Ou hepatic mitochondria1 monoamine oxidasc activity in Lipid deficiency Can. J. Biochem. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 11/10/14 For personal use only.
C. ] E ~ A N D A S W A M I ~
AND A. D'IORIO
Dcp.g~irannarclJ Uioclaenaistr)~,L'~fivrrsity(g Ortuwa, Ortawcr, Om., CarrnrrcfLl K I N Q N c
Keceia~edSsaiauary 29, 1979 Kaadaswami, C . & D'ilorio, A. (8979) On hepatic mitochondrial rnonozbrniime oxidase activity in lipid deficiency. Cmr. J. Biocheim. 57, 588-594 A marked decrease in liver rnitschondrial n~onoa~nine oxidase activity was noticed in rats fed a fat-free diet as compared with that of their controls. 811 lipid-deprived rats, the specific activity of this enzyme was very BOW towards difl'erent substrates studied. The activity of kynurenine 3-monooxygenase,which like monoamine oxidase is localizeci on she n~itochondriakouter membrane, was similarly depressed under conditions of lipid deprivation. On the other hand no major changes were observed in the activity of the inner mernbrane enzyme, kynurenine aminotransferase. Mitochondria from fat-free daet-fed rats were ciefieielat in essential fatty acids whereas no appreciable variations were found in the relative proportions of ishsspholipids in comparison with those of control mitochondria. Mitochoi~drialmonoamine oxidase activity of the deficient rats retained its sensitivities to inhibitor drugs Bike clorgyline and deprenyl. No changes were noticeable in the substrate specificity of monoamine oxidase in these rats. When we switched the fat-free diet-fed rats to a diet supplemented with a source of essential fatty acids, there was ana elevation in the activities of both monoamine oxidase and kynurenine 3-monooxygenase, their levels approaching those of the control rats. Kandaswarni, C. bL D'Iblirio, A. (1979) Qn hepatic mitochondria1 naonoamine oxidase activity in lipid deficiency. Can. JT. Biorlaerza. 57, 588-594 Chez Hes rats recevamt une ration alimentaire sans graisse, l'activite de la monoamine oxydase des mitochondries hdpatiques diminue de f a ~ o nrnarqude qua~ndon la compare avec celle des anira~auxcoritrdles. Chnez les rats privCs de graisse, l'activitd spkcifiqaie de cette enzyme est trks faibHe vis-his les ditlFdreaats substrats Ctudiks. Dans ees conditions de ddficience lipidique, I'activitC de Iss cynurenine hydroxylase, une autre enzynse logee, conline la monaoamine oxydase, sur la surface rnitschondriale externe, dianinue de la m$me fqon. En revanche, nous m'observons aucune variation importante de 19activitkcle la cynurdnine aminotransfkrase, enzyme de la menzbrane interne. Les mitochondries des rats privCs de graisse sont dCAcientes en acides gras essentiels, alors qu'aencura changement important n'est rernarqmk dans Bes proportions relatives des phosgholipides quand on les conapare B celles des mitochondries des animaux contrbles. L9activitCde la monoallpine oxydase mitocho~adrialeQes rats deficients rnai~atiemt sa seasibillte aux substances inhibitrices telles que la clorgyHime et le deprenyl. Chea ces rats, la spCcificitC de la nasnsamine oxydase % B'Cgard du substrat ale subit aucun changement notable. Qualad on remplace la ration alimentaire dClipidCe par une autre supplirnentee en acides gras essentiels, les activites de la ano~aoanlineoxydase et de la cynurknine hyclroxylase augmentent jmsqu'h des niveaux s9approchantde ceux des rats contriples. [Traduit par le jsurwal]
The enzyme monoamine oxidase (rnonoamine: oxygen oxidc~lreductase (deaminating) (EC 1A.3.4) ( M A O ) ) catalyzes the oxidative deamination of a bewildering variety oC amines to their corresponding aldehydes, thereby regulating intracellular levels of biogenic amines. This enzyme, described by Hare d I ) about 50 years ago, plays a central role in the mctabolisrn of 5-hydroxytryptamine and the catecholamines ABBREVIATIONS: MAO, rnonoamine oxidase; 5-HT, 5hydr~xytryptamine~ 'This paper is dedicated to the memory of the late Dr. Charles H. Best. This research was supported by grants from the Medical Research Council sf Camadn. "uthsr to whom reprint requests should be addressed.
aard also in the action s f amine-depleting drugs (2). Intracellular MAO: characterized by its location in the mitochoa~drion,is a dlavoprstein and is active against N-rnethylated arnines in addition to primary anaines, esnlike diarnine oxidase (2, 3 ) o r histarninase, originally described by Best (4). Besides its role in the oxidative degradation sf biogenic monoamimes, mitochsndrial M A 8 seems to possess varied and multifarious physiological functions (2, 3, 5 , 6 ) . 14 has also been implicated in several pathological states and mental disorders (5-7) and could be one of the mediating factors in certain disorders of ageing ( 8 ) . Nevertheless, there have been major gaps in saar understanding of the bi~chernicaliaspects of this enzyme and therefore its study is becoming an area of intensive and continuing invsstigatisn. Over the years several reports have accumulated in-
0008-4018/79/060588-07$01 -OCl/O
$? 1979 National Research Council of CanadznjCoraseil national de reckerches du Canada
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KANDASWAMB AND D'IORHB
dicating the possibility that a multiplicity of enzymes which oxidize different monoarnines occurs in the mitochondrion (9-1 1 ). Attention in our laboratory was focussed on the heterogeneity of M A 0 in rat liver mitochondria following the initial observations on the eliectrophoretic separation of different bands of this activity from rat liver and brain 6 12. 13). The possibility of occurrence of two different M A 0 activities in the mitochondrion, one mediating the oxidative dearnination of benzylarnine, and the other that of 5kydroxytryptamine, respectively, was suggested in subsequent findings ( 14, 15). However, during the p~irification of M A 8 from the mitochondrial fraction, differing modes of fractionation seem to influence the relative deaminating activities towards various substrates. ConsequentIy, the predominance of one of the activities is encountered, while the other activity is very Bow or even absent (1 1, 16-20). The question arises whether multiple activities associated with M A 0 could be ascribed to allotcepic changes accompanying the isolation of membrane-bound MAO. In this context it has been contended that the apparent multiplicity of M A 0 is due to a single enzyme species existing in different membrane environments (24 ) . Accordingly, the qraest for multiple rnonoamine-oxidizing activities has recently diverted to the question of membrane constituents contributing to the possible diversify of M A 8 , originally posed by Veryovkina et aF. ( 2 2 ) . Inasmuch as M A 8 is intricately bound to the mitochondrial outer membrane (23, 241, it may be expected to interact with membrane components. FOPinstance, pig liver M A 0 can bind to acidic phospholipids in solution ( 2 5 ) . Preliminary studies from our Baboratory have projected some evidence for an involvement sf a lipid component in the expression of M A 0 activity (26). This prompted us to explore further the question of lipid dependency of mitochondrial MAO. Thc mitochondrial outer membrane is rich in phospholipids (27) and it would be instructive to investigate whether the activity and substrate specificity sf M A 0 are influenced by the quality and content of phospholipids. It has been suggested that altering the fatty acid coanposition of phospholipids in cell membranes could have a noticeable effect on the activities of some membranebound enzymes (28). The fatty acid composition of cell membranes in animals can be modified to a great extent, by feeding a diet that lacks essential fatty acids ( 2 9 ) . Therefore, significant changes in these mernbranebound enzyme activities can be expected in cell membranes from fatty acid deficient rats if thcse activities were t o be influenced by modifications in the fatty acid composition of membranes. With this idea we have investigated the efTect s f Eat-free diet on the hepatic mitochondria1 M A 8 activity in rats and present these results in this communication. We have also examined the activity of kynurenine 3-monooxygenase ( L k y n ~ ~ r s n i n eNADPH:oxygen , caxidcpredalctase (3-hydroxylating) (EC 1 .I 4.1 3.9) ) , another rnitochoardri-
589
a1 outer membrane enzyme (301, under the same experimental situation.
Experimeartal Procedures Tyramine hydrochloride was purchased from Calbiochem., La Jolla, California. Ficoll was obtained from Pharmacia Canada Ltd., Montreal, Quebec. Phospholipid standards were procured from Serdary Research Labosatories, London, Ontario, OF Sigma Chemical Company, St. Louis, Missouri. Silica gel H was supplied by E. Merck AG, Darmstadt. All other chemicals were from Sigma Chemical Company or Fisher Scientific Company, Fair Lawn, New Jersey. Clorgyline (N-methyl-N-propargyl-3-(Z7-4-dich1~r~phenoxy) -propyBamine hydrochloride) was a gift from May & Baker Ltd., Dagenham, U.K, Deprenyl (phenylisopropyln~ethylpre~pionylaminehydrochloride) was kindly provided by Prof. 9. Knoll, Department of Pharmacology, Semmelweiss University of Medicine, Budapest, Hungary. Arlkrnuis and Diegs
Pregnant Sprague-Dawley rats supplied by Bis Breeding Laboratories (Ottawa, Ontario) were caged individually in suspended, wire-bottomed, galvanized cages, approximately 2 weeks after the gestational period. They were given tap water ad bibiturn and were fed the following diets: ( a ) a fat-free diet. and ( b ) Purina laboratory chow. The fat-free diet obtained in pellet form from United States BiochemicaEs Corporation (Cleveland, Ohio) was a modification of the test diet of Wooley and Sebrell ( 3 1 ) . The diet fortified with a total vitamin supplement (vitamin diet fortification mixture7 catalog Ko. 23430, I kg per 1881b ( 1 lb .= 0.45359237 kg) of diet) had the following composition: cellufil ( 16.4% ), salt mixture United States Phamacopoeia (USP) (40% ), sucrose (58.4% ) , and vitamin-free casein (21 .I % ) . The group receiving Purina laboratory chow diet was the control, Litters (males) were weaned at 25 days of age and placed on the same diet as that of their mothers. All the animals were individraally caged and received water and diet ad libitum. They were weighed and examined weekly far manifestations of fatty acid deficiency. They were kept on this diet for a period sf 8-16 weeks. At the end of a specified experimental period, a portion of the colony was fed a diet supplemented with a source of essential fatty acids. The fat-supplemented diet (supplied by the United States Bicrchemicals Corgssration) contained 5% corm oil and its composition was the same as that of the test diet except for the non-nutritive cellufil ( 1 1.45% ) . Livsr Mifscbaond~.fa arrd ibfcmhrane
Male Sprague-Dawley rats from each dietary group were starved overnight and killed by neck fracture, and their livers were excised. The liver tissue was homogenized immediately with ice-cold 0.25 -44sucrose in 5 mAd Tris-HCl buffer containing B rnM EBTA ( p H '7.4). The mitochondrial fraction was isolated by differential centrifugation as detailed by Colbeau ef n!. (27). Mitochondria1 subfra~tions were prepared by digitonin extraction as described bv Greenawalt (32). Microsornal contmination of the rnitochondrial fractions was assessed by monitoring the activity of glucose-6-phssphatase. Lipid E.xtracrisn und Analysis Extraction of membrane lipids was performed (27) as follows: the mitochondrial suspension ( 1-2 mL, 28-30 rng
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CAN. J. BIOGMEM. VOL. 57, 19199
sf protein per rnillilitre) was homogenized with 7 vol~ln~essubstrate. The reaction mixture in a total volume of 1.6 mE of methanol, and then 14 volumes sf chloroform were added contained 10 miW pstassium phosphate buffer (pH 7.21, to the suspension and it was again homogenized and filtered. 3 m.44 benzylamine, and an appropriate amount of enzyme. The tissue residue was rinsed with chloroform-methanol A unit of activity is a change in absorbance of 0.001 at ( 2 ~ 1 v/v) , which was then added to the lipid extract, the 250 nm during a 10-min incubation period. The disappearextract was then washed with 0.2 volumes of 0.956 NaCl ance of kynuramine was measured spectrophstometrically and kept overnight at 2°C. The lower phase was collected, (38). The incubation mixture consisted of 25 m M sodium dried over anhydrous Na2S04,evaporated to dryness under phosphate buffer (pH 7.4). 0.5 m.34 kynuran~ine, and envacuum, made to volume with chloroform, and stored. The zyme in a total volume of 3.0 mL. A decrease in optical mitochondrial lipid extract was subsequently analyzed for density of 0.008 at 360 nm d ~ r i n ga 10-min incubation fatty acids. Fatty acid methyl esters were prepared by re- p e r i d is taken as one unit of enzyme activity in this ease. acting aliquots of the lipid extract with methanolic HCl Kynurenine 3-rnonosxygenase was assayed spectrophoto(33) as follows: 5.8 mL of 2.5% methanolic HCI (w/v) metrically by measuring the disappearance of reduced was added to an aliquot sf the lipid extract in a centrifuge nicotinamide adenine dinucleotide phosphate (NADPH) as tube. The tubes were heated to boiling until the volume detailed by Okamoto (30). One unit of this enzyme activity was reduced to 0.5 mL. After the tubes were cooled, 1.0 ~ n b , is a change in absorbance of 0.001 at 340 nrn during a 10of water and 2.0 mL of petroleum ether were added. The min incubation period. Kynurenine aminotransferase was tubes were then shaken vigoroilsly to extract the methyl assayed at 37°C according to the method of Mason (39) esters into the petroleum ether phase. The methyl esters in a total volume of 1.8 mL containing 6 pmol of a-ketowere analyzed by gas-liquid chromatography as described glutarate, 3.5 pmol od L-kynurenine, $0 pmol of potassium by Kates (33). Portions from the lipid extracts of the phosphate buffer (pH 6.31, and 8.12 rcrnol of pyridoxal mitochondrial fraction were also analyzed for phospholipid phosphate. Glucose-6-phosphatase was assayed as described somposition according to the method of Skipski and Barclay by Wilson et a / . (40). Protein estimations were performed (34). Samples were applied to thin-layer plates coated with by the method of Lowry et aI. (41 ) with crystalline bovine a 8.5-mm layer of silica gel H made in a slurry of 1 mA2 serum albumin as the standard. Na2C03solution. The chromatoplate was washed first with wetone-hexane ( 1 :3, v/v) and dried at room temperature. The phospholipids were separated by developing the plate in a solvent system consisting of chloroform - methanol Differences in the body weight between the fat-free acetic acid - water (25: 15:4: 1, v/v). The separated phos- diet-fed (designated as deficient, lipid-deprived, or expholipids were visualized by expostare to iodine vapor and perimental) and the control diet-fed rats were noticed the bands were scraped off and eluted twice with the developing solvent (see above). Two additional extractions after 6 weeks sf age. The experimental rats began to were made with methanol and methanol - acetic acid - show typical signs of essential fatty acid deficiency after water (94: 1 :5, v/v) , respectively. The extracts were com- 8 weeks, namely roughiaess of hair, hair loss, and scaIibined and then concentrated in a stream of nitrogen. The ness of the tail. N o appreciable variation was found in the compssilipid phosphorus content of the eluates was estimated as described by Ames (35), following the method s f Chen tion of liver mitochondsial phospholipids between the et al. (36). control and fat-free diet-fed rats, Lecithin and phosphatidylethanolamine constituted the bulk sf the Assays and Other Estirnati~rts M A 0 was estimated polarographically with a YSI oxygen phospholipids in these two groups of rats. These two monitor (Yellow Springs Tnstmments Co., Tnc., Yellow phaspholigids were followed by cardislipin and phosSprings, Ohio), as described in an earlier communication phatidylinositd in the order of decreasing concentra(16). The incubation mixture contained 280 pmsl of sodium tion. The phospholipid/psotein ratios of mitochondria phosphate buffer (pH 7.41, 201*mo1 of neutralized semi- in these two groups of rats were also similar. As could carbazide, substrate ( 5 hydroxytryptmine (5-HT) . 3 mLW; be expected, the fat-free diet caused an altered fatty tyrarnine, 5 m M ; ben~yl~amine. 3 rnlV), and enzyme in a acid composition of liver mitochondria as iliustrated total volume of 1.2 mL. One unit of enzyme activity is the amount of protein required for the consumption of one in Table 1. Most noticeable among the changes in the nanoatom of oxygen per hour under the standard experi- mitochondrial fatty acids were the law percentages of n-6 series s f fatty acids in the deficient group. For exmental conditions. M A 0 activity was also determined by the spectrophots- ample the proportion s f linoleate (18:2) in the fatty metric method of Tabor et al. (37) with benzylarnine as a acid deficient mitochondria represented less than 28%
TABLE1. effect of fat-free diet on total fatty acid composition of rat liver mitochondria Total mitochondrial fatty acids,Q94
aThe values reported are averages for three rats. 01,
mitochondriafrom control rats; 11, mitochondriafrom fat-free diet-fed rats.
59 1
KANDASWAMI AND D'IORJO
TABLE 2. M A 0 activity in liver mitochondria from
control and lipid-deprived ratsQ
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Specific activityb, unitslrng of protein - -
-
mitochondria from control and lipid-deprived ratsa Sample
Specific activityb, units/rng s f protein
-
Substrate
Control mitochondria
Tyranline 5-Hydroxytryptamine Kynuramine Benzylamine
956 i6 681 5.4 553 & 8 723 184
+
TABLE 3. Kynurenine 3-monooxygemase activity in liver
Control mitochondria Exptl. Experimental mitochondriaC mitochondriaC 448 k 5 208 5. 2 240 f7 31135 8
a M A 8 activity was determined by the polarographic method, with tyramine or 5-hydroxytryptarnine as the substrate. The disappearance of benzylr~mineand kynuramine were followed spectrophstornetricallyas described in the Experimental section. bThe reported values are means standard deviation for four to eight rats. T h e experimental rats were on the fat-free diet for a period of 8 weeks.
+
of the value in the control mitochondria, after a period of 8 weeks of lipid deprivation (Table 1). At this point the percentage of arachidonate (2034) in the experimental mitochondria was about 30% of that of the control mitochondria. While there was a reduction En B 8 :2 and 20: 4, there were substantial increases in 1 6 :1, 18: 1, and 20:3 in the deficient mitochondria. In particular 20:3 (n-9) was quite high whereas it was found only in negligible proportions in the control mitochondria. Experiments with fat-free diet-fed rats revealed that mitochondrial M A 8 activity was markedly affected by lipid deprivation. Changes in mitochondrial M A 0 activity were apparent even within a period of 4-6 weeks of feeding the diet. After a period of 6 weeks of lipid deprivation, a 50% decrease in mitochondrial M A 0 activity (as assayed with benzylamine) was detectable in experimental rats in comparison with that of controls. The extent of microsornal contamination in the mitochondrial fraction was similar in these two groups of rats. In Table 2, the M A 0 activities of mitochondria isolated from these two groups of rats is depicted. After a period of 8 weeks on the fat-free diet, the mitochondrial M A 0 activity towards 5hydroxytryptamine in the lipid-deprived rats represented only 30% of that in the controls. Benzylamineoxidizing activity was diminished by 43% in the mitochondria of fat-deficient rats. Lipid-deprived rat liver mitochondria exhibited 46% of the specific tyramine-deaminating activity of the control mitochondria. Kynuramine-deaminating activity in the lipid-deprived rats was reduced by 43%. The effect of Zipid deprivation on the activity of kynurenine 3-monooxygenase was also studied. This activity was depressed in fat deficiency as illustrated in Table 3. The specific activity of mitochondrial kynurenine 3-msnooxygenase in the lipid-deprived rats was only about 40% of that of the control rats, after a period of 8 weeks of lipid deprivation (Table 3 ) . The levels of both M A 8 (benzylamine oxidation) and
471-4 89k2
QTheincubation mixturc contained 0.6 mL of 0.5 12-f Tris acetate buRer, pH 8.1 ; 0.1 mE of 0.3 M potassium chloride; 0.1 mL of 0.0042 ,VNADPH; 0.01 m L of 0.03 ML-kynurenine;and enzyme preparation in a final volume of 3.0 mL. The reaction was followed spectrophotometri~I1yby measuring the kynurenine-dependentdecrease in optical density at 340 nm at 23% &Thereported values are means +_ standard deviation for four to eight rats. eThe experimental rats were on the fat-free diet for a period of 8 weeks.
TABLE 4. M A 0 activity in liver mitochondria from control and fat-supplemented diet-fed ratsa
Origin sf rnitochondrial sample
Specific activityb, unitslmg of protein
Control Deficient Fat-supplemented aMAO activity was estimated with brnzylamine as a substrate by the spestrophotsmetric method of assay as described under the Experimental section. bThe reported values are means f standard deviation for four to eight rats.
kynurenine 3-monooxygenase decreased to the same extent in the outer membrane preparations of liver mitochondria from the lipid-deprived rats. Since both the mitochondrial outer membrane enzyme activities studied are depressed in fatty acid deficiency, the question arises whether various mitochondrial enzymes are affected in a similar manner under conditions of lipid deprivation. T o clarify this point the activity of kynurenine aminotransferase, am enzyme localized in the mitcochondrial inner membrane (421, was examined in the two groups of rats. This activity was found to be reduced only by 15 to 20% in the deficient mitochondria. Lipid deprivation did not cause any appreciable change in the substrate specificity of mitochondrial M A 0 activity. Also there was no change in Km towards different monoamines. After prolonged periods of lipid deprivation (16 weeks), mitschondrial M A 0 activity decreased to about 20% of the control values. Supplementation of the fat-free test diet with a source of essential fatty acids resulted in augmentation sf mitochondrial M A 0 activity in the experimental rats, showing that the effect of lipid deprivation on mitochondria1 M A 6 activity was due to the deficiency of essential fatty acids in the mitochondria. When the fat-supplemented diet was fed to the experimental rats for 10 days, the mitochondrial M A 0 activity in these rats was elevated almost to the level of the control rats (Table 4). Mitochondria1 kynurenine 3-monooxygenase activity level also revealed a similar pattern in lipid-deprived rats switched to the fat-supplemented
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CAN. H. BIQCMEM. VOL. 57, 1979
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TABLE 5. Kynrarenane 3-monooxygenase activity in Biver mitochondria from control and fat-supplemented dietfed ratsa
Origin of rnitoehondrial sample
Specific activityb, unitsj'mg of protein
Control Deficient Fat-supplemented GKynurenine 3-rnsnooxygennse activity was determined spectrophotometrically a s described in Table 3. bTke reported values are means zk standard deviation for four to eight rats.
FIG.2. Inhibition sf M A 0 activity by clorgyline (substrate, tryamine). The inhibitor was incubated with the mitochondria8 suspension for 5 min before adding the substrate. M A 8 activity was estimated by the polarographic method, The reported values are averages 0% four experiments. ( X ) , control mitochondria; ( @ ) , experimental mitochondria.
inhibition by deprenyl (Fig. 1 ) . Nevertheless, there was a slight shift in the concentration of the drug needed for 50% inhibition of benzylamine oxidation in the lipid-deprived mitochondria. A similar eRect Concentration sf deprewyt , M was observed with 5-hydro~yt~yptamine as a substrate FIG. 1. Inhibition of M A 0 activity by deprenyl (sub- in presence of clorgyline (not shown). Tyrarnine strate benzylamine). The inhibitor was incubated with the elicited a biphasic response to clorgyline inhibition in mitochondrial suspension for 48 min prior lo the addition both control and lipid-deprived mitochondria (Fig. 2 ) . of the substrate. The reported values are averages of four Such an inhibition pattern sf MAO is characteristic of experiments. M A 0 activity was assayed by the spectrophoto- tyramine as a substrate in rat liver mitochondria and metric method. ( @ ), control mitochondria; ( 0 1 , experi- is attributed to it being attacked by both types of MAO. mental mitochondria. Tt would appear that there is no variation in the substrate-selective inhibition of mitochondria1 M A 0 under conditions of essential fatty acid deficiency. diet. After a period s f 10 days on this diet, the specific activity of this enzyme in these rats was closer to control values (Table 5 ) . Discussion It was of interest to examine whether there was The avid association between lipids and proteins any change in the susceptibility of mitochondria1 M A 0 to selective inhibitors under conditions of lipid depriva- seems to be essential far the efficient functioning of tion. Deprenyl preferentially inhibits the oxidation of many membrane-bound enzymes (45). Even though benzylamine in rat liver mitochondria (43) while lipids have been implicated in the activity m d clorgyline is considered to be a specific inhibitor s f 5- specificity of mitochondrial M A 0 (1 1 , I $ ) , little is hydroxytryptamine oxidation (44). Based on differ- understood regarding the contribution of membrane ences in the sensitivities to the inhibitor drug lipids in modulating mitochondria1 M A 0 activity. clorgyline, and can the basis of varying substrate Tipton ( 1 1 ) reported that prolonged dialysis of a parspecificities (21, 441, two types of M A 0 activities have tially purified rat liver M A 0 preparation against Triton been discerned in the mitochondrion M A 0 A, possibly X-100 as well as treatment of the enzyme with phosmediating the oxidative deantination of 5-hydroxytryp- pholipase A resulted in loss of activity. Therefore, it is tarnine and M A 0 B, that of benzylarnirne. Tt has to be apparent that bound lipid may be required not only to noted that suck a classification is deemed to be rather stabilize the enzyme but also for its activity. In the arbitrary and the relative proportions of these activities course af our studies on the fractionation of rat liver may vary from tissue to tissue (21). Even though the mitochondria1 MAO. we observed that a water-soluble benzylamine-oxidizing activity was suppressed in lipid- enzyme fraction seemed to depend upon detergent and deprived rats, this activity was highly susceptible to presumably a lipid component for its activity ( 2 6 ) .
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KANDASWAMI AND D'IOREO
The idea that lipids influence the expression of mitochondria! MAO activity is substantiated in the present study. The observed effect of lipid deprivation upon mitochondrial M A 0 activity is consistent with the finding of Century and Horwitt (28), who reported that liver M A 0 was significantly influenced by dietary lipid. Their results showed that liver M A 0 activity was highest in rats fed 7% levels of Linseed Oil or Corn Oil in comparison with those fed beef fat or low levels of Corn Oil. Differences due to the dietary lipid were reflected in the Patty acids from the liver phospholipid fraction but no significant changes were found in liver lipid content. In this study there was no noticeable change in the brain M A 0 activity between the different groups. However, it has recently been observed that brain hfAO activity as assayed by kynuramine oxidation is depressed during lipid deprivation (46). It has been suggested that fatty acid changes in the tissue structures can affect mabcellular function ( 2 8 ) . To alter the fatty acid composition of the lipoproteins on enzyme-supporting structures is likely to affect some of the membrane-bound enzymes, such as MAO. Both M A 8 and kynurenine 3-monooxygenase are firmly bound to the mitochondrial outer membrane and are regarded as integral membrane proteins (23, 42, 4%). Such enzymes are considered to function fully embedded in their native lipoprotein structure (45, 48) and are held by hydrophobic interactions. It has keen shown that the hydrophobic bonding between proteins and lipids may be influenced by the fatty acid composition of the phospholipids involved (49). It is possible that clifferences in activities reported here could reflect changes in enzyme properties resulting from altered fatty acid compositions of the lipoproteins. In contrast with M A 8 and kynurenine 3-monooxygenase, kynurenine aminstransferase activity appears to be weakly bound to the mitochondrial inner membrane (42). Only a modest change in its activity is encountered in the fatty acid deficient mitochondria. It would appear that fatty acid deficiency affects rnitochondrial enzymes diEerently. A recent study by Aithal et a6. (501, on lipid-protein interactions in the mitochondria1 outer membrane, focusses its attention om two representative enzymes, namely kynurenine 3-monooxygenase and MAO. These authors report that acetone extraction of rat Iiver mitochondria impairs the activity of kynurenine 3-monooxygenase, while that sf M A 0 towards benzylamine is augmented. They tend to think that MAO, in contrast to kynurenine 3-monooxygenase, is not directly dependent on membrane lipids for the manifestation of its fhmll activity. However, under the conditions used for lipid depletion in this report. it is difficult to compare the lipid requirements of the two outer membrane activities. Treatment of the mitochondrial particles with aqueous acetone is designed to remove essentially
593
neutral phospholipids, while a major portion of the anionic phospholipids would remain unextracted (51 ). Besides, the extent of lipid removal from the mitochondria by this manipulation has not been determined in the above report. In the context of lipid dependency of mitochondrial MAO, one has to take into account recent attempts to differentiate the two proposed types of mitochondrial M A 0 activities by investigating the role of membrane Bipids in the oxidation of different substrates by M A 0 ($2, 53). It has been shown (52) that extraction of mitochondria with methylethylketone, which results in the removal of neutral phospholipids, leads to a substantial loss of 5-hydroxytryptamine-oxidizing activity ( M A 0 A ) , thereby implying that M A 0 A may be dependent ow membrane components for its activity unlike M A 0 B. However, it is illustrated in the present study that the fatty acid deficiency of the mitochondrial membrane influences the oxidation of both 5hydrsxytryptamine and benzylamine, characteristic of A and B types of M A 0 activities, respectiveIy. Therefore it does not seem possible to distinguish between these activities on the basis of such lipid dependency. This point is reinforced by the observation that there are no differences in the inhibitor susceptibilities sf M A 0 towards different substrates in the lipid-deficient mitochondria. On the other hand methylethylketone extraction of mitochondrial particles, as alluded to above, has been reported to change the sensitivity of tyramine oxidation to cIorgyline inhibition. The present observati~nthat fatty acids could influence the activity of membrane-bound M A 0 raises the question of lipid requirement for this activity is2 vkbro. There is some indication in the literattare to show that lipid depletion diminishes M A 0 activity. For example, Houslay and Tipton (18) have reported that prolonged incubation of a rat Iiver M A 0 preparation with sodium perchlorate abolishes all M A 0 activity. Sodium perchlorate being a chaotropic agent causes a weakening of the bonds between proteins and lipids (54). As already stated this bonding may be influenced by the fatty acid composition of the phospholipids involved. It has been shown in sttidies in our laboratory ( G . Kandaswami and A. D910rio, unpublished observations) that extraction of mitochondria with aqueous acetone and ammonia results in a marked reduction of M A 0 activities towards various amines like benaylamine, 5-hydroxytryptamine, tyramine, and kynurenine. This procedure removes most of the zwitterionis and anionic phospholipids from the mitochondria. However, only a limited extent of restoration of MAO activity is observed when the lipid-depleted mitochondria are incubated with mitochondrial lipid extracts. In this context it has to be reckoned that the nature of the apolar portion of the phospholipid used may be an important factor in influencing reactivation. It is known that some enzymes require a particular fatty acid pattern for their activity, in addition to a specific polar group 645). Further investigations to elucidate lipid-protein inter-
594
CAN. 9. BIBCHEM. VOL. 57, 1949
26. Kandaswami, @. & D'Iorio, A. (1978) Arclr. Biocltc~m. B i u p h s . 190,847-849 27. Colkau, A., Nachbaur, I. & Vignais, P. M. (19'9 1) Bioclli~?~. Ba'ophys. A c r ~249, 462-492 28. Century, B. & Hoiwitt, M. T. (1968) I. Nurl-. 95, 509Acknowledgments 5 16 We gratefully acknowledge the expert teshnical 29. Stancliff, R. C., Williams, M. A., Itsumi, K. %G Packer, assistance of Mrs. B. Betz. Our thanks are due t o the E. ( 1969) Arclr. Bioclrc~m.Bioplrys. 136, 629-648 in Enzya~zolo,qy(Tabor, members of the Animal Care Service s f the University 30. Okarnoto, H. (1970) in Mc~tilod.~ H . & Tabor, C. W., eds), vol. 17, part A. pp. 460-463, of Ottawa for their help. We are grateful t o Mr. P. Academic Press, New York Pilon for his help icn lipid analysis. We appreciate the 31. Wooley, J. G. & Sebrell, W. H. (1945) I. NUPI..29, secretarial assistance of Mrs. Hil&ne Amyot. 191-199 32. Greenawalt, J. W. (1974) in ~Wetlaocisira Ettzynaoiogy (Fleischer, S. & Packer, L., eds), vol. 3 1. part A, pp. 1. Hare, M. L. C. (1928) Bioclaem. J . 22, 968-979 3 10-323, Academic Press, New York 2. Kapellar-Adler, R. ( 1970) Amirle Oxiduses arad Meshsds for their Study, pp. 3 1-7 1, Wiley-Interscience, 33. Kates, M. ( 1972) Techniques o f Lipidology (Luhoratory Techniques in Biochemistry and Mslecarlar New York Biology Series) (Work, 2'. S. & Work, E., eels), Ameri3. Tipton, K. F. (1973) Br. fife$. Bull. 29, 116-119 can Elsevier Publishing Cs. Tnc.. New York 4. Best, C . H. (1929) J . Physiol. 67, 256-263 in 5. Gorkin, V. Z. ( 1973) A d v . Phariazacol. Chemot1mc.r. B I , 34. Skipski, V. P. & Barclay, M. (1969) in ~Wc~thods Enzyrno/ogy (Lowenstein, 9. M., ed), vol. 14, pp. 5301-50 597, Academic Press, New York 6. Jain, M. (1977) Life Sci. 20, 1925-1934 7. Sourkes, T. L. (1972) in Basic Nuuroc1aernistr.y 35. Arnes, B. N. (1966) in Methods in Enzynaology (NeufeBd, E . & Ginsburg, V., eds) , vol. 8, pp. 185-1 18, (Albers, R. W., Siegel, G. S., Katzman, R. & Agranoff, Academic Press, New York B. W., eds), pp. 565-606, Little, Brown, and Company, 3 6 Chen, P. S., Jr., Tsribara, T. Y. & Warner, M. (1956) Boston Anal. Clmeno. 28, B 756-1758 8. Robinson, D. S. (1975) Fed. Proc. 34, 103-107 37. Tabor, C . W., 'Tabor, H. & Rosenthal, S. M. (1954) 9. Dim Borges, J. M. & D'Horio, A. (1972) A c ~ IBio~l161?1. ). J s Biol. Cherza. 208, 645-66 I Psychopharmacol. 5,79-$9 10. Sandler, M. & Youdim, M. B. H. ( 1972) Plaarnz. Rev. 38. Weisbach, H., Smith, T. E., Daly, I. W., Witkop, B. & Udenfriend, S. (1960) J. Biol. Chern. 235, 116024,33 1-348 1164 11. Tipton, K. F. (1972) Ad11. Biochem. Psyehoplmarmacol. 39. Mason, M. (1954) 1. Biol. Citens. 211, 839-844 5, 11-24 12. K h , H. C. & D'Porio, A. ( 1967) Proc. Can. Fed. Biol. 40. Wilson, M. A., Cascarano, J., Wooten, W. L. & Pickett, C. B. (1978) Anal. Bioclaem. 85, 255-264 Soc. 10,235 13. Kim, H. C. & D'Iorio, A. (1968) Can, 1. Biochern. 46, 41. Lswry, 0.H., Wosebrough, N. J., Farr, A. L. & Randall, W. 3. (1951) I. Biol. Cllem. 193, 265-275 295-297 14. Sierens, L. & D9Hsrio, A. (1970) Can. J. Bdocheriz. 48, 42. Bkamoto, H. & Hayaishi, 0. (1970) J. B i d . Chern. 245,3603-3605 659-663 15. Diaz Borges, I. M. & D'lsrio, A. (1973) Can. I. 43. Knoll, 3. & Magyar, K. (1972) A d v . Biochem. Psyclmoplaarmacol. 5, 393-408 Biochem. 51, 1089-1095 16. Kandaswmi, C., Diaz Borges, J. M. %G D'Iorio, A. 44. Johnston, J. P. (1968) Biochen~.Pharmacol. 17, 12851297 (1977) Arch. Biochena. Bioplzys. 183, 273-280 17. D'Ioris, A*, Diem Borges. J. M. & Kandaswami, C . 45. Coleman, W. (1973) Biochirn. Biophys. Acta 380, 1-30 (1973) in Prorzdier~ira Cadechslanaine Research (Usdin, E . & Snyder, S. H., eds), pp. 145-146, Pergamon 46. Bemsshn, J. & Spitz, H. 3. (1974) BioCl~em.Bispltys. Res. Commun. 57,293-298 Press, New York 18. HousIay, M. D. & Tipton, K. F. (1973) Bioclrem. I. 47. Nisimsto, Y.. Takeuchi, F. & Shibata, Y. (1975) J. Biochern. 78,573-58 1 135, 173-186 19. Dennick, W. 6. & Mayer, It. J. (1977) Biochena. I. 48. Helenius, A, & Simons, K. (1975) Bioclzim. Biophys. Acta 4I5,29-79 161, 167-174 20. Hsu, M. (1973) Ph.D. dissertation, Northwestern Uni- 49. De Pury, G. G. & Collins, F. B. (1966) Chern. Phys. Lipid$ I , 1-3 1 versity, Evanston, P1linois, U.S.A. 21. Tipton, K OF., Houslay, M. D. & Mantle. T. J. (1976) 50. Aithal, H. N., Janki, W. M., Gushulak. B. D. & TustanoE, E. W. (1976) Arcla. Biochem. Biophys. 176, in Msnormnzirze Oxidase and its Irtlribirion (Ciha Foun1-1 1 dotion Symposiusa? 39), pp. 5-3 1, Elsevier, Amsterdam 22. Veiyovkha, I. V., Gorkin, V. Z., Mityushin, V. M. & 51. Fleischer, S. & Fleischer, B. (1967) in M e t l ~ o d s isz Ertzyrnology (Estabrook, 8. W. & Pullman, M. E., eds), Elpiner, I. E. ( 1964) Bioplmysics U.S.S.W. 9, 583-506 vol. 10, pp. 406-413, Academic Press. New York 23. Greenawalt, J. W. ( 1972) Adv. Biochem. P~l)lcho52. Ebstedt, B. & Oreland, L. (1976) Bioclmrrn. Plraraacol. pharmacol. 5,207-226 25, 119-124 24. Sottocasa, G. L. (1976) in Biochenzical Analysis of i%fera~bmnes (Maddy, A. H., ed), pp. 55-78, Chapman 53. Tret'yakov, A. V., Mukhlenov, A. G. & Gurkolo, V. K. (1976) Do&&. Bischetn. 228,23 1-234 and Hall, London 25. Olivecrona, T. & Oreland, %. (1971) Bioclaernisrry 68, 54. Hanstein, W. G., Davis, K. A. & Hatefi, Y. (1971) Arch. Biochern. Biophys. 147,534-544 332-340
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actions sf mitochondria1 RfAO should take these factors into consideration.
C. ] E ~ A N D A S W A M I ~
AND A. D'IORIO
Dcp.g~irannarclJ Uioclaenaistr)~,L'~fivrrsity(g Ortuwa, Ortawcr, Om., CarrnrrcfLl K I N Q N c
Keceia~edSsaiauary 29, 1979 Kaadaswami, C . & D'ilorio, A. (8979) On hepatic mitochondrial rnonozbrniime oxidase activity in lipid deficiency. Cmr. J. Biocheim. 57, 588-594 A marked decrease in liver rnitschondrial n~onoa~nine oxidase activity was noticed in rats fed a fat-free diet as compared with that of their controls. 811 lipid-deprived rats, the specific activity of this enzyme was very BOW towards difl'erent substrates studied. The activity of kynurenine 3-monooxygenase,which like monoamine oxidase is localizeci on she n~itochondriakouter membrane, was similarly depressed under conditions of lipid deprivation. On the other hand no major changes were observed in the activity of the inner mernbrane enzyme, kynurenine aminotransferase. Mitochondria from fat-free daet-fed rats were ciefieielat in essential fatty acids whereas no appreciable variations were found in the relative proportions of ishsspholipids in comparison with those of control mitochondria. Mitochoi~drialmonoamine oxidase activity of the deficient rats retained its sensitivities to inhibitor drugs Bike clorgyline and deprenyl. No changes were noticeable in the substrate specificity of monoamine oxidase in these rats. When we switched the fat-free diet-fed rats to a diet supplemented with a source of essential fatty acids, there was ana elevation in the activities of both monoamine oxidase and kynurenine 3-monooxygenase, their levels approaching those of the control rats. Kandaswarni, C. bL D'Iblirio, A. (1979) Qn hepatic mitochondria1 naonoamine oxidase activity in lipid deficiency. Can. JT. Biorlaerza. 57, 588-594 Chez Hes rats recevamt une ration alimentaire sans graisse, l'activite de la monoamine oxydase des mitochondries hdpatiques diminue de f a ~ o nrnarqude qua~ndon la compare avec celle des anira~auxcoritrdles. Chnez les rats privCs de graisse, l'activitd spkcifiqaie de cette enzyme est trks faibHe vis-his les ditlFdreaats substrats Ctudiks. Dans ees conditions de ddficience lipidique, I'activitC de Iss cynurenine hydroxylase, une autre enzynse logee, conline la monaoamine oxydase, sur la surface rnitschondriale externe, dianinue de la m$me fqon. En revanche, nous m'observons aucune variation importante de 19activitkcle la cynurdnine aminotransfkrase, enzyme de la menzbrane interne. Les mitochondries des rats privCs de graisse sont dCAcientes en acides gras essentiels, alors qu'aencura changement important n'est rernarqmk dans Bes proportions relatives des phosgholipides quand on les conapare B celles des mitochondries des animaux contrbles. L9activitCde la monoallpine oxydase mitocho~adrialeQes rats deficients rnai~atiemt sa seasibillte aux substances inhibitrices telles que la clorgyHime et le deprenyl. Chea ces rats, la spCcificitC de la nasnsamine oxydase % B'Cgard du substrat ale subit aucun changement notable. Qualad on remplace la ration alimentaire dClipidCe par une autre supplirnentee en acides gras essentiels, les activites de la ano~aoanlineoxydase et de la cynurknine hyclroxylase augmentent jmsqu'h des niveaux s9approchantde ceux des rats contriples. [Traduit par le jsurwal]
The enzyme monoamine oxidase (rnonoamine: oxygen oxidc~lreductase (deaminating) (EC 1A.3.4) ( M A O ) ) catalyzes the oxidative deamination of a bewildering variety oC amines to their corresponding aldehydes, thereby regulating intracellular levels of biogenic amines. This enzyme, described by Hare d I ) about 50 years ago, plays a central role in the mctabolisrn of 5-hydroxytryptamine and the catecholamines ABBREVIATIONS: MAO, rnonoamine oxidase; 5-HT, 5hydr~xytryptamine~ 'This paper is dedicated to the memory of the late Dr. Charles H. Best. This research was supported by grants from the Medical Research Council sf Camadn. "uthsr to whom reprint requests should be addressed.
aard also in the action s f amine-depleting drugs (2). Intracellular MAO: characterized by its location in the mitochoa~drion,is a dlavoprstein and is active against N-rnethylated arnines in addition to primary anaines, esnlike diarnine oxidase (2, 3 ) o r histarninase, originally described by Best (4). Besides its role in the oxidative degradation sf biogenic monoamimes, mitochsndrial M A 8 seems to possess varied and multifarious physiological functions (2, 3, 5 , 6 ) . 14 has also been implicated in several pathological states and mental disorders (5-7) and could be one of the mediating factors in certain disorders of ageing ( 8 ) . Nevertheless, there have been major gaps in saar understanding of the bi~chernicaliaspects of this enzyme and therefore its study is becoming an area of intensive and continuing invsstigatisn. Over the years several reports have accumulated in-
0008-4018/79/060588-07$01 -OCl/O
$? 1979 National Research Council of CanadznjCoraseil national de reckerches du Canada
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KANDASWAMB AND D'IORHB
dicating the possibility that a multiplicity of enzymes which oxidize different monoarnines occurs in the mitochondrion (9-1 1 ). Attention in our laboratory was focussed on the heterogeneity of M A 0 in rat liver mitochondria following the initial observations on the eliectrophoretic separation of different bands of this activity from rat liver and brain 6 12. 13). The possibility of occurrence of two different M A 0 activities in the mitochondrion, one mediating the oxidative dearnination of benzylarnine, and the other that of 5kydroxytryptamine, respectively, was suggested in subsequent findings ( 14, 15). However, during the p~irification of M A 8 from the mitochondrial fraction, differing modes of fractionation seem to influence the relative deaminating activities towards various substrates. ConsequentIy, the predominance of one of the activities is encountered, while the other activity is very Bow or even absent (1 1, 16-20). The question arises whether multiple activities associated with M A 0 could be ascribed to allotcepic changes accompanying the isolation of membrane-bound MAO. In this context it has been contended that the apparent multiplicity of M A 0 is due to a single enzyme species existing in different membrane environments (24 ) . Accordingly, the qraest for multiple rnonoamine-oxidizing activities has recently diverted to the question of membrane constituents contributing to the possible diversify of M A 8 , originally posed by Veryovkina et aF. ( 2 2 ) . Inasmuch as M A 8 is intricately bound to the mitochondrial outer membrane (23, 241, it may be expected to interact with membrane components. FOPinstance, pig liver M A 0 can bind to acidic phospholipids in solution ( 2 5 ) . Preliminary studies from our Baboratory have projected some evidence for an involvement sf a lipid component in the expression of M A 0 activity (26). This prompted us to explore further the question of lipid dependency of mitochondrial MAO. Thc mitochondrial outer membrane is rich in phospholipids (27) and it would be instructive to investigate whether the activity and substrate specificity sf M A 0 are influenced by the quality and content of phospholipids. It has been suggested that altering the fatty acid coanposition of phospholipids in cell membranes could have a noticeable effect on the activities of some membranebound enzymes (28). The fatty acid composition of cell membranes in animals can be modified to a great extent, by feeding a diet that lacks essential fatty acids ( 2 9 ) . Therefore, significant changes in these mernbranebound enzyme activities can be expected in cell membranes from fatty acid deficient rats if thcse activities were t o be influenced by modifications in the fatty acid composition of membranes. With this idea we have investigated the efTect s f Eat-free diet on the hepatic mitochondria1 M A 8 activity in rats and present these results in this communication. We have also examined the activity of kynurenine 3-monooxygenase ( L k y n ~ ~ r s n i n eNADPH:oxygen , caxidcpredalctase (3-hydroxylating) (EC 1 .I 4.1 3.9) ) , another rnitochoardri-
589
a1 outer membrane enzyme (301, under the same experimental situation.
Experimeartal Procedures Tyramine hydrochloride was purchased from Calbiochem., La Jolla, California. Ficoll was obtained from Pharmacia Canada Ltd., Montreal, Quebec. Phospholipid standards were procured from Serdary Research Labosatories, London, Ontario, OF Sigma Chemical Company, St. Louis, Missouri. Silica gel H was supplied by E. Merck AG, Darmstadt. All other chemicals were from Sigma Chemical Company or Fisher Scientific Company, Fair Lawn, New Jersey. Clorgyline (N-methyl-N-propargyl-3-(Z7-4-dich1~r~phenoxy) -propyBamine hydrochloride) was a gift from May & Baker Ltd., Dagenham, U.K, Deprenyl (phenylisopropyln~ethylpre~pionylaminehydrochloride) was kindly provided by Prof. 9. Knoll, Department of Pharmacology, Semmelweiss University of Medicine, Budapest, Hungary. Arlkrnuis and Diegs
Pregnant Sprague-Dawley rats supplied by Bis Breeding Laboratories (Ottawa, Ontario) were caged individually in suspended, wire-bottomed, galvanized cages, approximately 2 weeks after the gestational period. They were given tap water ad bibiturn and were fed the following diets: ( a ) a fat-free diet. and ( b ) Purina laboratory chow. The fat-free diet obtained in pellet form from United States BiochemicaEs Corporation (Cleveland, Ohio) was a modification of the test diet of Wooley and Sebrell ( 3 1 ) . The diet fortified with a total vitamin supplement (vitamin diet fortification mixture7 catalog Ko. 23430, I kg per 1881b ( 1 lb .= 0.45359237 kg) of diet) had the following composition: cellufil ( 16.4% ), salt mixture United States Phamacopoeia (USP) (40% ), sucrose (58.4% ) , and vitamin-free casein (21 .I % ) . The group receiving Purina laboratory chow diet was the control, Litters (males) were weaned at 25 days of age and placed on the same diet as that of their mothers. All the animals were individraally caged and received water and diet ad libitum. They were weighed and examined weekly far manifestations of fatty acid deficiency. They were kept on this diet for a period sf 8-16 weeks. At the end of a specified experimental period, a portion of the colony was fed a diet supplemented with a source of essential fatty acids. The fat-supplemented diet (supplied by the United States Bicrchemicals Corgssration) contained 5% corm oil and its composition was the same as that of the test diet except for the non-nutritive cellufil ( 1 1.45% ) . Livsr Mifscbaond~.fa arrd ibfcmhrane
Male Sprague-Dawley rats from each dietary group were starved overnight and killed by neck fracture, and their livers were excised. The liver tissue was homogenized immediately with ice-cold 0.25 -44sucrose in 5 mAd Tris-HCl buffer containing B rnM EBTA ( p H '7.4). The mitochondrial fraction was isolated by differential centrifugation as detailed by Colbeau ef n!. (27). Mitochondria1 subfra~tions were prepared by digitonin extraction as described bv Greenawalt (32). Microsornal contmination of the rnitochondrial fractions was assessed by monitoring the activity of glucose-6-phssphatase. Lipid E.xtracrisn und Analysis Extraction of membrane lipids was performed (27) as follows: the mitochondrial suspension ( 1-2 mL, 28-30 rng
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590
CAN. J. BIOGMEM. VOL. 57, 19199
sf protein per rnillilitre) was homogenized with 7 vol~ln~essubstrate. The reaction mixture in a total volume of 1.6 mE of methanol, and then 14 volumes sf chloroform were added contained 10 miW pstassium phosphate buffer (pH 7.21, to the suspension and it was again homogenized and filtered. 3 m.44 benzylamine, and an appropriate amount of enzyme. The tissue residue was rinsed with chloroform-methanol A unit of activity is a change in absorbance of 0.001 at ( 2 ~ 1 v/v) , which was then added to the lipid extract, the 250 nm during a 10-min incubation period. The disappearextract was then washed with 0.2 volumes of 0.956 NaCl ance of kynuramine was measured spectrophstometrically and kept overnight at 2°C. The lower phase was collected, (38). The incubation mixture consisted of 25 m M sodium dried over anhydrous Na2S04,evaporated to dryness under phosphate buffer (pH 7.4). 0.5 m.34 kynuran~ine, and envacuum, made to volume with chloroform, and stored. The zyme in a total volume of 3.0 mL. A decrease in optical mitochondrial lipid extract was subsequently analyzed for density of 0.008 at 360 nm d ~ r i n ga 10-min incubation fatty acids. Fatty acid methyl esters were prepared by re- p e r i d is taken as one unit of enzyme activity in this ease. acting aliquots of the lipid extract with methanolic HCl Kynurenine 3-rnonosxygenase was assayed spectrophoto(33) as follows: 5.8 mL of 2.5% methanolic HCI (w/v) metrically by measuring the disappearance of reduced was added to an aliquot sf the lipid extract in a centrifuge nicotinamide adenine dinucleotide phosphate (NADPH) as tube. The tubes were heated to boiling until the volume detailed by Okamoto (30). One unit of this enzyme activity was reduced to 0.5 mL. After the tubes were cooled, 1.0 ~ n b , is a change in absorbance of 0.001 at 340 nrn during a 10of water and 2.0 mL of petroleum ether were added. The min incubation period. Kynurenine aminotransferase was tubes were then shaken vigoroilsly to extract the methyl assayed at 37°C according to the method of Mason (39) esters into the petroleum ether phase. The methyl esters in a total volume of 1.8 mL containing 6 pmol of a-ketowere analyzed by gas-liquid chromatography as described glutarate, 3.5 pmol od L-kynurenine, $0 pmol of potassium by Kates (33). Portions from the lipid extracts of the phosphate buffer (pH 6.31, and 8.12 rcrnol of pyridoxal mitochondrial fraction were also analyzed for phospholipid phosphate. Glucose-6-phosphatase was assayed as described somposition according to the method of Skipski and Barclay by Wilson et a / . (40). Protein estimations were performed (34). Samples were applied to thin-layer plates coated with by the method of Lowry et aI. (41 ) with crystalline bovine a 8.5-mm layer of silica gel H made in a slurry of 1 mA2 serum albumin as the standard. Na2C03solution. The chromatoplate was washed first with wetone-hexane ( 1 :3, v/v) and dried at room temperature. The phospholipids were separated by developing the plate in a solvent system consisting of chloroform - methanol Differences in the body weight between the fat-free acetic acid - water (25: 15:4: 1, v/v). The separated phos- diet-fed (designated as deficient, lipid-deprived, or expholipids were visualized by expostare to iodine vapor and perimental) and the control diet-fed rats were noticed the bands were scraped off and eluted twice with the developing solvent (see above). Two additional extractions after 6 weeks sf age. The experimental rats began to were made with methanol and methanol - acetic acid - show typical signs of essential fatty acid deficiency after water (94: 1 :5, v/v) , respectively. The extracts were com- 8 weeks, namely roughiaess of hair, hair loss, and scaIibined and then concentrated in a stream of nitrogen. The ness of the tail. N o appreciable variation was found in the compssilipid phosphorus content of the eluates was estimated as described by Ames (35), following the method s f Chen tion of liver mitochondsial phospholipids between the et al. (36). control and fat-free diet-fed rats, Lecithin and phosphatidylethanolamine constituted the bulk sf the Assays and Other Estirnati~rts M A 0 was estimated polarographically with a YSI oxygen phospholipids in these two groups of rats. These two monitor (Yellow Springs Tnstmments Co., Tnc., Yellow phaspholigids were followed by cardislipin and phosSprings, Ohio), as described in an earlier communication phatidylinositd in the order of decreasing concentra(16). The incubation mixture contained 280 pmsl of sodium tion. The phospholipid/psotein ratios of mitochondria phosphate buffer (pH 7.41, 201*mo1 of neutralized semi- in these two groups of rats were also similar. As could carbazide, substrate ( 5 hydroxytryptmine (5-HT) . 3 mLW; be expected, the fat-free diet caused an altered fatty tyrarnine, 5 m M ; ben~yl~amine. 3 rnlV), and enzyme in a acid composition of liver mitochondria as iliustrated total volume of 1.2 mL. One unit of enzyme activity is the amount of protein required for the consumption of one in Table 1. Most noticeable among the changes in the nanoatom of oxygen per hour under the standard experi- mitochondrial fatty acids were the law percentages of n-6 series s f fatty acids in the deficient group. For exmental conditions. M A 0 activity was also determined by the spectrophots- ample the proportion s f linoleate (18:2) in the fatty metric method of Tabor et al. (37) with benzylarnine as a acid deficient mitochondria represented less than 28%
TABLE1. effect of fat-free diet on total fatty acid composition of rat liver mitochondria Total mitochondrial fatty acids,Q94
aThe values reported are averages for three rats. 01,
mitochondriafrom control rats; 11, mitochondriafrom fat-free diet-fed rats.
59 1
KANDASWAMI AND D'IORJO
TABLE 2. M A 0 activity in liver mitochondria from
control and lipid-deprived ratsQ
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Specific activityb, unitslrng of protein - -
-
mitochondria from control and lipid-deprived ratsa Sample
Specific activityb, units/rng s f protein
-
Substrate
Control mitochondria
Tyranline 5-Hydroxytryptamine Kynuramine Benzylamine
956 i6 681 5.4 553 & 8 723 184
+
TABLE 3. Kynurenine 3-monooxygemase activity in liver
Control mitochondria Exptl. Experimental mitochondriaC mitochondriaC 448 k 5 208 5. 2 240 f7 31135 8
a M A 8 activity was determined by the polarographic method, with tyramine or 5-hydroxytryptarnine as the substrate. The disappearance of benzylr~mineand kynuramine were followed spectrophstornetricallyas described in the Experimental section. bThe reported values are means standard deviation for four to eight rats. T h e experimental rats were on the fat-free diet for a period of 8 weeks.
+
of the value in the control mitochondria, after a period of 8 weeks of lipid deprivation (Table 1). At this point the percentage of arachidonate (2034) in the experimental mitochondria was about 30% of that of the control mitochondria. While there was a reduction En B 8 :2 and 20: 4, there were substantial increases in 1 6 :1, 18: 1, and 20:3 in the deficient mitochondria. In particular 20:3 (n-9) was quite high whereas it was found only in negligible proportions in the control mitochondria. Experiments with fat-free diet-fed rats revealed that mitochondrial M A 8 activity was markedly affected by lipid deprivation. Changes in mitochondrial M A 0 activity were apparent even within a period of 4-6 weeks of feeding the diet. After a period of 6 weeks of lipid deprivation, a 50% decrease in mitochondrial M A 0 activity (as assayed with benzylamine) was detectable in experimental rats in comparison with that of controls. The extent of microsornal contamination in the mitochondrial fraction was similar in these two groups of rats. In Table 2, the M A 0 activities of mitochondria isolated from these two groups of rats is depicted. After a period of 8 weeks on the fat-free diet, the mitochondrial M A 0 activity towards 5hydroxytryptamine in the lipid-deprived rats represented only 30% of that in the controls. Benzylamineoxidizing activity was diminished by 43% in the mitochondria of fat-deficient rats. Lipid-deprived rat liver mitochondria exhibited 46% of the specific tyramine-deaminating activity of the control mitochondria. Kynuramine-deaminating activity in the lipid-deprived rats was reduced by 43%. The effect of Zipid deprivation on the activity of kynurenine 3-monooxygenase was also studied. This activity was depressed in fat deficiency as illustrated in Table 3. The specific activity of mitochondrial kynurenine 3-msnooxygenase in the lipid-deprived rats was only about 40% of that of the control rats, after a period of 8 weeks of lipid deprivation (Table 3 ) . The levels of both M A 8 (benzylamine oxidation) and
471-4 89k2
QTheincubation mixturc contained 0.6 mL of 0.5 12-f Tris acetate buRer, pH 8.1 ; 0.1 mE of 0.3 M potassium chloride; 0.1 mL of 0.0042 ,VNADPH; 0.01 m L of 0.03 ML-kynurenine;and enzyme preparation in a final volume of 3.0 mL. The reaction was followed spectrophotometri~I1yby measuring the kynurenine-dependentdecrease in optical density at 340 nm at 23% &Thereported values are means +_ standard deviation for four to eight rats. eThe experimental rats were on the fat-free diet for a period of 8 weeks.
TABLE 4. M A 0 activity in liver mitochondria from control and fat-supplemented diet-fed ratsa
Origin sf rnitochondrial sample
Specific activityb, unitslmg of protein
Control Deficient Fat-supplemented aMAO activity was estimated with brnzylamine as a substrate by the spestrophotsmetric method of assay as described under the Experimental section. bThe reported values are means f standard deviation for four to eight rats.
kynurenine 3-monooxygenase decreased to the same extent in the outer membrane preparations of liver mitochondria from the lipid-deprived rats. Since both the mitochondrial outer membrane enzyme activities studied are depressed in fatty acid deficiency, the question arises whether various mitochondrial enzymes are affected in a similar manner under conditions of lipid deprivation. T o clarify this point the activity of kynurenine aminotransferase, am enzyme localized in the mitcochondrial inner membrane (421, was examined in the two groups of rats. This activity was found to be reduced only by 15 to 20% in the deficient mitochondria. Lipid deprivation did not cause any appreciable change in the substrate specificity of mitochondrial M A 0 activity. Also there was no change in Km towards different monoamines. After prolonged periods of lipid deprivation (16 weeks), mitschondrial M A 0 activity decreased to about 20% of the control values. Supplementation of the fat-free test diet with a source of essential fatty acids resulted in augmentation sf mitochondrial M A 0 activity in the experimental rats, showing that the effect of lipid deprivation on mitochondria1 M A 6 activity was due to the deficiency of essential fatty acids in the mitochondria. When the fat-supplemented diet was fed to the experimental rats for 10 days, the mitochondrial M A 0 activity in these rats was elevated almost to the level of the control rats (Table 4). Mitochondria1 kynurenine 3-monooxygenase activity level also revealed a similar pattern in lipid-deprived rats switched to the fat-supplemented
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TABLE 5. Kynrarenane 3-monooxygenase activity in Biver mitochondria from control and fat-supplemented dietfed ratsa
Origin of rnitoehondrial sample
Specific activityb, unitsj'mg of protein
Control Deficient Fat-supplemented GKynurenine 3-rnsnooxygennse activity was determined spectrophotometrically a s described in Table 3. bTke reported values are means zk standard deviation for four to eight rats.
FIG.2. Inhibition sf M A 0 activity by clorgyline (substrate, tryamine). The inhibitor was incubated with the mitochondria8 suspension for 5 min before adding the substrate. M A 8 activity was estimated by the polarographic method, The reported values are averages 0% four experiments. ( X ) , control mitochondria; ( @ ) , experimental mitochondria.
inhibition by deprenyl (Fig. 1 ) . Nevertheless, there was a slight shift in the concentration of the drug needed for 50% inhibition of benzylamine oxidation in the lipid-deprived mitochondria. A similar eRect Concentration sf deprewyt , M was observed with 5-hydro~yt~yptamine as a substrate FIG. 1. Inhibition of M A 0 activity by deprenyl (sub- in presence of clorgyline (not shown). Tyrarnine strate benzylamine). The inhibitor was incubated with the elicited a biphasic response to clorgyline inhibition in mitochondrial suspension for 48 min prior lo the addition both control and lipid-deprived mitochondria (Fig. 2 ) . of the substrate. The reported values are averages of four Such an inhibition pattern sf MAO is characteristic of experiments. M A 0 activity was assayed by the spectrophoto- tyramine as a substrate in rat liver mitochondria and metric method. ( @ ), control mitochondria; ( 0 1 , experi- is attributed to it being attacked by both types of MAO. mental mitochondria. Tt would appear that there is no variation in the substrate-selective inhibition of mitochondria1 M A 0 under conditions of essential fatty acid deficiency. diet. After a period s f 10 days on this diet, the specific activity of this enzyme in these rats was closer to control values (Table 5 ) . Discussion It was of interest to examine whether there was The avid association between lipids and proteins any change in the susceptibility of mitochondria1 M A 0 to selective inhibitors under conditions of lipid depriva- seems to be essential far the efficient functioning of tion. Deprenyl preferentially inhibits the oxidation of many membrane-bound enzymes (45). Even though benzylamine in rat liver mitochondria (43) while lipids have been implicated in the activity m d clorgyline is considered to be a specific inhibitor s f 5- specificity of mitochondrial M A 0 (1 1 , I $ ) , little is hydroxytryptamine oxidation (44). Based on differ- understood regarding the contribution of membrane ences in the sensitivities to the inhibitor drug lipids in modulating mitochondria1 M A 0 activity. clorgyline, and can the basis of varying substrate Tipton ( 1 1 ) reported that prolonged dialysis of a parspecificities (21, 441, two types of M A 0 activities have tially purified rat liver M A 0 preparation against Triton been discerned in the mitochondrion M A 0 A, possibly X-100 as well as treatment of the enzyme with phosmediating the oxidative deantination of 5-hydroxytryp- pholipase A resulted in loss of activity. Therefore, it is tarnine and M A 0 B, that of benzylarnirne. Tt has to be apparent that bound lipid may be required not only to noted that suck a classification is deemed to be rather stabilize the enzyme but also for its activity. In the arbitrary and the relative proportions of these activities course af our studies on the fractionation of rat liver may vary from tissue to tissue (21). Even though the mitochondria1 MAO. we observed that a water-soluble benzylamine-oxidizing activity was suppressed in lipid- enzyme fraction seemed to depend upon detergent and deprived rats, this activity was highly susceptible to presumably a lipid component for its activity ( 2 6 ) .
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KANDASWAMI AND D'IOREO
The idea that lipids influence the expression of mitochondria! MAO activity is substantiated in the present study. The observed effect of lipid deprivation upon mitochondrial M A 0 activity is consistent with the finding of Century and Horwitt (28), who reported that liver M A 0 was significantly influenced by dietary lipid. Their results showed that liver M A 0 activity was highest in rats fed 7% levels of Linseed Oil or Corn Oil in comparison with those fed beef fat or low levels of Corn Oil. Differences due to the dietary lipid were reflected in the Patty acids from the liver phospholipid fraction but no significant changes were found in liver lipid content. In this study there was no noticeable change in the brain M A 0 activity between the different groups. However, it has recently been observed that brain hfAO activity as assayed by kynuramine oxidation is depressed during lipid deprivation (46). It has been suggested that fatty acid changes in the tissue structures can affect mabcellular function ( 2 8 ) . To alter the fatty acid composition of the lipoproteins on enzyme-supporting structures is likely to affect some of the membrane-bound enzymes, such as MAO. Both M A 8 and kynurenine 3-monooxygenase are firmly bound to the mitochondrial outer membrane and are regarded as integral membrane proteins (23, 42, 4%). Such enzymes are considered to function fully embedded in their native lipoprotein structure (45, 48) and are held by hydrophobic interactions. It has keen shown that the hydrophobic bonding between proteins and lipids may be influenced by the fatty acid composition of the phospholipids involved (49). It is possible that clifferences in activities reported here could reflect changes in enzyme properties resulting from altered fatty acid compositions of the lipoproteins. In contrast with M A 8 and kynurenine 3-monooxygenase, kynurenine aminstransferase activity appears to be weakly bound to the mitochondrial inner membrane (42). Only a modest change in its activity is encountered in the fatty acid deficient mitochondria. It would appear that fatty acid deficiency affects rnitochondrial enzymes diEerently. A recent study by Aithal et a6. (501, on lipid-protein interactions in the mitochondria1 outer membrane, focusses its attention om two representative enzymes, namely kynurenine 3-monooxygenase and MAO. These authors report that acetone extraction of rat Iiver mitochondria impairs the activity of kynurenine 3-monooxygenase, while that sf M A 0 towards benzylamine is augmented. They tend to think that MAO, in contrast to kynurenine 3-monooxygenase, is not directly dependent on membrane lipids for the manifestation of its fhmll activity. However, under the conditions used for lipid depletion in this report. it is difficult to compare the lipid requirements of the two outer membrane activities. Treatment of the mitochondrial particles with aqueous acetone is designed to remove essentially
593
neutral phospholipids, while a major portion of the anionic phospholipids would remain unextracted (51 ). Besides, the extent of lipid removal from the mitochondria by this manipulation has not been determined in the above report. In the context of lipid dependency of mitochondrial MAO, one has to take into account recent attempts to differentiate the two proposed types of mitochondrial M A 0 activities by investigating the role of membrane Bipids in the oxidation of different substrates by M A 0 ($2, 53). It has been shown (52) that extraction of mitochondria with methylethylketone, which results in the removal of neutral phospholipids, leads to a substantial loss of 5-hydroxytryptamine-oxidizing activity ( M A 0 A ) , thereby implying that M A 0 A may be dependent ow membrane components for its activity unlike M A 0 B. However, it is illustrated in the present study that the fatty acid deficiency of the mitochondrial membrane influences the oxidation of both 5hydrsxytryptamine and benzylamine, characteristic of A and B types of M A 0 activities, respectiveIy. Therefore it does not seem possible to distinguish between these activities on the basis of such lipid dependency. This point is reinforced by the observation that there are no differences in the inhibitor susceptibilities sf M A 0 towards different substrates in the lipid-deficient mitochondria. On the other hand methylethylketone extraction of mitochondrial particles, as alluded to above, has been reported to change the sensitivity of tyramine oxidation to cIorgyline inhibition. The present observati~nthat fatty acids could influence the activity of membrane-bound M A 0 raises the question of lipid requirement for this activity is2 vkbro. There is some indication in the literattare to show that lipid depletion diminishes M A 0 activity. For example, Houslay and Tipton (18) have reported that prolonged incubation of a rat Iiver M A 0 preparation with sodium perchlorate abolishes all M A 0 activity. Sodium perchlorate being a chaotropic agent causes a weakening of the bonds between proteins and lipids (54). As already stated this bonding may be influenced by the fatty acid composition of the phospholipids involved. It has been shown in sttidies in our laboratory ( G . Kandaswami and A. D910rio, unpublished observations) that extraction of mitochondria with aqueous acetone and ammonia results in a marked reduction of M A 0 activities towards various amines like benaylamine, 5-hydroxytryptamine, tyramine, and kynurenine. This procedure removes most of the zwitterionis and anionic phospholipids from the mitochondria. However, only a limited extent of restoration of MAO activity is observed when the lipid-depleted mitochondria are incubated with mitochondrial lipid extracts. In this context it has to be reckoned that the nature of the apolar portion of the phospholipid used may be an important factor in influencing reactivation. It is known that some enzymes require a particular fatty acid pattern for their activity, in addition to a specific polar group 645). Further investigations to elucidate lipid-protein inter-
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CAN. 9. BIBCHEM. VOL. 57, 1949
26. Kandaswami, @. & D'Iorio, A. (1978) Arclr. Biocltc~m. B i u p h s . 190,847-849 27. Colkau, A., Nachbaur, I. & Vignais, P. M. (19'9 1) Bioclli~?~. Ba'ophys. A c r ~249, 462-492 28. Century, B. & Hoiwitt, M. T. (1968) I. Nurl-. 95, 509Acknowledgments 5 16 We gratefully acknowledge the expert teshnical 29. Stancliff, R. C., Williams, M. A., Itsumi, K. %G Packer, assistance of Mrs. B. Betz. Our thanks are due t o the E. ( 1969) Arclr. Bioclrc~m.Bioplrys. 136, 629-648 in Enzya~zolo,qy(Tabor, members of the Animal Care Service s f the University 30. Okarnoto, H. (1970) in Mc~tilod.~ H . & Tabor, C. W., eds), vol. 17, part A. pp. 460-463, of Ottawa for their help. We are grateful t o Mr. P. Academic Press, New York Pilon for his help icn lipid analysis. We appreciate the 31. Wooley, J. G. & Sebrell, W. H. (1945) I. NUPI..29, secretarial assistance of Mrs. Hil&ne Amyot. 191-199 32. Greenawalt, J. W. 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actions sf mitochondria1 RfAO should take these factors into consideration.
