498

Biochimica et Biophysica Acta, 437 (1976) 498--504

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27952 MONOAMINE OXIDASE IN O U T E R MEMBRANE OF S K E L E T A L MUSCLE M I T O C H O N D R I A

JEAN HIMMS-HAGEN and CLARE IRWIN Department of Biochemistry, University of Ottawa, 275 Nicholas St., Ottawa, Ontario, K I N 9A9 (Canada)

(Received December 16th, 1975)

Summary (1) Monoamine oxidase (EC 1.4.3.4) is present in rat skeletal muscle mitochondria. (2) A radioassay p r o c e d u r e f or the assay o f m o n o a m i n e oxidase in muscle m i t o c h o n d r i a is described. It is based on t he p r o c e d u r e using side-chain [2-14C] t r y p t a m i n e as substrate described by Wurtman, R.J. and Axelrod, J. {1963) Biochem. Pharmacol. 12, 1439--1441 and employs a pH of 8.0 and a substrate c o n c e n t r a t i o n o f 0.25 mM. (3) Th e Km o f the muscle mitochondrial e n z y m e at pH 8.0 is 1.34 • 10 -s M and t h a t o f the liver e n z y m e under the same conditions is 2.5 • 10 -s M. Muscle m i t o c h o n d r i a contain only one quarter of the activity o f e n z y m e present in liver mitochondria. (4) Mo n o amin e oxidase is shown to be in t he o u t e r m e m b r a n e o f skeletal muscle m i t o c h o n d r i a and thus to be a suitable marker e n z y m e for use in the f r a c tio n atio n o f these mitochondria.

Introduction In th e course o f experiments in which skeletal muscle m i t o c h o n d r i a were being fractionated it was necessary to use an enzymic marker for the o u t e r m e mb r an e. Monoamine oxidase (monoamine: oxygen oxidoreductase, deaminating, EC 1.4.3.4), c o m m o n l y used as a marker for t he o u t e r m em brane of liver m i t o c h o n d r i a [ 1 , 2 ] , is usually considered to be absent from skeletal muscle [ 6 , 7 ] . Indeed, our preliminary studies in which we used a spectrophotometric m e t h o d f o r the assay o f this e n z y m e [8] also showed the e n z y m e to be absent f r o m muscle mitochondria. However, the only ot her e nz ym e we have d e t e c t e d in the outer membrafle of muscle mitochondria, rotenone-insensitive N A D H - c y t o c h r o m e c reductase

499 (EC 1.6.99.3), is difficult to measure accurately because of the much greater proportion of the rotenone-sensitive enzyme in the inner membrane, and is thus n o t a sensitive marker for the purification of the inner membrane fraction. A report that a low activity of monoamine oxidase could be d e t e c t e d in muscle mitochondria [9] by use of a radioassay [10] led us to study this enzyme further. The m e t h o d as originally described [10] and used [9] was found n o t to be optimal for the measurement of monoamine oxidase in skeletal muscle mitochondria. This paper describes a m e t h o d suitable for the assay of monoamine oxidase in small amounts of muscle mitochondria, makes a comparison of the activity o f the enzyme in muscle mitochondria with the activity in liver mitochondria and provides evidence that the enzyme is localized in the outer membrane of the muscle mitochondria. Methods Male Holtzman rats (obtained from Canadian Breeding Laboratories, St. Constant, Quebec) weighed 350--450 g and were kept at 24--28°C with a 12-h lighting schedule. They were killed by decapitation. Mitochondria were isolated from mixed leg and back muscles in a medium containing 0.21 M mannitol, 0.07 M sucrose, 0.01 M EDTA and 0.01 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 7.4 by a m e t h o d involving proteolytic digestion in a medium containing heparin and ATP as described previously [ 1 1 ] . Liver mitochondria were isolated in a mediu m containing 0.25 M sucrose by a m e t h o d which did not involve proteolytic digestion [12]. For separation of the outer membrane fraction muscle mitochondria were suspended in 20 mM potassium phosphate buffer, pH 7.2 (56.9 mg in 5 ml), and stirred gently on ice for 20 min. Three samples of 1.5 ml each were layered on three discontinuous sucrose gradients (containing 1.4 ml 1.0 M sucrose and 1.0 ml 0.6 M sucrose, both in 20 mM potassium buffer pH 7.2) and centrifuged at 140 000 X g for 60 min in a Beckman model L2-65B centrifuge (rotor SW 56). The outer membrane bands, located at the boundary of the t w o sucrose layers, were removed with a fine-tipped pipette, pooled, diluted to 4 ml with 20 mM potassium phosphate buffer pH 7.2 and recentrifuged at 92 000 X g for 30 min. The pellet was resuspended in 0.3 ml of 0.5 M potassium phosphate buffer at pH 7.9. Protein c o n t e n t of the fractions was estimated by the Lowry m e t h o d [ 1 3 ] . C y t o c h r o m e oxidase (EC 1.9.3.1) was measured polarographically as described previously [ 1 1 ] . NADH-cytochrome c reductase was measured as described by Sottocasa et al. [14] in the presence and absence of 1.5 • 10 -6 M rotenone. Malate dehydrogenase (EC 1.1.1.37) was measured spectrophotometrically [1,15]. Samples of whole mitochondria and of outer membrane were treated with Lubrol (0.3 mg/mg protein) for assay of c y t o c h r o m e oxidase, monoamine oxidase and malate dehydrogenase or by sonication (10 X 5 s, setting 10, microprobe, Biosonik III) for assay of NADH-cytochrome c reductase. Radioactive tryptamine (side-chain [ 2J4C] tryptamine bisuccinate, purchased from New England Nuclear Corporation at a specific activity of 53 Ci/mol) was diluted with unlabeled tryptamine (hydrochloride, Calbiochem) to achieve a specific activity of 1.25--1.75 Ci/mol for all the concentrations of tryptamine

500 studied. The actual specific activity was measured for each concentration used. The assay for monoamine oxidase was performed essentially a s described by Wurtman and Axelrod [ 1 0 ] . Mitochondria and 0.5 M potassium phosphate buffer, pH 8.0 (volume 300 pl) are added to 13-ml glass-stoppered centrifuge tubes on ice. [14C] Tryptamine (25 #l of 3.25 mM tryptamine, specific activity about 1.5 Ci/mol) is added and the tubes incubated at 37°C for 20 min. The reaction is stopped by the addition of 0.2 ml 2 M hydrochloric acid. Blanks contain the same c o m p o n e n t s except that the tubes are boiled before the addition of the tryptamine. Toluene is then added (6 ml) and after shaking the tubes are centrifuged (900 X g, 5 min). 4 ml of the toluene layer is transferred to a glass vial containing 10 ml of toluene with dissolved PPO, 6 g/1. Counting is done at room temperature in a Beckman LS-250 counter at an efficiency of 95%. In the experiments described, substrate concentration was varied over the range 0.0015 mM to 0.96 mM. The amount of mitochondrial protein usually used was 50 pg for the whole mitochondria and 20 pg for the outer membrane fraction. The reaction rate was proportional to the amount of protein added and was linear for at least 20 min. Results The m o n o a m i n e oxidase of isolated skeletal muscle mitochondria was found to have a pH o p t i m u m of 8 (Fig. 1). The Km for tryptamine was 1.34 • 10 -s M and V was 1.0 nmol/min per mg protein (Fig. 2). This rate is almost double

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502 TABLE I SPECIFIC ACTIVITIES OF MARKER SKELETAL MUSCLE MITOCHONDRIA Experiment

ENZYMES

IN

OUTER

MEMBRANE

FRACTIONS

OF RAT

Monoamine oxi-

NADH-cytochrome c reductase (nmol/min per mg protein)

Cytochrome oxidase

Malate dehydro-

dase * * * (nmol/min per mg protein)

Rotenoneinsensitive

(ttmol/min per mg protein)

genase (nmol]min p e r rng

Rotenonesensitive

protein) Whole mitochondria

1 2

0.44 0.38 (9.9%) *

3.42

3 Outer memb r a h e fraction

1 2 3

1.82 1 . 5 2 (7&7%) * X 4 . 1 4 **

0.54

13.04 10.01 10.09

45.2

2.01 0.47 5.24

2.9

X 4 . 0 **

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c r e d u c t a s e w h i c h is

m o n o a m i n e oxidase is present in mitochondria because the specific activity of the enzyme in the mitochondrial preparation was approximately t w e n t y times greater than that in the homogenate and the proportion of total homogenate monoamine oxidase recovered in the mitochondrial fraction was the same as the proportion of homogenate c y t o c h r o m e oxidase recovered in the same fraction. The specific activity of m o n o a m i n e oxidase in an outer membrane fraction of muscle mitochondria was four times greater than that of the enzyme in whole mitochondria (Table I). Another outer membrane enzyme, rotenoneinsensitive NADH-cytochrome c reductase, was also enriched 4-fold in this same fraction (Table I). In contrast the specific activities of inner membrane enzymes (cytochrome oxidsae and rotenone-sensitive NADH-cytochrome c reductase) were reduced by 85--95% in the same fraction and the specific activity of a matrix enzyme, malate dehydrogenase was also low (Table I). Discussion Monoamine oxidase is shown to be present in skeletal muscle mitochondria, contrary to previous reports that this enzyme is lacking in skeletal muscle [6,7]. The enzyme appears to be in the outer membrane, as it is in liver [1,2] and heart [3--5], since its activity is increased in a fraction which contains a similarly increased specific activity of rotenone-insensitive NADH-cytochrome c reductase but a reduced specific activity of inner membrane and matrix enzymes. The activity of the enzyme in muscle mitochondria is about one quarter of t h a t in liver mitochondria. This could be because of the presence of a smaller proportion of outer membrane in muscle mitochondria compared with liver mitochondria, because of a lower concentration of skeletal muscle mitochondrial outer membrane or because of the use of a proteolytic enzyme in the

503 preparation of the muscle mitochondria. The purification of skeletal muscle mitochondrial outer membrane is technically difficult, as it is with other mitochondria with a high concentration of cristae, such as those of heart [4,5] and brown adipose tissue [ 1 6 ] . We have e m p l o y e d m e t h o d s used successfully for fractionation of liver mitochondria, such as osmotic swelling [ 1 7 ] , osmotic swelling plus sonication [14] and digitonin [18] without success. The m e t h o d described is a modification of the osmotic swelling technique and we have not y e t succeeded in preparing outer membranes of sufficient purity to be able to compare the specific activities of the liver and muscle mitochondrial amine oxidases directly. The procedure r e c o m m e n d e d for the assay of monoamine oxidase employs a final substrate concentration of 0.25 mM and a pH of 8.0. This pH o p t i m u m is lower than that reported b y others [19] for the liver enzyme (9.2--9.4). The Km of the muscle and liver enzymes at pH 8 are lower than those reported by others for the liver enzyme using benzylamine as a substrate at pH 7.4 (reported as 0.175 mM [20] or 0.182 mM [ 1 9 ] ) . This may in part be because the enzyme uses the n o n p r o t o n a t e d form of the substrate [ 2 1 ] , and with a pK of 10.2 [ 22] the amount of tryptamine required to produce a given concentration of this form would be decreased approximately 4-fold when the pH is increased from 7.4 to 8.0, and in part because of the use of a different substrate. Numerous methods for the assay of monoamine oxidase are available [23] b u t none appears to take the observed pH dependence into account. The low Km for oxygen (0.125 mM [20] ) would suggest that assay in air (providing 0.227 mM oxygen in solution) also does not provide optimal conditions. For simplicity, however, we have chosen to use the assay in air. The assay provides a sensitive m e t h o d which can be used for small amounts of mitochondrial or outer membrane protein. Monoamine oxidase would appear to be a suitable marker for the outer membrane of muscle mitochondria. Palmitoyl CoA synthetase (EC 6.2.1.3) is not a useful marker [4,5] in our experiments because its activity is destroyed b y the proteolytic digestion essential for obtaining a good yield of mitochondria from skeletal muscle. Rotenone-insensitive N A D H - c y t o c h r o m e c oxidase is also not a useful marker because it is not easy to measure in the presence of the large excess of the rotenone-sensitive enzyme of the inner membrane (about 90% of the total) and thus is not suitable for assessing the purity of mitoplasts. A recent report that muscle mitochondrial NADH-cytochrome c reductase is largely rotenone-insensitive [25] is contrary to our findings. A possible explanation for this discrepancy is that the rotenone-sensitive enzyme was not activated in the reported experiments. In our experience, considerable sonication is necessary for full expression of the activity of the inner membrane enzyme; Lubrol activation is not useful because it inhibits the activity of this enzyme [2]. References 1 2 3 4

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