Multiple forms of monoamine oxidase in human plasma

BIOCHEMICAL

MEDICINE

Multiple

(1975) 13, 141-156

Forms of Monoamine in Human Plasma1

Oxidase

ADA WEN-SHUNG MA LIN AND DONALD 0. CASTELL Clinical

Investigation Philadelphia,

Center, U. S. Naval Hospital, Pennsylvania 19145

Received February 25. 1975 The presence of multiple forms of monoamine oxidase (MAO) in mitochondria has been suggested by many workers. Support for this hypothesis comes from kinetic studies including effects of selective inhibitors, substrate specificity, and thermal inactivation (l-7), and separation studies by chromatography or gel electrophoresis (g-13). Recently, McCauley and Racker (14) have demonstrated the separation of two immunologically distinct forms of the mitochondrial enzyme from bovine brain. However, the question of the true multiplicity of mitochondrial MAO remains unsettled due to controversies over methods of solubilization of the enzyme. To date, there has been little reported on possible multiplicity of soluble plasma MAO. Our recent investigation on human plasma MAO (15) has shown that MAO from a patient with hepatic fibrosis and hemochromatosis has different kinetic properties than the enzyme isolated from pooled normal human plasma. This finding suggested the possibility that human plasma may contain isoenzymes of MAO. In this communication, we report that three forms of MAO have been isolated in human plasma by hydroxyapatite column chromatography, and discuss some of their physical chemical properties.

MATERIAL

AND

METHODS

Materials. Benzylamine-free base was purchased from Matheson Coleman and Bell Manufacturing Chemists. It was redistilled under vacuum before use. Semicarbazide hydrochloride was purchased from Fisher Scientific Company. Crystallized serum bovine albumin, recrystallized ammonium sulfate, nitroblue tetrazolium (grade III), o-dianisidine (3, 3-dimethylbenzidine) fast blue base, tryptamine hydrochloride, tyramine hydrochloride, hydroxyapatite suspension (Type I), diethyl amino ethyl (DEAE) cellulose (medium mesh), and beef liver catalase (twice crystallized), were all purchased from Sigma Chemical. DEAE-Sephadex A-50, Sephadex G-200 and the calibration kit for 1 This work was supported Program Grant 5-05-532R.

by the Department

141 Copyright 0 1975 by Academic press, Inc. All rights of reproduction in any form reserved

of the Navy Clinical Investigation

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protein molecular weight determination were purchased from Pharmacia Fine Chemicals, Inc. Horseradish peroxidase was purchased from Worthington Biochemical Corporation. Method Activity of monoamine oxidase. Monoamine oxidase activity was routinely assayed by directly measuring benzaldehyde formation from benzylamine (16). One unit of enzyme activity was defined as the amount of enzyme catalyzing a change in absorbance of 0.01 per 90 minutes at 250 nm in a 1.5 nrl reaction mixture containing 2.2 mM benzylamine, 0.2 M phosphate buffer (pH 7.2) at 37” (15). The molar absorptivity of benzaldehyde at 250 nm was assumed to be 0.12 x 10“ M-r cm-’ (17). One unit of enzyme corresponds to the formation of 1.25 nmoles of benzaldehyde in 90 minutes at 37”. When tryptamine and tyramine were used as substrates, the peroxide liberation was measured by coupling with horseradish peroxidase (18) and the change in absorbance at 420 nm was measured. The details of the reaction mixture was described in the legends of Table 1. Protein was determined by the method of Lowry et al. (19) with bovine serum albumin as standard. Purification of the enzyme. All operations were carried out at 5” unless TABLE SUBC~T~~TE

1

SPE~~FICITY~

(Activity = H202 Production) (A) Constant activity of tryptamine Benzylamine

Y

72.5 29.5 44.8

(B) Constant activity of benzylamine Benzylamine

Y

1000 loo0 1000

Tryptamine 1.0 1.0 1.0 Tryptamine 13.8 33.9 22.3

Tyramine 15.2 33.2 23.9

a Suitable amount of (Y,p. and y forms of MAO were incubated with 2.5 mM of substrate in 0.05 M phosphate buffer pH 7.2 (final volume 0.5 ml) at 37” for 3 hours. Amount of enzyme used in tryptamine and tyramine oxidation is about tenfold greater than in benzylamine oxidation. At the end of incubation 300 pg of horseradish peroxidase and 50 pg of o-dianisidine in 25 ~1 of absolute methanol were added to the system. The absorbence at 420 nm was measured against a blank containing all the components except MAO which was substituted with buffer. The ratio obtained was normalized either with tryptamine (A) or with benzylamine (B) value.

MAO

IN

HUMAN

PLASMA

143

otherwise noted. All centrifugation was done at 5” and 10,OOOg for 10 minutes. Plasma monoamine oxidase was purified according to a modification of McEwen’s method (20). Briefly, the freshly frozen plasma was defrosted at 5” overnight. Any precipitation was removed by centrifugation. The clear plasma was then subjected to ammonium sulfate fractionation, alcohol fractionation, and repetitive ammonium sulfate fractionation as previously described in detail (15). Enzyme prepared in this way usually has a purification of 100 to 200-fold. This partially purified enzyme was then used for column chromatography. Hydroxyapatite column chromatography. Hydroxyapatite suspension (Sigma Type 1) was gently mixed and suspended in 2 mM phosphate buffer (pH 6.8). The fine particles were removed by decantation and resuspension several times. The thin slurry was then poured into a 1.6 x 40 cm column through the Pharmacia packing funnel. The column was first settled by gravity overnight, followed by equilibrating with 2 mM phosphate buffer with a LKB peristaltic pump. The conductivity of the eluate was measured to assure the equilibration. The final packed column was 16 x 60 mm with a flow rate of 1.4 ml/hour. Approximately 200-300 mg of enzyme with specific activity of 30-50 units/mg in 5-7 ml was applied on the column. The enzyme was first eluted with starting buffer (3 bed vol of column). Afterward, it was connected to an exponential gradient with a known fixed mixing volume at the top of the column. DEAE-Sephadex A-50 column. DEAE-Sephadex A-50 was swollen in a large amount of water at 100” for 2 hours. The fine particles suspended in the top were removed by repeated decantation and resuspension. The swollen ion exchange Sephadex was then washed thoroughly on a Buchner funnel with 5 mM phosphate buffer (pH 7.5) and then equilibrated in the refrigerator for 2 days with several changes of the buffer. The equilibrated DEAE-Sephadex was then poured into a column of 0.9 x 10 cm (Pharmacia column K9/10) and further equilibrated with 5 mM phosphate buffer. MAO activity was eluted with a linear gradient of NaCl (O-O.3 M) in 5mM phosphate buffer. Gel jiltration on Sephadex G-200. Sephadex G-200 was swollen in a large amount of water at room temperature. The fine particles were removed by repeated decantation and resuspension. The gel was then allowed to continue swelling in a refrigerator (5”) for at least 2 weeks. It was followed by equilibration with 0.2 M phosphate buffer (pH 7.2) for 2 days with several changes of buffer. After this, the gel was poured into a 16 x 40 cm column (K 16/40) and allowed to settle by gravity. The column was washed upward with a 3-5 bed vol of the same buffer. Enzyme was applied at the bottom of the column with the flow rate adjusted to 0.15 ml/minute (15 drops/l.1 ml). The eluate was collected at 15 drops/fraction at the top of the column.

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RESULTS Separation of three forms of MAO from

hydroxyapatite

column.

Three activity peaks, designated as (Y, p, and y, were obtained by hydroxyapatite (HA) column chromatography, as indicated in Fig. 1. Alpha form was eluted by the starting buffer (2 mM phosphate buffer, pH 6.8). Beta and y forms were eluted by phosphate buffer with a conductance reading of 20 and 80 corresponding to 10 and 40 mM phosphate buffer (pH 6.8). By this step CYform was purified to 18-fold whereas j3 and y forms were purified to five- and threefold, respectively. The overall yield from the HA column is 60%. The concentration of the phosphate ion is a very important factor in the separation of the multiple forms. Separate experiments revealed that LYform was not adsorbed by HA, with its elution point affected only by the column length. On the other hand, p and y were weakly adsorbed by HA and were not eluted by 2 mM phosphate buffer up to 300 ml (30 bed vol). In addition, they were not eluted by increasing the ionic strength with the addition of NaCl gradient up to 0.2 M of NaCl. However, at 80 mM phosphate buffer, all three forms were eluted simultaneously with a shoulder on each side. The conductance of the solution as a function of phosphate ion concentration is shown in Fig. 2.

FRACTION

FIG. 1. Elution profile of MAO on hydroxyapatite column chromatography. Monoamine oxidase activity, 0; absorbance at 280 nm 0; column size 16 x 60 mm; 370 mg of MAO (16, 148 units, specific activity, 44) was put on the column. Mixing volume was 29 ml on the top of the column. Alpha was eluted with 2 mM phosphate buffer (PH 6.8). Beta and y were eluted with a convex gradient of phosphate ion concentration. Flow rate was 1.5 ml/hour.

MAO

IN

HUMAN

145

PLASMA

240 220 200 3a 180 2 180 e 140 5F 120

.04

.06 .08 PHOSPHATE pH 6.8

10 SUFFER,

d2

.14

.16

M

FIG. 2. Conductivity reading of phosphate buffer at pH 6.8. Conductivity was measured by a Wescan model 210 conductivity meter with a microflow cell. Conductivity = Cell factor x Reading. Since the cell factor is not constant over a range of concentration a correlation graph as shown is more meaningful than a simple reading in micromhokm.

FIG. 3. DEAE A-SO column chromatography of (Yform MAO. 8.2 mg of (Yform of MAO (sp act 800) was put on column (0.9 X 10 cm). It was eluted with 100 ml linear gradient of 0.1-0.3 M NaCl in 5 mM phosphate buffer pH 7.5. MAO activityo; protein as measured by absorbance at 280 nm 0; flow rate, 25 ml/hour.

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protein patterns of multiple forms of MAO isolated from FIG. 4. Disc electrophoretic hydroxyapapite (HA) column. Polyacrylamide gel (7.5%) electrophoresis was carried out at 4” for 1 hour with a current of3 mA/tube at pH 8.9, according to the method of Davis. Protein was stained with amido block. A. Crude preparation (200 fold purification; sp act 60). B. Alpha form of MAO isolated from HA column (sp act 1080). C. Gamma form of MAO isolated from HA column (sp act 150). D. Alpha form of MAO further purified by DEAESephadex Column (lO,OOO-fold purification, sp act 3000). E. Beta form of MAO isolated from HA column (sp act 310).

The major activity peak of (Y form was further purified on DEAESephadex A-50 column as shown in Fig. 3. All the MAO activity were eluted at 0.16 M of NaCl. Combining fractions 44-61 represented 65% of recovery of the activity put on the column. The combined effects of HA

MAO

IN

HUMAN

147

PLASMA

i d I I

1(I , 1

0

b

200-

I

-5.0 -4.0

0 I

-1o

looI”

0b

0.. 0-O I

0

l

---

10

/#/yy 20

0-o

20

40

-2.01

x.

I

50

8 t

1.0 _

I

0-y

I

0

50

FRAClKm

FIG. 5. Typical elution profile of enzyme on Sephadex G-200. 200 mg of MAO (sp act 29) was put on the column (1.6 X 40 cm) MAO activity, 0; absorbance at 280 nm, 0; Elution was performed with 0.2 M phosphate buffer (pH 7.2), flow rate 9 mUhour with upward flow.

and DEAE-Sephadex A-50 column resulted in a lO,OOO-fold purification for (Y. The fractions contained in each form of isoenzyme as shown in Fig. 1 were further examined by electrophoresis on 7.5% polyacrylamide gel. The electrophoretic patterns of the original sample and of a, p, and y are shown in Fig. 4. It is noted that although each form of these isoenzymes is not homogenous, the protein patterns of these forms are distinctly different . Molecular Weight of MAO To examine the molecular weights of the multiple forms of MAO, Sephadex G-200 gel filtration was performed. Figure 5 shows that the partially purified MAO preparation which included all three forms of the enzyme was eluted as one symmetrical activity peak. Comparison with proteins of known molecular weight eluted under the same conditions revealed that the molecular weight of human plasma MAO was approximately 150,000 (Fig. 6). Spectrum of Enzyme Partially purified MAO (lOO- to 200-fold purification) has a typical protein uv absorption at 279 nm. The visible spectrum, as shown in Fig. 7, has a maximal absorption of 410 nm and two minor ones at 545 and 575 nm. The isoenzymes, a, @, and y, which were purified by hydroxyapatite column chromatography show no significant change in the ultraviolet

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FIG. 6. Estimation of molecular weight of monoamine oxidase by gel filtration. The elution volume of monoamine oxidase is indicated by the solid arrow.

30.

.25.

E 0 e

15.

.lO.

.05.

350

440

WAVELENGTH

530

520

no

nm

FIG. 7. Visible spectrum of MAO. Solid line represents the spectrum of partially purified MAO before hydroxyapatite column (sp act 29). Broken line indicates the spectrum of o(, /I, and y forms of MAO (sp act: a, 420; j3, 150; y, 80).

MAO

IN HUMAN

PLASMA

range. However, the absorptions in the visible removed by this purification step.

149 range are completely

Substrate Speci$city As shown in Table 1, the oxidation ratios of benzylamine: tryptamine: tyramine for the three forms of MAO are different. Alpha form is about 2.5 times more active than p form and 1.5 times more active than y form in the oxidation of benzylamine (Table 1A) whereas j3 and y are more active than CYin the oxidation of tryptamine and tyramine (Table 1B). Inhibition by Semicarbazide Both cw and /3 forms are inhibited by semicarbazide. The Lineweaver-Burk plots of benzylamine oxidation in the presence and absence of this inhibitor are shown in Fig. 8. The apparent Michaelis-Menten constants (K,) calculated from the plots for both forms are the same in the absence of inhibitor (0.30 x 10-3~). However, in the presence of 133 PM of semicarbazide their apparent K, value change differently (a:0.69 x 10V3, @1.27 x 10-3~).

FIG. 8. Lineweaver-Burk plots of semicarbazide inhibition on (Y (sp act 1080) and j3 (sp act 310) forms of MAO. 60-120 Units of enzyme were incubated in 1.5 ml of 0.2 M phosphate buffer, pH 7.2, containing amount of benzylamine as indicated. (A) Without semicarbazide, the plots for both (Y(O) and /3(x) are not separable. (B) Alpha form with 133 PM of semicarbazide (0) (C) Beta form with 133 &CMof semicarbazide (-O-).

150

LIN

I

I

I

AND

CASTELL

I

I

I

1 IS

4 so

-

--7

jz?

Oh/

’ 37

I 40

I 4s

I so

, ss

70

oc

FIG. 9A. Effect of incubation temperature on MAO activity. 100 units of 01,p, or y, with protein concentration of 0.01-2.0 mg/ml in 0.1 M phosphate buffer (PH 7.2) were all incubated for 30 minutes at noted temperature. At the end of incubation enzyme was immediately transferred to an ice bath to stop the reaction.

9B. Effect of incubation time on MAO activity. Alpha, p, and -y forms of MAO was incubated at 65.5” for various periods of time. Other conditions same as Fig. 7.

FIG.

MAO

IN HUMAN

151

PLASMA

Thermal Stability

All three forms of the enzyme were shown to be stable to heat in 0.1 M phosphate buffer (pH 7.2) at protein concentrations of O.Ol-to 2.0 mg/ml. They do not lose any activity by incubation at 55” up to 30 minutes. Beyond this point, loss of activity occurs with an increase in either the temperature or the duration of incubation. At temperatures above 6O”C, the thermal stability of QI, p, and y forms were significantly different. The temperature at which the enzyme starts to lose activity after 30 minutes incubation is defined as the critical temperature. As shown in Fig. 9A, the critical temperature of (Yis W, whereas for /3 and y it is 60”. Moreover, the rate of activity loss with further increase of temperature is much sharper for j3 and y than for (Y. Figure 9B shows the time curves of activity loss of the three forms of enzyme at 65.5”. Alpha form loses its activity faster in the initial phase but approaches an equilibrium after 10 minutes incubation. Beta and y forms their activities with a slower rate than LY, but continue to lose their activity and approach an equilibrium much slower. &or

I

I

I

I

I

,

1

, 6.0 I

2.0c

FIG. 9C. To show that in thermal inactivation studies (Yform of MAO can be fitted by a third order kinetic plot, l/(Activity)* = kt + C.

152

LIN

AND CASTELL

FIG. 9D. To show that in thermal inactivation studies p and y forms of MAO can be fitted by first order kinetic plot, Log (Activity) = kr + C.

The kinetic orders of the thermal inactivation studies were analyzed. As shown in Fig. 9C, a plot of (activity) -2 versus time is linear only for (Yform of MAO. On the other hand, plots of log activity versus time are linear for j3 and y forms (Fig. 9D). The results indicate, therefore, that during the first 30 minutes the loss of activity of (Y is similar to a third order reaction whereas those of /3 and y are similar to first order reactions . DISCUSSION

For some time the existence of isoenzymes for mitochondrial MAO isolated from a number of sources has been suggested. Our study shows that three forms of MAO can be clearly separated from human plasma MAO. This conclusion is based on the results of hydroxyapatite column chromatography, which has the capability to separate proteins according to their molecular properties (21). A typical chromatogram is shown in Fig. 1, the three distinct peaks indicates molecular difference among the three enzyme forms. This is further supported by the different protein patterns revealed by polyacrylamide gel electrophoresis. The three forms of MAO are eluted with other proteins, all having different elec-

MAO

IN

HUMAN

PLASMA

153

trophoretic mobility. It is not likely that HA column separates all other proteins except MAO which distributed over 70 fractions. Several characteristic features of these isoenzymes of serum MAO are shown, namely, their substrate specificity, inhibition by semicarbazide. thermal stability, affinity to hydroxyapatite, and molecular weight. The (Y form is the major component. It is characterized by a much stronger activity toward the oxidation of benzylamine than both the /3 form and y form. It is stable to heat up to 55” and the rate of activity loss at higher temperature (65.5’) appears to fit third order reaction kinetics. It shows no affinity to hydroxyapatite. Beta and y forms of MAO are similar to each other in many properties. They are more active toward the oxidation of tryptamine and tyramine than is the (Yform. They are more heat stable, having a critical temperature at 60”. Above this temperature, the loss of activity with increasing incubation time are much faster than for (Y form and the thermal inactivation process resembles first order kinetics. Both p and y forms are readily adsorbed by hydroxyapatite, with y form showing stronger adsorption than p form. This adsorption is probably not due to the charge difference only. Increasing NaCl concentration did not elute the enzymatic activity. Inhibition of semicarbazide revealed that both CYand p forms of MAO are inhibited. The Lineweaver-Burk plots become different when 133 PM of semicarbazide is present. This may suggest that the binding of inhibitor modified the substrate-enzyme affinity differently in the two forms. Thus the geometry of the activity center of (Yand p forms may be different. Despite these differences, all three forms of MAO have practically the same molecular weight, estimated to be 150,000 according to gel filtration. This finding appears also to suggest that the multiple forms arise not from aggregation or dissociation of subunits but rather from certain structural differences. However, it is possible that they may be a result of different hybrids of different subunits such as in the case of lactic dehydrogenase. The nature of multiple form, i.e., whether they are different in primary sequence or in hybrids of subunits may not be analyzed until purified forms are achieved. It is of interest to compare the estimated molecular weight of human plasma MAO to that of MAO isolated from other sources. As shown in Table 2, the molecular weight of human plasma MAO falls into the same range with the MAO from pig and beef plasma. It is also not far from the molecular weight of the monomeric form of mitochondrial MAO. Thus, it seems likely that the three isoenzymes reproted here (CX,p, and 7) are themselves in an unaggregated state. The (Y form was further purified by DEAE-Sephedex A-50 to give a total of lO,OOO-fold purification. Further purification of /? and y was not

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TABLE ESTIMATED

MOLECULAR

Source Plasma

Mitochondria

WEIGHTS

2 OF MONOAMINE

OXIDASE

MW

Method

Pig Beef Human

195,000 170,000 (monomeric) 150,000

Gel filtration Sedimentation Gel filtration

(this work)

Pig liver

108,000-117,000 (unaggregate) 275,000-290,000 (aggregate form) 102,000 (monomer) 435,000 (tetramer)

Gel filtration

(24)

Pig brain

Reference

(22) (23

(24 Gel fiiltration

(25)

sought due to small recovery. The isoenzymes, although not yet homogeneous, are free of any absorption in the visible range. This is different from pig plasma MAO which has an absorption at 480 nm (22) and beef plasma MAO which has absorption at both 410 nm and 480 nm (26). The small absorptions at 410 nm, 545 and 575 nm for the partially purified enzyme (loo- to 200-fold) are completely removed by hydroxyapatite column chromatography. It should be mentioned that this enzyme, MAO, is very difficult to purify. It is not separable by many chromatographic columns such as DEAE and carboxyl methyl cellulose, phosphocellulose or sephadex. The purification by any of these methods was poor (two- to fivefold). The key to successful separation in the hydroxyapatite column is phosphate ion concentration in the eluant. With proper adjustments of the molarity of phosphate ion, separation of the three forms becomes possible. A question that needs to be answered is whether the cy, p, and y forms of MAO as reported here represents true multiplicity in vim, or are merely an artifact from purification process. Our suggestion of true multiplicity is based on many lines of experimentations and thinking. We recognize that to totally resolve this question more work needs to be done including immunological response, amino acid composition analysis, prosthetic group analysis, and subunit analysis. These cannot be completed until further purification of each form to a homogeneous state is achieved. If the observed multiple forms of MAO were a result of a single protein that had been modified secondarily, e.g., by minor proteolysis or a deamidation process, then it is expected that certain positive centers (or negative centers, if decarboxylation) would be removed from the protein moiety. This would change the electrostatic charge of the protein leading to a fairly large difference in electrophoresis or in ion exchange column

MAO

IN

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PLASMA

155

chromatography. This was not observed. In fact, the electrophoretical mobility of (Y, p, and y forms are not separable by electrophoresis at pH 8.9 on 7.5% acrylamide gel (with electrophoresis at acidic condition all activity was denatured; thus no information is available). In addition, the three forms are not separable by ion exchange columns. In an earlier communication (15), we have reported that MAO isolated from normal subjects and from hemochromotosis patients have different kinetic properties (different pH optimum range and different inhibition by semicarbazide and kynuramine). Those initial observations had suggested the possibility of the presence of isoenzymes in plasma MAO. The present observations of the three forms of MAO which differ in many physical and chemical properties support our previous hypothesis. SUMMARY Multiple forms of monoamine oxidase (MAO) have been isolated for the first time from normal human plasma. Three forms of the enzyme designated as (Y, p, and y were separated by means of hydroxyapatite column chromatography. The cx form was further purified to lO,OOO-fold by DEAE-Sephadex A50 column chromatography. All three forms have a molecular weight of 150,000, which is similar to the unaggregated form of mitochrondrial MAO isolated from pig liver and brain and is in the same range as pig and beef plasma MAO. Substrate specificity, semicarbazide inhibition, and thermal stability of the three forms are different. Alpha form is more specific on the oxidation of benzylamine, whereas /3 and y forms are more specific on the oxidation tryptamine and tyramine. The critical temperature is 55” for (Yform and 60” for /3 and y forms. At elevated temperature (65.5”) CYform lost its activity according to third order kinetics while p and y forms follow first order kinetics. ACKNOWLEDGMENTS Our sincere appreciation to Linda M. Davis for her technical help in preparation of enzyme, and to Antoinette Dalessandro, Linda Yeck, and Aleksandras Radzius, Jr. for their editorial and illustrative assistance.

REFERENCES 1. Oswald, E. O., and Strittmatter, C. F., Proc. Sot. Exp. Biol. Med. 114, 668-673 (1963). 2. Hardegg, W., and Heilbronn, E., B&him, Biophys. Acta 51, 553-559 (1961). 3. Werle, E., and Roewer, F., Biochem. J. 50, 32&326 (1952). 4. Fellman, J. H., and Roth, E. S., Can. J. Biochem. 43, 909-914 (1W5). 5. Sierens, L., and D’Iorio, A., Can. J. Biochem. 48, 659-663 (1970). 6. Sandler, M. and Youdim, M. B. H., Pharmacol. Rev. 24, 331-348 (1972). 7. Fuller, R. W., in “Advances in Biochemical Psychopharmacology” (E. Costa, and M. Sandier, Eds.), Vol. 5 p. 339. Raven Press, NY 1972. 8. Go&in, V. Z., Nature (London) 200, 77 (1963). 9. Youdim, M. B. H. and Sandler, M., Biochem. J. 105, 43p (1%7).

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10. Shik, J. H. C. and Eiduson, S., Nature (London), 224, 1309-1310 (1%9). 11. Raglans, J. B., B&hem. Biophys. Res. Commun. 31, 203-208 (1968). 12. Youdim, M. B. H., in “Advances in Biochemical Psychopharmacology” (E. Costa, and M. Sandler, Eds.), Vol. 5, p. 67. Raven Press. New York. N.Y., 1972. 13. Diaz Borges, J. M. and D’Iorio, A., in “Advances in Biochemical Psychopharmacology” (E. Costa, and M. Sandler, Eds.), Vol. 5. p. 79. Raven Press, New York, N. Y., 1972. 14. McCauley, R., and Racker, E., Mol. Cell. Biochem.. 1, 73-81 (1973). 15. Lin, A. W. S. M. and Caste]], D. O., Biochem. Med. 9, 373-385 (1974). 16. Tabor, C. W., Tabor, H., and Rosenthal, S. M., J. Biol. Chem. 208, 645-661 (1954). 17. Dearden, J. C., and Fohes. W. F., Can. /. Chem. 36, 1362 (1958). 18. Guidotti, G.. Columbo. J. P., and Foa, P. P., Anal. Chem. 33, 151-153 (1961). 19. Lowry, 0. H., Rosebrough, N. J., Farr. A. L., and Randall, R. J., /. Biol. Chem. 193, 265 (1951). 20. McEwen. C. M., Jr., J. Biol. Chem. 240, 2003-2010 (1965). 21. Bernardi, G., in “Methods in Enzymology” (W. Jakoby. Ed.), Vol. 22 p. 325. Academic Press, N.Y., 1971. 22. Buffoni, F., and Blaschko, H., in “Methods in Enzymology” (H. Tabor, and C. W. Tabor, Eds.), Vol. 17, pp. 682-686. Academic Press, N.Y.. 1971. 23. Achee, F. M., Chervenka, C. H., Smith, R. A. and Yasunobu, K. T., Biochemistry 7, 43294335 (1968). 24. Oreland, L.. Arch. Biochem. Biophys. 146, 410-421 (1971). 25. Tipton, K. F., Eur. J. Biochem. 4, 103-107 (1968). 26. Yasunobu, K. T., and Smith, R. A., in “Methods in Enzymology” (H. Tabor, and C. W. Tabor, Eds.), Vol. 17, p. 698, Academic Press. N.Y.. 1971.