum albumin as the standard.

RESULTS

Sroichiomerry of rhe M A 0 reaction Based on the investigations of numerous workers since the initial observation of HARE(1928), the stoichiometry of the deamination catalyzed by amine oxidases (mono-, di-, a n d polyamines) h a s been formulated as

RCHINHZ

+

0 2

+ HzO+

RCHO + NH3

+ HZO2.

Using the same mitochondrial preparation for all determinations and assay procedures as described in

SABITGABAY. FRANCES M ACHEE and GULRII.MENTES

418

effect on M A 0 activity in brain mitochondria using concentrations of pyrophosphate up to ionic strengths of 0.50. Thus, the nature of the inhibition by phosphate seemed to be a specific ion effect, rather than just the effect of increased ionic strength of the medium. In addition to phosphate, chloride and sulfate ions were also found to influence M A 0 activity in brain mitochondria. As with the case of phosphate, there was a differential influence of the salts on M A 0 activity. dependent on the substrate. In general, kynuramine deamination showed the greatest sensitivity to the presence of ions. Two-hundred mM-chloride ( I 0.23)caused an inhibition of approx 509.; and 150 mMsulfate ( I 0.47) an inhibition of 4076 with kynuramine. The inhibition with the biogenic amines was n o greater than 25?, with these same concentrations. The phenylalk ylamines appeared to be more affected by phosphate ions than by chloride, whereas the opposite was true for the indolalkylamines. There was little Effect of ions on M A 0 activity or n o apparent difference due to the cationic species Following the early studies by VAN WOERT& COT- of the salt with both sodium and potassium acting in a similar fashion. These data also indicated that ZIAS (1966) on the inhibitory effect of anions on the activity of rat liver MAO, only a few reports have the ionic strength was of less significance than the appeared on the prevalence of this phenomenon on presence of a particular anion. Dialysis of the mitoother M A 0 systems. It had been observed early in chondrial preparation before use did not alter the the investigation of the properties of brain mitochon- result, indicating that the varying levels of inhibition drial M A 0 with kynuramine in this laboratory thai observed were not due to any endogeneous factor there was a noticeable diminution of activity with in- present in the mitochondria1 fraction. creasing concentration of phosphate, used a s the buffering ion. This effect was further investigated with Kinetics of inhibition by chloride ions The differences in the properties of M A 0 with reother substrates. In general, concentrations of phosphate up to approximately 5 0 m ( I 0.135) did not spect to substrate was also reflected in the type of significantly affect M A 0 activity. However, with inhibition produced by chloride ions. Non-competi200 mhf-phosphate (I 0.54), kynuramine deamination tive inhibition was found in the deamination of serowas inhibited 457, the phenylalkylamine substrates, tonin and kynuramine, indicated by a decrease in V,,, tyramine, dopamine and phenylethylamine, approx and no change in K , (Table 1). For tyramine and 15-257; and the indolalkylamines, serotonin and t r y p phenylethylamine deamination, although the V,, also tamine l0-15%. In contrast t o the effect of phosphate decreased with increased chloride concentration, the ions. pyrophosphate appeared to have little or no K , increased. indicative of a mixed type of inhibition.

the Methods, from tyamine: 5.68nmol hydrogen peroxide. 5.59nmol ammonia and 5.18 nmol amine product were formed per mg protein per min. From t r y p tamine: 2.28 nmol peroxide. 2.52 nmol ammonia and 2.41 nmol amine product. and from kynuramine: I .73 nmol peroxide. I .89 nmol ammonia and 1.40 nmol 4-hydroxyquinoline were formed. It was thus evident that a satisfactory stoichiometric relationship was achieved with the bovine brain mitochondria1 fraction. The aldehyde produced by the action of M A 0 is rapidly metabolized further by aldehyde dehydrogenase or aldehyde reductase to form the corresponding acid or alcohol. I n the radiometric assays, all these constituents are measured together as the oxidized product. In the case of the kynuramine assay. the aldehyde undergoes a spontaneous condensation to form 4-hydroxyquinoline. a reaction which is faster than the further oxidation processes (WEISSBACH et al., 1960).

TABLE1 THEEFFFCT OF

Kynuramine (3) K. V k X

Phenylethylamine (2) K. Vm,,

CHLORIDE ON KlhETIC CONSTANTS FOR MOWOAMINL OXIDASF IN B I LF BRAIN MITOCHOYORIA

(0)

Chloride concentration (100 mM)

4.38 + 0.16 x lo-’ 3.52 k 0.32

4.44 0.22 x 10-5 2.74 i-0.26

4.31 f 0.17 x lo-’ 2.22 + 0.14

2.34 0.28 x lo-’ 2.72 5 0.12

2.78 + 0.22 x 10-5 2.43 f 0.05

3.37 + 0.16 x lo-’ 2.24 f 0.01

1.61 x 10-4 4.43

1.62 x 3.52

1.62 x 10-4 2.93

*

(200 mM)

Serotonin K. V,,

Tyramine (4) K, V L X

7.30

+ 0.33 x

7.18 k 0.65

lo-’

9.27 f 0.21 x 10-5 6.90 k 0.70

10.86 k 0.92 x 6.27 f 0.42

All assays were carried out in 50mM-phosphate buffer, pH 7.4 at 37’C. K , is expressed i n molar concentration is in terms of nmol of product per rnin/rng protein. Values given are means ~ s . E . M .The numbers in brackets beside substrate names are the number of replicate determinations for each constant. No estimates of variation are given for serotonin since there was only a single kinetic study performed on the effect of chloride with this substrate. (M); V,,,

Parameters affecting MA0 activity in brain mitochondria TABLE 2. THEEFFECT

OF

pH

MA0

ON THE KINETIC PARAMETERS OF

x Substrate

pK, (37'C)

pH

Tyramine

10.13

7.4 8.2 9.1 7.4 8.2 9.1 7.4 8.2 9.1 7.4 8.2 9.1 7.4 8.2

Tryptamine

9.83

Serotonin

9.60

Kynuramine

9.58

Phenylethylamine

9.43

0.19 1.16 8.54 0.37 2.29 15.70 0.63 3.83 24.02 0.66 4.02 24.88 0.92 5.56

IN BOMNE BRAIN MITOCHONDRM

Apparent

Un-ionized

K,

(5) (2) (2) (3) (2) (3) (5) (2) (2) (7) (2) (4) (5) (3)

419

Corrected K , (P)

(PM)

0.134 k 0.006 0.404 k 0.015 1.640 + 0.111 0.044 If: 0.004 0.183 f 0.002 1.334 0.220 0.654 + 0.167 7.334 + 0.498 9.944 1.057 0.343 + 0.031 0.699 f 0.040 3.832 0.299 0.224 + 0.016 0.906 +_ 0.050

70.6 f 3.2 34.8 f 1.3 19.2 1.3 12.0 f 1.1 8.0 f 0.1 8.5 f 1.4 103.8 f 26.5 191.5 f 13.0 41.4 f 4.4 52.0 f 4.7 17.4 f 1.0 15.4 f 1.2 24.4 f 1.7 16.3 f 0.9

+

+

+

Vl,,

7.22 f 0.50 6.88 f 0.32 4.92 f 0.48 2.39 f 0.22 2.53 f 0.15 1.67 f 0.25 3.61 f 0.24 5.86 0.36 3.97 f 0.54 3.87 f 0.20 5.20 +_ 0.03 5.77 f 0.19 2.37 f 0.15 3.47 f 0.22

Kinetic constants were determined using standard assay procedures at 37'C and calculated as described in the Methods section. p H 7.4 assays were carried out in 50m~-K-phosphatebuffer; pH 8.2 and 9.1 assays were carried out in lOmM or SO mM Na-pyrophosphate buffers. The 'corrected K , values represent the experimentally determined apparent K , x % un-ionized amine. V,,, is expressed as nmol of product/mg proteinimin at 37'C. Values given are the means ~ s . E . M . The number of replicate determinations are given in brackets,

pH Optima of MA0 activity in houine brain mitochondria In the previous paper in this series (ACHEEet al., 19741, the pH optimum for the phenylalkylamines, tyramine and dopamine, was reported to be in the region of pH 7.4, while the indolalkylamines, serotonin and tryptamine, had a higher optimum, closer to pH 8.2. Kynuramine differed from the physiological amines with a pH optimum of 9.1. In line with this apparent distinction between the phenyl and indolyl substrates, the activity of phenylethylamine also appeared to be optimal around pH 7.3, using a constant substrate concentration for the activity measurements at all pHs. On the other hand, m-iodobenzylamine was found to have an optimum of 8.6.

in correcting the K,s for the ionization in this way, one observes that the lowest Kks are obtained at pH 7.4 for all substrates which might be of physiological significance. A comparison of V,,, at the three pHs under consideration showed that the pH for optimal activity was in the same area as found when one compared Y versus pH for all the substrates, except phenylethylamine.

"I

/

0.9

I

/

0,''

Effect of pH on the kinetic parameters of M A 0 The kinetic parameters of M A 0 were determined at the three pH values observed to be optimal for the various substrates. Results are given in Table 2. It has been suggested by several investigators that the effect of pH on the activity of M A 0 is related to the ionization of the substrate, so that the true substrate for M A 0 i s the amine in the unprotonated form (MCEWENer ul., 196X, 1969; WILLIAMS. 1974). If one corrects for the ionization of the substrate. then 'true' K , values ( K , ) are obtained which have been shown for solubilized and purified liver M A 0 not to vary with pH, in contrast to the apparent K , values (MCEWENet al., 1968, 1969; WILLIAMS, 1974). The apparent K, values determined for the beef brain mitochondria were corrected for ionization using the pK, values for the aliphatic amino group of each substrate at 37-C. The same constancy of corrected K , with pH was not found for the membrane-bound enzyme as seen From the data in Table 2. However,

a2 0.1 .

10

20

y

30

40

IO-~Y

FIG. I . Effect of substrate concentrations on the activity of beef brain mitochondria1 MAO. [The activity with serotonin as substrate was determined using assay conditions as described in 'Methods'. The buffer systems were 50mMphosphate buffer. pH 7.4 (t-+) and IOmwpyrophosphaie buffer, pH 9.1 (& --0).

SABIT GABAY. FRANCES M. ACHEE and GULRIZMENWS

420

1

1

2

If x

3

4

-

5

x10-4

10-4

FIG.2. Effect of pH on high substrate inhibition of MAO. (a) Deamination of phenylethylamine in 50mM-phosphate buffer. pH 7.4 (-0) and IOmht-pyrophosphate buffer, pH 8.2 ( D - 0 ) .(b) Deamination of tyrarnine in 50 mht-phosphate buffer, pH 7.4 (-0) and 10 mM-pyrophosphate buffer. Assays were performed as described in ‘Methods’. pH 9.1 (O---O).

High substrate inhibition of M A 0 activity

Effect of heating on M A 0 activity

In the kinetic studies of M A 0 in the bovine brain mitochondria at pH 7.4, no substrate inhibition was M. This was not observed up to about 1 to 2 x found to be the case for studies at the higher pHs. In particular, it was observed that inhibition by high substrate concentrations became more prominent as the pti was increased. with a decrease in activity being observed at concentrations that did not appear ‘to have any inhibiting effect at pH 7.4 (Figs. 1 and 2). Of the substrates tested, phenylethylamine deamination appeared to be the most vulnerable in this respect, exhibiting no inhibition at concentrations up to 2 x M at pH 7.4, but showing substrate inhiM at pH bition at concentrations just above 1 x 8.2 (Fig. 2a). The deamination of serotonin (Fig. 1) was markedly inhibited by high substrate concentrations at pH 9.1 but with tyramine, at pH 9.1, little or no inhibition was observed at 2 x M (Fig. 2b).

Previous thermostability studies with liver mitochondrial preparations from rat (YOUDIM& SOURKES, 1965) and pig (ORELAND & EKSTEDT, 1972) had shown that M A 0 was relatively heat stable. Likewise, M A 0 in intact, purified mitochondria from both beef and rabbit brain have been found to be thermostable (GABAYet al., 1973). For beef brain mitochondria, while there appeared to be minor differences in the amount of activity remaining when tested with the various substrates, in general, less than 50% of the activity was lost after heating for 20 min at 50°C. Activity is quickly lost above this temperature with less than 50% of the activity remaining after 20 min heating at 60°C. With the rate of heat inactivation at 50°C (Table 3, Fig. 3), again some variation with respect to the amount of activity remaining with any given substrate was apparent, but there did not appear to be any marked differences among any of the substrates as has been reported with other M A 0

TABLE 3. RATEOF

HEAT INACTlVATlON AT

50°C

OF

MA0

IN B O W M BRAIN MITOCHONDRIA

yo Activity remaining after heating at 5OC for the following length of time (min) Substrate Dopamine (3) Kynuramine (2) rn-lodobenzylamine (1) Phenylethylamine (2) Serotonin (4) Tryptamine (3) Tyramine (3)

5

10

97 f 0.6 103 f 3.0

86 f 2.5 97 f 1.0 82 96 f 4.5 82 f 7.0 90 f 2.5 91 f 0.9

100 i 7.5

92 f 4.9 97 f 1.5 95 5 2.5

15

20

81 f 4.4 74 f 6.2 90 f 0.5 84 5 4.5 78 90 f 1.5 84 i 0.5 76 f 5.8 70 f 2.4 82 f 3.3 77 f 4.2 81 f 3.6 75 f 3.4

30

40

50

62 f 4.9 71 5 4.0 60 76 i 1.5 57 i 4.9 64 Itr 4.0 68 f 3.2

50 ~tr3.2 61 +_ 3.0 52 69 f 2.0 50 f 6.4 52 f 3.9 54 f 5.8

47 k 3.2 54 S_ 7 , 5 50 63 f 1.0 48 f 6.8 50 f 4.0 49 f 5.2

60 38 f 3.5 46 5 5.0

45 53 38 40 42

k 2.0 k 7.5 & 1.2 f 3.1

The mitochondria1 suspension (approx 1 rngiml) in 0.27 M-sucrose was heated at 50 C for the indicated time Intervals and assayed by standard procedures in 0.05 M-phosphatebuffer, pH 7.4 at 37°C. Activities were compared to an unheated control sample and values are the means ~ s . E . M . The number of replicate determinations are given in brackets.

Parameters affecting M A 0 activity in brain mitochondria 2.00,

t-. Phcnylclhylamine D--U Kynuramine

Hlyraminc u Tryptaminc &-*Dopamine 0-0 Serotonin

1.90 i

I

10

20

30

40

50

60

LENGTH Of HEATING (mid

FIG.3. Thermostability of M A 0 at 50’C. The mitochondrial fraction was heated at 50°C for the time intervals and assayed with the different substrates as described in ‘Methods’.The percentage of activity remaining was determined by comparison with an unheated control sample. Data points are the mean of 2-4 determinations.

42 I

M A 0 has a broad substrate specificity, these substrate differences have been taken as an indication of the multiple nature of MAO. In the present study on M A 0 in purified bovine brain mitochondria, the stoichiometry of product formation was found to be in good agreement with the general enzyme reaction stated earlier, using tyramine, tryptamine and kynuramine as typical M A 0 substrates. Thus, in this respect there is no substrate variation and it can profitably be discussed in the context of the net catalytic reaction. However, it is apparent from other data that each substrate or structurally similar substrate group, in general, does respond to environmental conditions in a different fashion which might reflect either a multiplicity of molecular forms, multiple active sites, or different affinity groups within the active site. Although the degree of inhibition by phosphate and other anions at the concentrations used in this study was not as pronounced in comparison to inhibitions generated by more specific inhibitors, still it is of interest to note the distinctive subtle variations with respect to the different substrates. The data presented here support the earlier report of VAN WOERT& COTZIAS (1966) on the anion inhibition of rat-liver MAO. Using serotonin and tyramine, they also found that

100

Pp

80 60

& RACKER,1973; YANG& NEFF, systems (MCCAULEY 1973a,b) which might point to the definite existence of multiple molecular forms in this system. The thermal inactivation of M A 0 was greatly affected by the pH of the solution in which heating takes place as shown in‘Fig. 4. However, both at pH 7.4 and at pH 8.2, the extent of the inactivation did not appear to vary with substrate, comparing serotonin and phenylethylamine.

\

r L 4 1

\

“t

DISCUSSION The results presented here show the many complexities of MAO. They also offer a possible explanation for the difficulties encountered in trying to correlate the data on the fundamental properties of M A 0 from the literature. Enzymatic heterogeneity is accepted as a general phenomenon and it should not be an exception that the properties of M A 0 vary significantly with respect to the species and tissue from which the enzyme is derived. It is not always possible to extrapolate from one species to another or from one tissue to another, although one would expect some basic similarities in the performance of the same enzyme-catalyzed process. However, within the same tissue preparation, it has been found that many properties of M A 0 are a function of substrate, and since

\

pH 8.2

I

I

5

I

I

I

I

10

15

20

25

M i n 01 Heating a t 50’

FIG. 4. Effect of pH on the thermostability of M A 0 at 50C. The mitochondria1 fraction was heated 50 C in either 50 mM-phosphate buffer, pH 7.4 or 10 mu-pyrophosphate buffer, pH 8.2. The remaining activity was measured using serotonin ( x ) or phenylethylamine (0)and the percentage remaining activity was determined by comparison to control samples in the same buffer systems which were not heated. The activities were measured in the same buffers

as the incubation using standard assay conditions as described in ‘Methods’.

422

SABITGABAY.FRANCES M. ACHEEand GULRIZMENES

tyramine deamination was affected more by phosphate than by chloride with the reverse situation for serotonin deamination. Whereas non-competitive inhibition by chloride had been found for both tyramine and serotonin in that previous study. the results here on bovine brain M A 0 showed a mixed type of inhibition for tyramine and non-competitive inhibition for serotonin. In a recent study by BROWNE ~t al. (1973) on rat heart and liver MAO, they also noted inhibition of tyramine deamination by increasing concentrations of phosphate buffer. but apparently only at pHs higher than 7.8. In this regard, as well as with optimal pH values, their results were somewhat different than those presented here, but they have also emphasized the complexities of interaction among the source of enzyme activity, the substrate and the buffer system. In comparison to all the other substrates tested, kynuramine oxidation was more strongly affected by the presence of salts. The marked difference in the inhibition of this substrate could very likely be interpreted as indicating that an entirely different enzyme might be involved. YOUDIMet al. (1969) separated four forms of M A 0 from rat brain using gel electrophoresis and found that kynuramine was deaminated very readily by MAOz and to a lesser extent by the three other forms. Another possible explanation, however, for the stronger inhibition with kynuramine might be made on the basis of its structure. Kynuramine contains two amino groups, although it is not oxidized by diamine oxidase, with one amino group being attached directly to the benzene ring. This additional ionizable group could conceivably lead to a stronger interaction in the presence of the chloride ions to provide for even less of a breakdown of the ES complex in comparison to the other substrates. The deamination of kynuramine, like serotonin, is inhibited non-competitively by chloride, whereas the inhibition is of a mixed type when tyramine and phenylethylamine were substrates. This might indicate a difference in binding groups for the various substrates within the active center. That pH markedly affects the activity of M A 0 is evident from the data in Table 2. The substrate specificity order one obtains from a comparison of V, is subject to variation dependent on the pH. Earlier studies of the pH optima for M A 0 activity, carried out using a constant substrate concentration at all pH values, indicated a distinct pattern whereby the phenylalkylamines, tyramine and dopamine, were optimally oxidized at pH 7.4, the indolealkylamines, serotonin and tryptamine, around pH 8.2 and the non-physiological substrate, kynuramine, at pH 9.1 (ACHEE et at., 1974). These optimal values derived from a comparison of v appear to be substantiated by the V,,, values in the present study. Phenylethylamine appears also to follow this general pattern. showing a pH optimum of 7.3 in a study of v vs pH. However, this optimum value does not appear to hold true when one looks at V,,,. tn-Iodobenzyla-

mine, like the other non-physiological substrate, has a higher pH optimum of 8.6. Studies by MCEWEYet a/. (1968, 1969) on solubilized, partially purified human liver M A 0 and by WILLIAMS (1974) on purified pig brain M A 0 have indicated that the true substrate for the enzyme is perhaps the unprotonated amine. Although the approach described in those studies may strictly apply only to model substrates such as benzylamine, with the only ionizable group being the reactive amino group, a simplistic view was taken here. Thus. only the ionization of the reactive amino group was considered with the thought that some relevant information might still be obtained. Phenylethylamine, of all the substrates, might be expected to give valid results, in that it has only the one ionizable group, but the ‘corrected’ K , values ( K k ) in Table 2 still show a slight variation with pH. However, the hypothesis of an unprotonated amine substrate is still tenable for the bovine brain mitochondria. since it might be expected that the microenvironment of M A 0 in the intact mitochondria affects its properties. It had been noted previously that membrane-bound enzymes may not have Michaelis constants identical to those of their soluble counterparts (ACHEE ef a/., 1974) and this was found to be indeed the case for M A 0 in rat liver (HOUSLAY & TIPTON. 1975).The observation that the K ; values are lowest at pH 7.4 may be a reflection of actual affinities under physiological conditions. While not proved here. the hypothesis of un-ionized ‘true’ substrate could explain some of the observations with regard to substrate inhibition. It had been previously stated that at pH 7.4, the marked high substrate inhibition reported for M A 0 on other systems was not found with the bovine brain mitochondria (ACHEEet a\., 1974). As with the case of the kinetic constants. it was thought that the point of consideration was the difference between a solubilized system and a membrane-bound enzyme, for where substrate inhibition had been reported. it seemed more often than not that the investigations were carried out using solubilized. partially purified systems ( G A B A Y & VALCOURT, 1968; NAGATSU el U/.. 1970). Although this may still be a valid factor, subsequent studies on mitochondria1 preparations at high pHs have led to observations of substrate inhibition. The extent of the inhibition appears to vary with the substrate and with pH and this is substantiated by the differences in ionization of the substrates as seen in Table 2. Thus, tyramine was found not to be markedly affected by high substrate concentrations, being the more ionizable of the substrates studied at all pHs, so that for any given concentration, there would be actually less of the effective substrate with tyramine than with the other amines. In this respect also, it is noteworthy that a higher concentration is required for optimal activity with tyramine than with the other substrates. In contrast, phenylethylamine shows a marked susceptibility to substrate inhibition, especially at the higher pHs and this probably

Parameters affecting M A 0 activity in brain mitochondria

423

accounts for the observation that measuring v with mental conditions in the determination of the enzya constant substrate concentration results in an matic activity of MAO. It is evident that the binding apparent pH optimum of 7.3 but one obtains a higher to the mitochondrial membrane must play a critical V,,, value at pH 8.2. role in the function of the enzyme in creating a relaIn the studies on the effect of pH as well as the tively non-polar environment which would favor the effect of ions on M A 0 activity, a parallelism in re- oxidation of the amine. Recently, it has been demonsponse is discernible among the substrates based on strated that the separation of multiple forms of M A 0 similarity of structure. Thus, the phenylalkylamines, by electrophoresis could be the result in the binding the indolalkylamines and the non-physiological sub- of different amounts of membrane lipid material to strates, kynuramine and m-iodobenzylamine are dis- a single enzyme species (HOUSLAY & TIPTON,1973a,b; tinguishable. However, the thermostability studies did TWTONe t a / . , 1973).Accordingly, it had been reported not follow the same pattern. A careful analysis of the that Triton X-100 solubilized mitochondrial M A 0 data revealed that while the activity involved in the preparations from both rat liver and human brain deamination of phenylethylamine and kynuramine yielded bands with polyacrylamide gel electrophoresis appeared more stable, heat inactivation observed with (YOUDIM & SANDLER,1968; YOUDIMet at., 1970). all the other substrates was nearly identical. These However, treatment of such a preparation with the results are in contrast to other reports of marked dif- chaotropic agent, sodium perchlorate, which disrupts ferences in thermal stability of M A 0 in the deamina- protein-lipid complexes (HANSEIN ef a/., 1971). tion of different amines which have been taken as resulted in abolition of these bands. leaving a single evidence of the existence of multiple forms of M A 0 band and in addition, eliminated some of the heat (YANG & NEFF,1973b; MCCAULEY & RACKER, 1973). and inhibitor differences previously observed with & TIPTON,1973a,b; TIPRecently, a number of publications have appeared various substrates (HOUSLAY in which it has been suggested that M A 0 exists as TON et a!., 1973). It was concluded by these authors two forms or types, designated A and B (JOHNSTON, that the multiple forms which were separated from 1968; YANG et al., 1972; SQUIRES,1972, 1975; the partially purified preparations were artefacts of & RACKER, 1973; YANG & NEFF, 1974; the solubilization procedure. Furthermore, the two MCCAULEY & TIPTON,1974; BATHINA forms distinguishable by clorgyline are believed to NEFFe t a / . ,1974; HOUSLAY et a/., 1975; R o w & GILLIS, 1975) distinguished pri- bear no obvious relationship to the electrophoretimarily by their sensitivity to the M A 0 inhibitors, cally separable forms. but arise from modification of clorgyline and deprenyl, and further characterized by a single enzyme species in 1:ii10 and are not preparatheir substrate preferences and heat stability. The A tive artefacts (K. TIPTON,Personal Communication). form is more readily inhibited by clorgyline and Concretely, the lipid environment surrounding the appears to have a preference for the substrates, sero- enzyme imposes certain restrictions on the enzymatic tonin and norepinephrine, while the B form is more activity reflected in substrate differences, which in sensitive to deprenyl with its preferred substrates turn is also influenced by surrounding conditions, being phenylethylamine and benzylamine. Other such as pH or ions. Since subtle differences in subamines, such as tyramine and tryptamine, dopamine strate reactivity have been the earmarks by which preand kynuramine, are thought to be substrates accept- sumed mitochondrial M A 0 isoenzymes are recogable to both types. However, in some reports, a so- nized, and since in both their demonstration and incalled 'common substrate' has been classified in either teraction with M A 0 inhibitors the sensitivity of sub& strates towards anions and pH have been overlooked. the A or the B group (SQUIRES,1972; HOUSLAY TIPTON,1974). In their study on rat brain MAO. the question of what conditions are necessary for the YANG & NEFF (L973a,b) reported that after heating proper evaluation of M A 0 activity and the identificathe mitochondria at 5 0 f for 20 min in 0.067 M-phos- tion of its isoenzymes is still of concern to many inphate buffer, pH 7.2, 90% of the serotonin deaminat- vestigators. ing activity remained, whereas there was only 20% activity left with phenylethylamine. The difference in Ac~non.ledgemenrs-The authors are indebted to Dr. K . of Cambridge University, England for his critical activity remaining observed here for purified beef TIPTON mitochondria under similar conditions was only reviewing of this manuscript. They also wish to thank Mrs. D.D.S. for their trchniabout between the same two substrates (Fig. 4) BESSIET S o K A s l s and M. DOHEKTY, with the serotonin activity being the more thermola- cal assistance in the preliminary phases of this work. Dr. was a trainee in the VA Dental Student Summer bile. When the same thermostability studies were car- DOHERTY Research Pfogram. ried out in pH 8.2 buffer, there was very neglible difference in the remaining activity for the two substrates. While the existence of multiple forms in beef REFERENCES brain cannot be definitely ruled out on the basis of these observations, it does indicate, however, a very ACHEE F. M.. TfficLci.4 G. & GARAY S. 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SABITGABAY.FRANCES M. ACHEE and GULRIZMENTES

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