Journal of Neural Transmission 38, 15--29 (1976) @ by Springer-Verlag 1976

Variation in Monoamine Oxidase Activity in Rat Brain Crude Mitochondrial Fractions Prepared by Rate Zonal Centrifugation* M. B. H. Youdim MRC Clinical Pharmacology Unit, Radcliffe Infirmary, Oxford, Great Britain With 6 Figures Received October 8, 1975

Summary Rat brain crude mitochondrial fractions were fractionated by rate zonal centrifugation using an iso-osmotic gradient of Ficoll and sucrose. The results demonstrated that the isolated fractions were biochemically heterogeneous with regard to the enzymes, monoamine, oxidase (MAO), NADH dehydrogenase and succinate dehydrogenase. When the activity of MAO was plotted as % of the highest specific activity towards tyramine, kynuramine oxidation remained fairly constant in fractions 10 to 30 but tyramine and dopamine showed separate peaks of activity in fractions 21 and 32 respectively. Sonic oscillations of separated particulate fractions did no: change the ratios of various monoamine deamination when compared to the intact particles. There are numerous reports that sotubilized mitochondrial monoamine oxidase (MAO) (monoamine-02; oxidoreductase [deaminating] EC 1.4.3.4.) of liver and brain exists in more than one form (Sandier, Youdirn, 1972; Youdim, 1975) as separated by gel electrophoresis. No adequate explanation has been put forward as to the basis of this multiplicity, although polymerization of solubilized basic active subunits (Gomes et al., 1969), conformational changes * MAO, Monoamine Oxidase; LDH, Lactate dehydrogenase; NADH, Nicotinamide-adenine dinucleotide reduced. Monoamine Oxidase (EC 1.4.3.4.), Lactate dehydrogenase (EC 1.1.1.27.), NADH dehydrogenase (EC 1.6.99.3.), Succinate dehydrogenase (EC 1.3.99.1.).

16

M . B . H . Youdim:

(Youdim, Collins, 1971) and attachment of membraneous material and phospho-lipids (Tipton, 1972; Tipton et al., 1972; Houslay, Tipton, 1973; Youdim, 1973 a) have been implicated. Because the methods used for bringing MAO into solubilized form are rather harsh, objections have been raised that multiple forms of MAO as separated by gel electrophoresis are the result of preparative procedures (Houslay, Tipton, 1973). The presence of monoamine oxidase attached to various populations of brain mitochondria was first envisaged by Youdirn (1972), and recent observations of Kroon and Veldstra (1972); Youdim (1973 b) and Yang and Neff (1973) suggest that this may indeed be so. Liver (Novikoff, Shin, 1964; Wilson, Cascarano, 1972) and brain mitochondria have been shown to exist in a range of sizes (Neidle et al., 1969; Salganicoff, Koeppe, 1968). It has recently been shown that different fractions of crude liver mitochondrial preparations separated by zonal centrifugation do indeed have different enzyme activity with regard to N A D H dehydrogenase, succinic dehydrogenase and a-glycerophosphate dehydrogenase (Wilson, Cascarano, 1972; Blokhlus, Veldstra, 1970). The present study was undertaken to investigate the substrate specificity of crude brain mitochondrial fractions separated by zonal rate centrifugation. It was considered essential that the mitochondria themselves, prepared during zonal centrifugation, be maintained as far as possible in a physiological state; therefore, an iso-osmotic gradient of Ficoll-sucrose was used according to the method of Wilson and Cascarano (1972). Materials and Methods Male Sprague Dawley rats weighing 150--200 grammes and kept on a diet of Spillers small animal diet were used. Ficoll was purchased from Pharmacia, U.K., Kynuramine was purchased from Sigma Chemical Co. Ltd., U.K., 1-14C-tryptamine hydrochloride were obtained from New England Nuclear, U.K., 1-I4C-Dopamine hydrochloride and 1-14C-tyramine hydrochloride were purchased from Amersham Radiochemical Centre, Amersham, U.K., 1-14C-Benzylamine hydrochloride was obtained from Isotope and Nuclear Division, ICN Pharmaceutical, Clevdand, U.S.A.

Preparation of Mitochondria All manipulations were performed at 4 ~ The rats were killed by decapitation and the brains were rapidly removed and placed on ice. The meninges were removed and the brains were washed with 0.32 M sucrose and dried by gentle blotting. A 10 % (W/V) brain homogenate in 0.32 M

Heterogeneity of Brain Monoamine Oxidase Activity

17

sucrose was prepared using a perspex-perspex homogenizer with a clearance of 0.1 ram. The homogenate was centrifuged at 8000 g-min in a MSE High Speed 18 centrifuge and supernatant collected. The pellet obtained (P 1), was washed once by recentrifugation aflcer resuspension in 0.32 M sucrose and the washing was added to the first supernatant. The supernatant was then centrifuged at 6X 105 g-min. The pellet formed (P 2) was resuspended in 0.32 M sucrose (1 ml/1 g of brain. This procedure is essentially as described by Gray and Whittaker (I962).

Rate Zonal Centrifugation Fractionation of the P2 fraction was carried out using a zonal rotor in an MSE 65 MK It ultracentrifuge by the adapted method of Wilson and Cascarano (1972). Gradients were made using 8 . 5 % (W/V) sucrose throughout but Ficoll varied from 0 to 11.5 % (W/V). The cushion was 80 ml of 45 ~ (W/V) sucrose. All solutions were prepared in 0.05 M trisHC1 buffer (pH 7.4) and gradient concentrations were measured using an Abbe refractometer and corrected for 4 ~ The cushion fottowed by the gradient (600 ml) was introduced with the rotor operating at 3000 rev/min and unloaded at rest aPcer deceleration. 30 ml of the resuspended P2 fraction were introduced rapidly through the core onto the gradient and then centrifuged for 20 rain at 9500 • g. The rotor was stopped completely by deceleration and fractions of 15 ml were collected. The pellets were isolated by centrifugation at 3X 105 g-rain and suspended in 15 ml 0.05 M phosphate buffer (pH 7.4) which was then divided into five 3 ml fractions of which one was used for the measurement of refractive index and the rest were kept at 4 ~ for enzyme assays.

Enzyme Assays Lactate dehydrogenase (LDH, EC 1.1.1.27.) as a marker for synaptosomes was assayed according to the method of Kornberg (1965). N A D H dehydrogenase (EC 1.6.99.1.) (Minakami et aI., 1962), and succinate deW hydrogenase (EC 1.3.99.1.) (Veeger et at., 1969) activities were determined by following the rate of reduction of ferricyanide at 420 nm. The final volume of the enzyme incubation mixtures was 3 ml containing 0.05 M Tris-HC1, p H 7.4, a volume of the particulate fraction containing 200 #g protein (suspended in 0.05 M phosphate buffer, p H 7.4) and 1 mM KCN. For N A D H dehydrogenase the assay medium also contained 0.3 mM N A D H and 2 mM KaFe (CN)r for succinate dehydrogenase, 5.0 mM succinate, 2.0 mM KaFe (CN)6. For the determination of MAO activity a range of substrates were used. With kynuramine as the substrate the spectrophotofluoremetric method of Krarnl (1965) was used. When 1-i4C-benzylamine, 1-14C-dopamine, tJ4Ctyramine and 1-C-tryptamine were employed as substrates the radioactive amines were diluted with non-radioactive amines to give a final concentraJournal of Neural Transmission 38/i

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18

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tion of 1 mM containing 60,000 dpm and M A O activity estimated by the method of Southgate and Collins (1969). Protein was determined by the method of Lowry et al. (1951) using crystalline bovine serum albumin as standard and corrected for interference by Ficoll.

Results The distribution of protein throughout the gradient is shown in figure 1. The protein concentration reached a peak at fraction 21 and a shoulder was observed between fractions 30 to 35. The Ficollsucrose gradient (density g/cm 3) is also shown in figure 1, with the lower density being at the higher fraction numbers.

Monoamine Oxidase Activity in ParticuIate Fractions To determine if there were differences in the activity of MAO between the fractions collected, five substrates were used. The total recovery of MAO activity using kynuramine and tyramine as substrates were respectively 85 and 78 ~ of the original activity. The 0.8

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Fig. 1. The distribution of crude brain mitochondriai protein on the Ficolt-sucrose density gradient after sonal rate centrifugation. The experimental details are described under Methods. The results are the mean of five separate experiments _+ SEM. Protein ( , , ) ; density of gradient ( . . . . . ). Protein was measured according to the method of Lowry et al. (t951) and corrections were made for interference with Ficoll

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Fig. 2. Specific activity of monoamine oxidase in crude brain mito&ondrlal fractions obtained by rate zonal centrifugation. The results obtained are shown when using tyramine, benzylamine and dopamine as substrates, and assayed according to the method of Southgate and Collins (1969). The fractions are numbered in order of decreasing density and the results are mean of five separate experiments. The results of when using kynuramine and tryptamine as substrates and the _+ SEM are excluded for the sake of clarity. However, the data _+ SEM are presented in table 1 and as per cent of highest tyramine monoamine oxidase activity in Fig. 3

results of MAO activity using i4C-benzytamine, i4C-dopamine, 14Ctyramine, i4C-tryptamine and kynuramine in the fractions collected aEer centrifugation are shown in figure 2 and table 1 +_ SEM. The distribution of tyramine and benzylamine MAO activities were similar, showing peaks at fraction 20--21 and 28--30. The pattern of "dopamine MAO" deviates from that of the tyramine and benzylamine curves, the highest specific activity being observed at the lower Ficoll-sucrose density (fractions 25--40), the results showing significant differences in MAO activity towards tyramine and benzylamine when compared to that towards dopamine in fractions 20 (P < 0.05) and 30 (P < 0.05). When the activities are expressed as per cent of the highest tyramine MAO specific activity (fraction 20), the following results were obtained (Fig. 3). Kynuramine MAO activity remained fairiy constant in fractions 7--32 at about 70--85 %. Benzylamine MAO (not shown for sake of clarity) was 90 % in fraction 20 and 50 % in 2*

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Heterogeneity of Brain MonoamineOxidase Activity

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fraction 32. A comparison with tyramine MAO showed that at lower fractions (16--23) activities were similar. However, at the higher fractions (e.g. 3.2) there is twice as much activity for tyramine as compared to benzylamine. The activity of "dopamine MAO" which was lower than that of tyramine MAO in fractions 5--26 showed a major peak in fractions 25--35 (Figs. 2 and 3). It increased from 35 ~ in fraction 5 to 93 % in fraction 32. In this fraction there was 35 ~ and 6 0 % greater activity for dopamine as compared to tyramine and benzylamine respectively. Tryptamine MAO activity showed to peaks of activities one at fraction 19 and the other at fraction 25--28.

Enzymes Other than Monoamine Oxidase The numbered fractions in which MAO activity was measured were also analysed for the enzymic activities of NADH-dehydrogenase, lactic dehydrogenase, and succinate dehydrogenase. The plot of specific activity against fraction number shows a different pattern

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Fig. 3. The distribution of specific activity of monoamineoxidase expressed as per cent of highest specific activity for tyramine, when using dopamine, tryptamine, tyramine, and kynuramineas substrates, obtained by zonal rate centris The results for benzylamine (see Fig. 2) are excluded for the sake of clarity. The fractions are numberedin order of decreasing density as described in the section of Results. TI~eresults are the mean of five separate experiments

22

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for each of the enzymes tested (Figs. 4, 5, 6). There was no sharp enzymic activity peak for NADH-dehydrogenase. However, succinate dehydrogenase showed a peak of activity at fraction 20 and a shoulder at about fraction 36 very similar to that observed for MAO activity towards tyramine and benzylamine. About 80 ~ of lactic dehydrogenase activity was observed between fractions 24 and 36

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