Eur. J. Biochem. 92, 165-174 (1978)

Effect of Phospholipid Depletion by Phospholipases on the Properties and Formation of the Multiple Monoamine Oxidase Forms in the Rat Liver Stephen P. BAKER and Brian A. HEMSWORTH Pharmacological Laboratories, Department of Pharmacy, University of Aston, Birmingham (Received August 26, 1977/June 23, 1978)

The effect of phospholipid depletion by phospholipases on the properties and formation of monoamine oxidase A and B been investigated. The enzyme was solubilized, partially purified, treated with phospholipases and subjected to gel filtration to reduce the amount of enzymeassociated phospholipids. Phospholipase A treatment of the purified monoamine oxidase fraction had no effect on the deprenil inhibition pattern or the observed transition temperatures in the Arrhcnius plots. However, the rate of enzyme inactivation by heat and trypsin were greatly increased but differences in rates of inactivation of monoamine oxidase A and B were still observed. Phospholipase C treatment of the enzyme fraction had no effect on the deprenil inhibition pattern, Arrhenius plots, heat stability or trypsin digestibility. The inhibition pattern of membrane-bound monoamine oxidase and the phospholipase-treated fractions by propargylamine showed a seduced substrate specificity compared to deprenil suggesting a hydrophobic region in the enzyme is a factor involved in the structural differences of monoamine oxidase A and B.

It is now widely accepted that the enzyme monoamine oxidase exists in more than one form in some tissues (for a review see [l]). Evidence for the existence of more than one form of the enzyme is based on sensitivity to inhibitors [2], electrophoretic mobility [ 3 ] and sensitivity to inactivation by heat [4] and proteolytic enzymes [ 5 ] . The most commonly used notation for the different monoamine oxidase forms is that proposed by Johnston [ti]using the inhibitor clorgyline. He suggested that an A form of the enzyme was highly sensitive to clorgyline and a B form which was less sensitive. More recently, Knoll and Magyar [7] showed that deprenil, a structurally similar compound to clorgyline, is a monoamine oxidase inhibitor selective for the B form of the enzyme. Part of the recent focus in the study of monoamine oxidase has been on the nature of the multiple forms of the enzyme. Houslay and Tipton [8] have found that treatment of partially purified monoamine oxidase from rat liver with the chaotropic agent sodium perchlorate reduced the amount of enzyme-bound phospholipids. This resulted in a loss of selective inhibition towards different substrates by clorgyline and a loss Abbreviations. DEAE, diethylaminoethyl ; AH, aminohexyl ; Nbsz, 5,5’-dithiobis(Z-nitrobensoate). Enryme. Monoarnine oxidase or monoaminc : 0 2 oxidoreductase (deaminating) (EC 1.4.3.4).

of differential resistance to heat inactivation of the A and B forms of the enzyme. The authors suggested that the outer mitochondria1 membrane environment, to which monoamine oxidase is strongly associated [9], was responsible for the formation of the different enzyme forms. Neff and Yang [lo] have expanded this hypothesis further by suggesting that the enzyme region with which the aromatic moiety of substrates and inhibitors interact are composed of or influenced by different phospholipids, thus forming monoamine oxidase A and B from enzyme protein with the same catalytic site. However, in an attempt to reproduce the findings of Houslay and Tipton [8], Ekstedt and Oreland [ l l ] inactivated most of the monoamine oxidase A activity while only a small portion of the B form was inactivated by organic solvent extraction. Since little change was observed in the enzyme’s sensitivity to inhibition by clorgyline and deprenil, the investigators findings did not support the hypothesis of Houslay and Tipton [8]. Therefore, it is still not clear whether the membrane environment is involved in the formation of the different forms of monoamine oxidase. The disposition of membrane components and the effects of lipids on membrane-bound enzymes has been investigated by the use of pure phospholipases [12,13]. PhospholipaseC enzymes catalyze the remov-

166

al of the phosphorylamine moieties from some phosphoglycerides and the resultant diglyceride has no detergent properties [12].Treatment of certain enzymes with phospholipase C has been shown to reduce their activity and a reactivation of the enzyme was produced by the addition of lipid [14]. These results showed the requirement of the enzyme for phospholipid for activity, however, it is not clear yet as to whether the lipid is required in the enzymatic reaction or for conformational stability. In contrast to phospholipase C, phospholipase A hydrolyzes specific ester bonds in phosphoglycerides to form monoacylphosphoglycerides [12]. These products of hydrolysis are effective detergents which disrupt membranes [15] and in some cases have been shown to inhibit enzymes unless albumin is present [16]. Phospholipase A has been used to release enzymes from membranes and to solubilize the enzymes [12,16]. In addition a number of enzymes have been reported which when depleted of the phospholipid by the action of phospholipase A lose activity and are reactivated by the addition of phospholipids [12]. As a result of these observations phospholipase A and phospholipase C are useful tools in solubilizing membranebound proteins and in studying lipid protein interactions during the investigation of lipid-dependent enzymes. In this paper we present some of our results obtained by reducing the phospholipid content of a partially purified monoamine oxidase preparation with phospholipases. Some properties of the enzyme were studied both before and after phospholipid depletion with respect to the characteristics of the A and B forms of the enzyme.

MATERIALS AND METHODS Chemicals The following chemicals were obtained from Sigma Chemical Co. Ltd (Surrey, U.K), tyramine, 5-hydroxytryptamine, benzylamine, bovine serum albumin, trypsin, phospholipase C (Clostridium welchii), phospholipase A (bee venom), d,l -a-phosphatidylcholine dipalmitoyl, d,1-a-phosphatidylethanolaminedipalmitoyl, sphingomyelin, niercaptoethanol, TritonX-100,lethyl-3-(3-dimethylaminopropyl)carbodiimide,DEAESephadex, and 5,5'-dithiobis-(2-nitrobenzoate).Aminohexyl-Sepharose was obtained from Pharmacia Fine Chemicals AB, Uppsala, Sweden. Tyramine hydrochloride labeled with 14C at C-2 of the side chain (50 pCi ; 55 Ci/mol) and 5-hydroxytryptamine creatinine sulphate labeled with 14C at C-2 of the side chain (50 pCi; 55 Ci/mol) were purchased from the Radiochemical Centre (Amersham, U.K.). [7-14C]Benzylamine hydrochloride (0.1 mCi; 5.6 Ci/mol) was ob-

Effect of Phospholipid Depletion on Monoamine Oxidase

tained from Mallinckrodt Chemical Works (St Louis, Mo., U.S.A.). Monopropargylamine hydrochloride was purchased from Aldrich Chemical Co. (Milwaukee, Wis., U.S.A.). Deprenil (phenylisopropylpropinylamine hydrochloride, E250) was a gift from Dr J. Knoll (Semmelweis University of Medicine, Budapest, Hungary). All other reagents of the highest purity were obtained from BDH Chemicals Ltd (Poole, U.K.). Isolation o j Mitochondria Mitochondria were prepared essentially by the method of Hunter et al. [17]. Male Wistar rats were decapitated by guillotine and the livers removed, weighed and placed in ice-cold 0.25 M sucrose. Portions of chopped liver were homogenized with about 9 vol. of 0.25 M sucrose in a glass homogenizer with a loose-fitting Teflon motor-driven pestle. The homogenate was made 10% with sucrose and centrifuged at 600 x g for 10 min and the supernatant was then centrifuged at 10000 x g for 10 min. The crude mitochondrial pellet was washed twice and recentrifuged with 0.25 M sucrose and the final pellet was treated as described in the following sections. Protein Determinations Protein concentrations were determined by the method of Lowry et al. [18] using bovine serum albumin as standard. Detemination of Enzyme Activity Monoamine oxidase activity was assayed by incubating 50 pl enzyme preparation (0.1 -2.5 mg protein/ml), 100 pl of 0.1 M sodium phosphate buffer, pH 8.0 containing 2 mM EDTA and 100 p1 of either tyramine, 5-hydroxytryptamine or benzylamine. Final substrate concentration was 1 mM containing 25 nCi of [14C]tyramine or 50 nCi of either 5-[14C]hydroxytryptamine or [14C]benzylamine.For inhibitor studies the 100 pl of buffer was replaced by 50 pl of 0.2 M buffer containing 4 m M EDTA and 50 pI of the inhibitor in water. Incubations were performed at 37 "C in a shaking water bath for 20 min. At the end of the incubation 0.3ml of 2 M HCl was added followed by 5 ml of toluene. The tubes were then vigorously shaken for 30 s and centrifuged at 500 x g for 2 min to separate the layers. Aliquots of the toluene layer were transferred to a vial or the tubes were kept at - 20 "C for 3 h and the toluene poured into a vial followed by 5 ml of scintillation fluid [5 g of 2,5-diphenyloxazole and 0.3 g of 1,4-di(2,5-phenyloxazole) benzene per liter of toluene]. The vials were then counted for 5-20 min in a Beckman LS-230 liquid scintillation spectrometer. The efficiency of counting

167

S. P. Baker and B. A. Hemsworth

was found to be 83% for I4C by internal standard and determinations of enzyme activity differed by less than 3%. Blank values were obtained by using a boiled enzyme (lOO°C, 5 min) or distilled water in place of the active enzyme and were subtracted from the enzyme counts. Enzyme activity was linear for at least 20 rnin and linear through 1.5 mg of protein/ ml final concentration with all substrates used.

added and the tubes shaken for 1 min followed by centrifugation at 500 x g for 3 min. The organic layer was then read at 710 nm against the blank. A calibration curve was prepared using phosphatidylcholine, phosphatidylethanolamine and sphingomyelin as standards which gave similar absorbance values and was linear through 300 pg of phospholipid. The presence of TritonX-100 or neutral lipids had no effect on the phospholipid determination.

Solubilization and Purification

The crude sucrose-washed mitochondria were washed with distilled water and suspended in 0.05 M phosphate buffer, pH 7.2. To the mitochondria1 suspension, solid ammonium sulphate was added to give a 10% (w/v) solution. The suspension was stirred on ice for 20 rnin and then centrifuged at 25000 x g for 20 min. The pellet was suspended in 0.1 M phosphate buffer, pH 8.0 to a protein concentration of 10 mg/ml and a solution of 10% Triton X-100 was added dropwise to give a final concentration of 0.18%. The suspension was stirred on ice for 30 min and centrifuged at 40 000 x g for 30 min. The supernatant was made 20% (w/v) in ammonium sulphate, stirred on ice for 20 min and centrifuged at 20000 x g for 20 min. The pellet was dissolved in 0.05 M phosphate buffer, pH 11.0and applied to a 5 x 2.2 cm column of Nbs2Sepharose prepared by the method of Lin and Foster [19] and pre-equilibrated with 0.05 M phosphate buffer, pH 8.0. The protein was eluted at 0.5 ml/min and between 80 % and 90 % of the monoamine oxidase activity freely eluted. Bound protein was eluted with 0.05 M mercaptoethanol. The eluted monoamine oxidase was then applied to a 12 x 2.2 cm column of DEAE-Sephadex pre-equilibrated with 0.01 M phosphate buffer, pH 8.0 and eluted at 0.5 ml/min. Monoamine oxidase was not bound but retained protein could be eluted with 0.1 M phosphate buffer, pH 7.2 containing 0.8 M NaC1. This procedure resulted in a 15- 20-fold purification over the original homogenate with a 30 - 40 % recovery of enzyme activity. Phospholipid Determination

Phospholipids were estimated by a slight modification of the method developed by Raheja et al. [20]. The chromogenic solution was prepared as described by Vaskovsky and Kostetsky [21] and phospholipids were extracted from the protein samples by the method of Folch et al. [22]. The chloroform-methanol extract was evaporated to dryness and 0.4 ml chloroform and 0.1 ml of chromogenic reagent was added to the tube. A blank was prepared with chloroform and chromogenic reagent only. The tubes were placed in a boiling water bath for 1 to 1.5 min and cooled to room temperature. After standing for 5 min, '1 ml of chloroform and 2 ml of 1,2-dichloroethane were

Treatment with Phospholipases

Suspensions of mitochondria and soluble preparations of monoamine oxidase (10 mg protein/ml) in 0.05 M Tris buffer, pH 7.8 were made 1 mM in CaClz and 0.5 mg of phospholipase C per 10 mg of protein or 0.05 mg of phospholipase A per 10 mg of protein were added. Bovine serum albumin (0.2 mg/ml) was also present when phospholipaseA was used. The mixtures were incubated at 37 "C for 20 rnin and with mitochondria, the suspension was centrifuged at 20000 x g for 20 min. The pellet was resuspended in 0.05 M phosphate buffer, pH 8.0 and assayed for enzyme activity. After incubation of the soluble enzyme preparation, EDTA was added to a concentration of 1 mM and enzyme assays or gel filtration experiments were performed. Gel Filtration Experiments

Gel filtration was carried out at 4 "C on a 40 x 2.5 cm column of Biogel A-150m with a flow rate of approximately 25 ml/h. Columns were equilibrated with 0.05 M phosphate buffer, pH 8.0 containing 0.05 % Triton X-100. Protein elution was detected with a Uvicord IT (LKB Instruments Ltd, Surrey, U.K.). Treatment with Trypsin

Mitochondria or soluble monoamine oxidase pseparations in 0.05 M phosphate buffer, pH 7.5 were incubated at 37°C with 100 or 200 pg trypsin/mg protein. At varying times up to 40 min, samples were removed, a 3-fold weight excess of soya bean trypsin inhibitor was added and the residual enzyme activity estimated.

RESULTS Reduction o j Monoamine-Oxidase-Associated Phospholipid by Phospholipases and Gel Filtration

The phospholipid content of the isolated mitochondria was 181 pg phospholipid/mg protein. After treatment of the mitochondria with phospholipase C, 66 %, of the phospholipid-phosphoryl groups were

168

Effect of Phospholipid Depletion on Monoamine Oxidase 0.4

E 6 0.3

-

m

N

2 0.2 m c

Table 1. Phospholipid content of mitochondria and soluble monoamine oxiduse prepurutions after treulment with phospholipuses Each value is the mean of four deterininatiotls which differed by less than 5 "/,. Percentages are either of mitochondrial control value or of soluble and partially purified monoamine oxidase control value Fraction

Phospholipid

D 0

2

0.1

4

Mitochondria Fig. 1. Ge1,fillrutionofpurtiully purified monoumine oxiduse on Biogel A-150m. A 40 x 2.5-cm column was equilibrated with 0.05 M phosphate buffer, pH 8.0 containing 0.05'x Triton X-100. Enzyme samples were applied to the column in the presence of 1 % (w/v) sucrose and 3.2 ml fractions were collected. (--) Protein elution pattern measured at 280nm and (0) enzyme activity measured with tyramine as substrate

found to be hydrolyzed without effecting monoamine oxidase activity. Longer incubation periods than that used (20 min) did not result in further hydrolysis of the phospholipids. Treatment of the mitochondria with phospholipase A resulted in a clearing of the mitochondrial suspension but 80% of the monoamine oxidase activity was centrifuged down at 20000 x g with no detectable enzyme activity in the supernatant. The phospholipid content of this pellet after centrifugation was found to be 110 pg phospholipid/mg protein and this was not reduced by longer periods of incubation with phospholipase A. Upon solubilization and partial purification of the enzyme, the associated phospholipids rose to 221 pg phospholipid/mg protein. Fig. 1 shows the elution profile to the partially purified enzyme preparations from a column of Biogel A-l50m in the presence of 0.05% Triton X-100. Two protein peaks were observed; an initial partially retarded peak which contained 83 % of the applied monoamine oxidase activity and a broad fully retarded peak which contained no detectable enzyme activity. The eluted enzyme activity peak had a phospholipid content of 162 pg phospholipid/mg protein which is a reduction of the phospholipid content from the soluble partially purified enzyme from 223 pg to 162 pg phospholipid/mg protein, i.e. 27 %. A summary of the phospholipid contents of the various enzyme fractions described above is shown in Table 1. Treatment of the soluble partially purified monoamine oxidase preparation with phospholipase C had no effect on the enzyme activity and gel filtration of this preparation resulted in a similar protein and enzyme activity elution pattern as observed with the untreated preparations (see Fig. 1). However, after phospholipaseC treatment and gel filtration, the eluted protein containing the monoamine oxidase activity

&ng protein

mito:$ soluble chondrial monoamine control oxidase control

181

100

Phospholipase-C-treated mitochondria

62

34

Phospholipase-A-treated mitochondria

110

61

Soluble and partially purified monoamine oxidase

22 1

122

100

Soluble monoamine oxidase after gel filtration

162

90

73

Soluble monoamine oxidase after treatment with phospholipase C and gel filtration

9

5

4

Soluble monoamine oxidase after treatment with phospholipase A and gel filtration (fully relarded peak)

14

X

6

was found to have lost 96% of the phospholipidphosphoryl groups. When the solubilized and partially purified preparations of monoamine oxidase were treated with phospholipase A, less than 20 % of the enzyme activity was lost with all substrates tested (benzylamine, tyramine and 5-hydroxytryptamine). The protein and enzyme activity elution profile from a column of Biogel A-l50m is shown in Fig. 2. In a similar manner to the untreated preparation, two protein peaks were observed; one a partially retarded peak and a second broad fully retarded peak. However, only 15 of the applied enzyme eluted with the first peak whereas 40 of the activity eluted in the second fully retarded peak. The phospholipid content of the partially and fully retarded peaks were 47 and 14 pg phospholipid/mg protein respectively. This represents a reduction of the phospholipid content of the enzyme fractions to 21 7; and 6 respectively compared to the soluble partially purified enzyme. Further attempts to reduce the phospholipid content of the second peak by treatment with organic solvents, chaotropic agents or phospholipase were unsuccessful. Therefore, this fraction was used in further studies and is referred to as phospholipid depleted monoamine oxidase.

S. P. Baker and B. A . Hemsworth

169 100

80

.2" 60

.-.--c 0

-0

r c c

40

20 E f f l u e n t volume ( m l )

Fig. 2. Ge1,filtration ofpartially purified monoamine oxiduse on Biogel A-150m after treatment bvithphospholipase A . A 40 x 2.5-cm column was equilibrated with 0.05 M phosphate butfer, pH 8.0 containing 0.OS'x Triton X-100. Enzyme samples were applied to the column in the presence of 1 "/, (w/v) sucrose and 3 . 2 ml fractions werc collected. (--) Protein elution pattern measured at 280 nm and (0)enzyme activity measured with tyramine as substrate

0

Properties of Soluble and Phospholipase- Treated Monoamine Oxidase The inhibition of soluble partially purified monoaniine oxidase by deprenil is shown in Fig. 3 A. Using the nomenclature of Johnston [6], deprenil is more selective for inhibiting monoamine oxidase B (benzylamine oxidation) than monoamine oxidase A (5-hydroxytryptamine oxidation) with the inhibition of tyramine oxidation showing a double siginoid curve indicating it is oxidized by both enzymes. There was no difference in the inhibition pattern observed with the soluble preparation compared to when the enzyme was bound to the mitochondria1 membrane. Recent studies have shown that the enzyme inhibition produced by deprenil and clorgyline are time-dependent varying with the substrate used [23,24]. In the present report, no further time-dependent enzyme inhibition was observed after 10 min with the 3 substrates used. The inhibition of the phospholipid-depleted enzyme preparation by deprenil is shown in Fig. 3 B. The same selectivity and inhibition pattern was observed as with the untreated preparation. Furthermore, a similar inhibition pattern was seen after treatment of the solubilized enzyme with phospholipase C and gel filtration (figure not shown). The effect of treating the soluble enzyme preparation at 50°C is shown in Fig.4A. Monoamine oxidase A lost 80% of its activity in 10 min whereas monoamine oxidase B was completely inactivated within 5 min. Enzyme activity towards tyramine was lost at a rate intermediate of the A and B form consistent with it being a substrate for both enzymes. A similar time course loss of activity was observed with the phospholipase-C-treated enzyme (figure not shown). Fig. 4B shows the effect of heat on the phospholipid-depleted enzyme preparation. Similar to the soluble untreated enzyme, phospholipid-depleted

l o g [ D e p r e n i l ] (-log M)

Fig. 3. inhibition of rnonoamine oxidasi? preparations by drprenil. Portions of (A) partially purified enzyme (50 1.11, 2.5 mg protein/ml) o r (B) partially purified enzyme after treatment with phospholipase A and gel filtration (50 pl, 2.0 mg protein/ml) were incubated at 37 "C for I0 min with 50 pl of deprenil in water to give the final concentration indicated before addition of substrate. After this incubation, no further time-dependent loss of activity was observed with any substrate. The substrates used were tyramine (e), 5-hydroxytryptamine (A)and benzylamine (H). Each point on the graphs is the mean of 3 enzyme determinations which differed by less than 3 %

monoamine oxidase still showed that the A form was more heat resistant than the B form. However, the overall rate of inactivation was greater as the A form was completely inactivated within 10 min and the B form within 1 min. The inactivation of solubilized monoamine oxidase by trypsin is shown in Fig. 5A. Over a 40-min period monoamine oxidase A lost 60 of its activity whereas monoamine oxidase B activity was decreased by only 20 %. Tyramine oxidation was decreased by 39 %. The phospholipase-C-treated preparation showed the same time course loss of activity with all 3 substrates (figure not shown). In comparison, Fig. 5B shows the effect of trypsin on the phospholipid-depleted enzyme. Still observable was a B form that was more resistant to inactivation by trypsin than the A enzyme with tyramine oxidation being inactivated at an intermediate rate. Using only half the trypsin concentration (100 pg trypsin/mg protein) to that which was

170

Effect of Phospholipid Depletion on Monoamine Oxidase

0

0

0 2

4

6

8

10

Time (min)

Fig.4. Effect of heat treatment on the activity of the monoamine o.uidasr preparations. Enzyme samples (2.5 mg protein/ml) of (A) partially purified enzyme or (B) partially purified enzyme after treatment with phospholipase A and gel filtration were incubated at 50 "C in 0.05 M phosphate buffer, p H 8.0. At the times indicated, samples (50 p1) were removed and assayed for enzyme activity at 37 "C. The substrates used were tyramine (a),5-hydroxytryptamine (A)and benzylamine (w). Each point on the graphs is the mean of three enzyme determinations which differed by less than 3

0

10

20

30

40

Time (rnin)

Fig. 5. Inactivation if monoarnine oxidase preparutions by trypsin. Enzyme samples (2.5 mg protein/ml) of (A) partially purified enzyme o r (B) partially purified enzyme after treatment with phospholipase A and gel filtration were incubated at 37 "C in the presence of 200 pg and 100 pg trypsin/mg enzyme protein respectively. At the times indicated, a 3-fold weight excess of soya bean trypsin inhibitor was added and the residual enzyme activity determined. Control incubation without trypsin lost no enzyme activity for over 40 min. The substrates used were tyramine (O),5-hydroxytryptamine (A) and benzylamine (m). Each point on the graph is the mean of three enzyme determinations which differed by less than 3

X

used with the untreated preparation, monoamine oxidase A was completely inhibited by 8 min whereas monoamine oxidase B activity was decreased by 95 % in 30 min. A number of membrane-bound enzymes have been shown to be influenced by the lipid environment that surrounds them, reflected by the appearance of transition temperatures in Arrhenius plots [25,26]. Since it has been suggested that lipids are responsible for the formation of monoamine oxidase A and B [8], then an Arrhenius plot of the enzyme activity may reveal some information regarding this hypothesis. Fig.6 shows an Arrhenius plot of the membranebound enzyme. A transition temperature of 26.8 "C was observed for 5-hydroxytryptamine, benzylamine and tyramine oxidation. The same transition temperature was observed for the soluble, phospholipaseC-treated and the phospholipid-depleted enzyme preparation (figures not shown).

Inhibition of Monamine Oxidase by Propavgylarnine If the hydrophobic region of monoamine oxidase that interacts with the aromatic moieties of inhibitors and substrates is the determining region of the A and B form of the enzyme as proposed by Neff and Yang [lo], then an irreversible acetylene inhibitor that lacks an aromatic moiety should have a greatly reduced or no selectivity. Propargylamine meets the above requirements in that it is a monoamine oxidase inhibitor that lacks an aromatic group [27]. Evidence for irreversible enzyme inhibition was obtained by incubating mitochondria with propargylamine until no enzyme activity was detectable. The mitochondria were then diluted in 50 mM phosphate buffer, pH 8.0 and centrifuged a t 20000 x g for 10 min. The washing procedure was repeated 6 times and no enzyme activity was detectable. Furthermore, propargylamine inactivated monoamine oxidase was dialyzed for 24 h at

171

S. P. Baker and B. A. Hemsworth

4.4

r

oxidation was observed over inhibition of monoamine oxidase A. Similar inhibition patterns were observed with the soluble, phospholipase-C-treated and phospholipid-depleted enzyme preparations (figures not shown).

DISCUSSION

I

2.4 32

I

33

34

,

b,

35

36

K-’) Fig. 6. Arrhenius plots o j membrane-bound monoumine oxidase. lo4/ T

(

Enzyme samples were incubated for S min at the indicated temperatures before addition of substrate. Activity was measured with tyramine (o),5-hydroxytryptamine (A)and benzylamine (m). Each point o n the graph is the mean of three enzyme determinations which differed by less than 3 ”/,

-log [ ~ r o p a r g y ~ a r n i n e ] ( - ~ M) og

Fig. I. Inhibition of membrane-bound monoamine oxiduse by pvopargjlarnine. Enzyme samples ( S O pl, 2.5 mg protein/ml) in 0.1 M phosphate buffer, pH 8.0 were incubated at 37 “C for 30 min with SO pi of propargylamine in water to give the final concentration indicated. After this incubation no further time-dependent loss of enzyme activity was observed. Control incubations in the absence of inhibitor showed no loss of enzyme activity over the preincubation period. Thc enzyme was assayed with tyramine (O),5-hydroxytryptamine (A) and benzylamine (m) as substrates. Each point on the graph is the mean of three enzyme determinations which differed by less than 3

4 ° C against 2 x 15 1 of 50 mM phosphate buffer, pH 8.0 and no enzyme activity was observed. Propargylamine inhibition was also found to be timedependent. A 30-min preincubation of enzyme with inhibitor was found necessary before complete inhibition was observed with the three substrates used. Fig. 7 shows the inhibition of membrane-bound monoamine oxidase by propargylamine. The inhibition pattern shows a greatly reduced substrate selectivity compared to deprenil (see Fig. 3A). However, significant selectivity for inhibiting monoamine oxidase B and tyramine

One experimental procedure to investigate the effect of the membrane environment on the properties and nature of the multiple forms of monoamine oxidase would be to compare the membrane-bound enzyme to a preparation after the environment had been altered. This approach has been used by Ekstedt and Oreland [ I l l , who compared the inhibition of membrane-bound monoamine oxidase by clorgyline before and after extraction of lipids with organic solvent. Houslay and Tipton [8] used the approach of solubilizing and partially purifying the enzyme and then reducing the lipid content by treatment with the chaotropic agent, sodium perchlorate. Chaotropic agents are however potent protein denaturants and can induce lipid peroxidation [28] which may result in inhibition of monoamine oxidase activity 1291. In the present work, advantage was taken of commercially available phospholipases in an attempt to reduce the phospholipids associated with the enzyme, Treatment of mitochondria with phospholipase A and C resulted in hydrolysis of the phospholipids to a level where useful comparisons of the enzyme properties could not be made (Table 1). This was most likely due to the structure of the membrane preventing access of the phospholipase to the phospholipids for complete hydrolysis. Therefore, monoamine oxidase was solubilized and partially purified before treatment with the phospholipases. Upon solubilization, the phospholipid environment becomes more accessible to hydrolysis by phospholipases. This is indicated by the finding that gel filtration of the soluble enzyme preparation only reduced the phospholipid content by 27 %, whereas after phospholipase C treatment and gel filtration, there was a 96 %, decrease in phospholipidphosphoryl groups. In addition, phospholipase A treatment resulted in a 79% and 94% reduction of phospholipids associated with the slightly and fully retarded peaks respectively after gel filtration (Fig. 2). The shift of enzyme activity after phospholipase A treatment, from a partially retarded to a fully retarded peak during gel filtration indicates a breakdown in size of soluble membrane units that contained the enzyme or a breakdown of an aggregated state of the enzyme (Fig. 2). Soluble units of membrane that contain the monoamine oxidase activity with a higher phospholipid to protein ratio than crude mitochondria may explain the increase in phospholipid content observed with the partially purified preparation

172

(Table 1) although the solubilizing detergent may have also caused the observed increase. Gel filtration of the partially purified preparation resulted in a recovery of 83% of the applied enzyme activity whereas after phospholipase A treatment only 6 5 % of the applied enzyme activity was recovered. Recently, some investigators have reported that Triton X-100 can inhibit monoamine oxidase [30,31] which could explain the 17 % loss of activity of the partially purified preparation during gel filtration in the presence of Triton X-100. Furthermore, the loss of 35% of enzyme activity on gel filtration after phospholipase A treatment may indicate that the action of the phospholipase renders the enzyme more sensitive to detergent inhibition. Phospholipase C treatment of the soluble preparation had no effect on the enzyme activity indicating that the phospholipid-phosphoryl groups are not required for enzyme activity. Treatment with phospholipase A resulted in less than a 20 % loss of monoamine oxidase activity and no further loss was observed with longer contact periods used in the present study (20 min) suggesting that the phospholipids that phospholipase A hydrolyzes are not required for enzyme activity, However, the residual amounts of phospholipids present in the enzyme preparation after phospholipase treatment may be required for activity. The loss of activity observed was most likely due to the detergent effect of the hydrolysis products as such an inhibition has been observed with other enzymes unless bovine serum albumin is present in the incubation media [16]. In contrast, Tipton [32] found that his preparation of rat liver monoamine oxidase lost all activity when treated with phospholipase A. This preparation was incubated for 6 h which in itself may have reduced the activity of the enzyme, or the hydrolysis products may have caused the inhibition. It has become increasingly clear that some membrane-bound enzymes are regulated by lipids that interact with the enzyme [12,33]. These regulatory lipids or annulus, may be only one molecule thick and are probably tightly bound to the protein [34]. Since. the pattern of gel filtration of the untreated enzyme preparation shows heterogeneity with respect to protein (Fig. l), it is difficult to determine if the phospholipd associated with monoamine oxidase was hydrolyzed by the phospholipase. However that the enzyme shifted from a partially to a fully retarded peak on gel filtration with a corresponding loss of 94% of the phospholipid suggests that at least some of the phospholipid was degraded. Furthermore, that some phospholipid barrier around the enzyme was hydrolyzed is indicated by the observation that the soluble monoamine oxidase fraction was not retained by Nbs2-Sepharose (see Methods section) since monoamine oxidase is known to contain reactive thiol groups [35,36]. However, after phospholipase A treat-

Effect of Phospholipid Depletion on Monoamine Oxidase

ment the enzyme is then bound to the Nbsz-Sepharose and easily released by mercaptoethanol (Baker and Hemsworth, unpublished observations). It has been suppested that lipids control the formation of monoamine oxidase A and B because extraction with perchlorate, which reduced the phospholipid content, resulted in the loss of selective inhibition and heat stability between the two forms of the enzyme [XI. From the present work no definitive statement can be made regarding whether or not phospholipds are responsible for the formation of monoamine oxidase A and B. Even though the phospholipid content of the enzyme fraction treated with phospholipase A and subjected to gel filtration was similar to that reported by Houslay and Tipton [8] for their perchlorate-treated preparation, selective inhibition by deprenil (Fig. 3) and differential heat stabilities and trypsin digestion were still observed. It may be possible, therefore, that the phospholipase did not digest the whole phospholipid annulus as there was still a small amount detectable in the phospholipid-depleted preparation (Table 1). Alternatively, lipids other than phospholipids may be responsible for the formation of monoamine oxidase A and B. Although 95% of the phospholipid-phosphoryl groups were removed by treatment with phospholipase C and gel filtration and no change was observed in any of the enzyme properties studies, phospholipid-phosphoryl group involvement in formation of the two forms of monoamine oxidase cannot .be ruled out due to the residual amount still observed. A transition in Arrhenius plots for membrane functions has been interpreted as a conformational change in the catalytic site which occurs as the result of a phase change in the lipid environment [37,38]. In some reports correlations have been found between transition temperatures of membrane lipids using electron paramagnetic resonance of spin-labelled lipids and breaks in Arrhenius plots of enzymes [39,40]. Transitions in Arrhenius plots have recently been reported for rat brain mitochondria1 monoamine oxidase [41]. If lipids were responsible for the transition temperature observed with monoamine oxidase in the present work (Fig.6), then since both the A and B enzyme have the same transition temperature, these two forms of the enzyme must be in similar lipid environments. However, if lipids constitute or control the region of the enzyme that interacts with aromatic constituents of substrates and inhibitors [lo] and if this region is outside the catalytic site [42] then the Arrhenius plot may not reflect changes in these lipids. Alternatively, the transition temperature for monoamine oxidase may reflect a conformational change within the enzyme protein independent of lipid thus indicating that monoamine oxidase A and B are similar proteins. These two enzyme forms have

S. P. Baker and B. A. Hemsworth

173

recently been shown to be immunologically identical [43]. The results presented here of the tryptic digestibility and heat stability of the lipid-depleted monoamine oxidase are in agreement with those of Oreland and Ekstedt [44] for. the pig liver enzyme. These results suggest that the membrane environment around the enzyme confers resistance to such modes of enzyme inactivation. As the phospholipids are removed, a larger area of the protein is exposed and thus the rate of inactivation by trypsin and heat increased (Fig.4 and 5). However, the increased rates of inactivation by heat and trypsin could also be due to the action of hydrolysis products on the enzyme thus increasing the enzyme’s susceptibility to heat and trypsin. Even though bovine serum albumin was present during phospholipase A treatment to remove hydrolysis products, local action of these productsregardless of the presence of albumin cannot be ruled out. The differences between the A and B forms of monoamine oxidase in resistance to heat and trypsin inactivation were unchanged after removal of 95 % of the phospholipid-phosphoryl groups suggesting that these groups do not stabilize the enzyme, although the residual groups not hydrolyzed may be involved. The results on the extent of enzyme inhibition by propargylamine (Fig. 7) as compared to deprenil (Fig. 3A) indicates that a major factor governing selectivity of inhibition between monoamine oxidase A and B is the hydrophobic region in the enzyme that interacts with the aromatic moiety of the inhibitor. Propargylamine lacks an aromatic moiety and its selectivity between the two enzyme forms is greatly reduced. The difference between monoamine oxidase A and B may be due therefore to the sequence of nonpolar amino acids or as suggested by others [10,45], lipids that constitute or control these regions. However, propargylamine does show some selectivity for inhibiting monoamine oxidase B over the A form of the enzyme suggesting other factors may be involved. For example, Severina [46] suggested that aromatic hydroxyl groups of monoamine oxidase substrates and inhibitors interact with a polar region in the enzyme. Perhaps, the lack of groups on the propargylamine molecule which could interact with such a polar region is the reason for its selectivity for monoamine oxidase B.

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S. P. Baker, Department of Pharmacology, University of Miami School of Medicine, P.O. Box 520875, Miami, Florida, U.S.A. 33152 B. A. Hemsworth, Pharmacological Laboratories, Department of Pharmacy, University of Aston, Gosta Green, Birmingham, Great Britain, B4 7ET