BIOCHEMICAL MEDICINE AND METABOLIC BIOLOGY 36, 1-7 (1986)

Purification of Human Blood Platelet Monoamine Oxidase

A. SZUTOWICZ, P. J. ORSULAK, AND R. D. KOBES

Department of Psychiatry, University of Texas Health Science Center, and Psychiatric Clinical Diagnostic Laboratory, Veterans Administration Medical Center, Dallas, Texas 75216

Received May 2, 1984

Human platelets contain an intramitochondrial form of monoamine oxidase (monoamine:O, oxidoreductase (deaminating), EC 1.4.3.4, MAO) that is similar in many physiochemical properties to one of the forms of MAO found in the central nervous system. Numerous studies have found differences in platelet MAO activity in various subgroups of schizophrenic and depressed patients (l- 5). These findings support the hypothesis that abnormalities in the metabolism of biogenic amines are involved in the pathophysiology of these disorders. However, studies of brain samples from deceased schizophrenics and normal subjects showed no differences in MAO activity (6-8). This raises the possibility that monoamine oxidase activity in brain in situ might be affected by alterations in the membrane structure or cellular environment of the enzyme. In addition, in the brain there are two molecular forms of this enzyme, designated MAO-A and MAO-B. MAO- A is located primarily intraneuronally while MAO-B is an intraglial enzyme (9,lO). The role of these enzyme forms in monoamine metabolism in healthy as well as in schizophrenic brain is unknown. Human blood platelets contain only the B form of monoamine oxidase (5). Therefore platelets provide a clinically useful source for selective studies of the properties of this molecular form of the enzyme in healthy subjects and in subjects with psychiatric disorders.

Few attempts to purify MAO from platelets have been made thus far. Those purifications which have been reported have provided enzyme of relatively low specific activity (11). This study reports purification of MAO-B from human platelets, by a procedure which provides an enyzme of much higher purity than has been reported before and partially characterizes the molecular properties of the purified enzyme.

MATERIALS AND METHODS

Materials

Outdated platelet rich plasma was obtained from a local blood bank. Bio-Beads SM-2, protein molecular weight standards, and the protein assay kit were purchased from Bio-Rad (Richmond, Calif.); DE-52 cellulose was obtained from Whatman (Maidston, England). All other reagents were from Sigma Chemical Company

1 088%4505/86 $3.00

Copyright D 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

2 SZUTOWICZ,ORSULAK,ANDKOBES

(St. Louis, MO.). [3H]Pargyline was obtained from New England Nuclear (Boston, Mass.).

Methods

MAO purijication. Platelet rich plasma preparations from 26 pints of blood were pooled and centrifuged at 400g for 5 min to decrease contamination of the preparations by red and white blood cells. The supernatant was centrifuged for 5 min at 6000g at 5-15°C. Plasma was discarded and the platelet pellet was suspended in a small volume of 0.02 M potassium phosphate buffer, pH 7.4 (1 .O- 1.5 ml per original pint of blood), and kept frozen at -20°C until used.

Immediately before use the preparation was thawed by addition of distilled water to restore the original volume of the sample and homogenized in glass homogenizer with a Teflon pestle for 1 min at 600 rpm. All subsequent steps were carried out at 0-4°C. The homogenate was centrifuged at 8OOOg for 30 min, the supernatant was discarded, and pellet membranes were suspended in 0.01 M KPi buffer, pH 7.4 (1.5 ml per original pint of blood). To this suspension of platelet membranes, a 20% (v/v) Triton X-100 solution was added to a final concentration of 0.5% (v/v). The suspension was then mixed for 30 min and centrifuged at 44,000g for 30 min after which the supernatant was discarded.

The resulting pellet was homogenized with 0.01 M potassium phosphate buffer, pH 7.4, containing 1.5% (v/v) Triton X-100, mixed for 40 min, and centrifuged as above after which the supernatant was collected.

Triton X-100 was removed by addition of Bio-Beads SM-2 (0.5 g per each 10 mg of the detergent present in solution) and mixed for 1 hr. The Bio-Beads were then separated by filtration through a sintered glass funnel and the supernatant was collected and mixed with an equal volume of 20% glycerol.

The enzyme preparation was next applied to a 2.5 x 17-cm DE-52 column equilibrated with 0.01 M potassium phosphate, pH 8.0, in 10% (v/v) glycerol at : a flow rate of 2.0-2.5 ml/min. The column was washed with 100 ml of 0.1 M potassium phosphate buffer in 10% (v/v) glycerol. The enzyme was eluted with ; 0.1 M potassium phosphate buffer, pH 8.0, containing 10% glycerol and 0.25% (v/v) Triton X-100. The fraction containing the most active enzyme was turbid and lightly yellow.

The sample was treated with sufficient Bio-Beads SM-2 to remove Triton X- 100 (see above) and concentrated overnight on a Micro-Con-FiIt concentration/filter using a filter membrane with a cutoff of 15,000 Da (Bio-Molecular Dynamics, Beaverton, Oreg.) to about 6 ml.

The concentrated enzyme preparation was mixed with Triton X-100 (final concentration 0.25% v/v), applied to a 2.2 x 90-cm Sepharose CL-6B column : equilibrated with 0.05 M potassium phosphate buffer, pH 8.0, with 10% glycerol [ and 0.25% (v/v) Triton X-100 at a flow rate of 36 ml/hr, and eluted with the same buffer. Fractions containing the highest MAO activities were pooled, treated with Bio-Beads SM-2, and dialysis-concentrated overnight on the Micro-Con- Filt device, against 2 liters of 0.01 M potassium phosphate buffer, pH 7.2, with

I ’

0.25% Triton X-100 containing no glycerol.

PLATELET MONOAMINE OXIDASE 3

The enyzme preparation was applied to a 1.5 x 12-cm tyramine-butyl-Sepharorose column (12) equilibrated with the same buffer at a flow rate of 2.0-2.5 ml/min. MAO was eluted with 240 ml of a continuous gradient of O-O.25 M KC1 in the same buffer. The most active fractions (9 ml each) were pooled and concentrated on the Micro-Con-Filt device after removal of Triton X-100 with Bio-Beads SM- 2.

Monomine oxidase assay. Activity of MAO in enzyme preparations was de- termined with 1 mM tyramine as a substrate by the calorimetric method of Szutowicz er al. (13). Protein content was determined by the method of Bradford (14) with human gamma globulin as a standard. Polyacrylamide gel electrophoresis was performed according to Laemmli (15) in 0.1% (v/v) sodium dodecyl sulfate, in the Model SE400 vertical electrophoresis unit (Hoefer Scientific Instruments, San Francisco, Calif.).

[3K]Pargyline binding. Twenty micrograms of enzyme with a specific activity of 9 nmole/min/mg of protein was incubated for 1 hr with 2 &I of [3H]pargyline (sp radioact 20 mCi/nmole) in 0.05 M KPi buffer, pH 7.4, in a final volume of 0.1 ml. The enzyme was precipitated with 10% (v/v) trichloroacetic acid solution, centrifuged, washed three times with the same amount of trichloracetic acid, solubilized in lysis solution (15), and subjected to electrophoresis on 7.5% poly- acrylamide gel in the presence of 0.1% (v/v) sodium dodecyl sulfate. After fixing in trichloroacetic acid one line was cut off the slab and cut into thirty 0.4-cm slices which were digested overnight in 30% H202 and counted in scintillation fluid. Another part of the gel was stained as described elsewhere (15).

RESULTS AND DISCUSSION

The freezing, thawing, and centrifuging of the crude platelet preparation in hypo-osmotic medium removed soluble platelet proteins, that formed about 70% of total platelet proteins, without loss of monoamine oxidase activity (Table l), thereby achieving a 3-fold increase of the enzyme specific activity. Also, preliminary solubilization of the platelet membrane fraction with a low (0.5% v/v) Triton X- 100 concentration appeared to remove about 50% of the membrane bound proteins with only a small loss of monoamine oxidase activity (Table 1). On the other hand the fraction solubilized between 0.5 and 1.5% (v/v) Triton X-100 contained almost 70% of the original enzyme which had a specific activity of 10.8 nmole/min/mg of protein (l&fold purification). Thus the fractional extraction with Triton X-100 appeared to be a much more efficient method of enzyme purification than straight one-step extraction, which yielded enzyme purified only 4-fold (5). This purification was also much higher than enrichment of the enzyme activity obtained by isolation of platelet mitochondria (16). Also removal of Triton X-100 by treatment with Bio-Beads SM-2 increased the amount of enzyme recovered in the extract (Table 1). Apparently Triton X-100 inhibited monoamine oxidase activity (unpublished data).

DE-52 column chromatography followed by Bio-Beads treatment provided a further 2-fold purification of the enzyme to 19.2 nmole/min/mg of protein with moderate loss of total activity (Table 1). In this case treatment with Bio-Beads apparently removed some contaminating proteins (Table 1).

4 SZUTOWICZ, ORSULAK, AND KOBES

TABLE 1 Purification of MAO from Human Blood Platelets

Specific Total activity

Protein activity (nmole/min/mg Yield Purification Step bg) (nmole/min) of protein) (%I (xl

Platelet homogenate 1925 1174 0.61 100 1 Platelet membranes 541 1170 2.17 100 3.6 0.5-1.5% Triton

X-100 extract nd 664 nd 57 nd Triton X- 100

free extract 74 798 10.8 68 18 DE-52 eluate 36 572 15.9 49 26 Triton X-100 free,

Micro-ConFilt concentrate 27 510 19.2 43 31 Sepharose CL-6B eluate 11.6 171 14.7 15 24 Tyramine-butyl-

Sepharose eluate 0.46 52 112.8 4.5 185 Micro-Con-Filt concentrate 0.46 33 72.3 3 119

Note. Enzyme was isolated from outdated platelets obtained from 26 pints of blood. 1 mM tyramine was used as substrate for all enzyme assays (see Materials and Methods).

Chromatography on CLdB Sepharose caused loss of enzyme activity. However, it separated MAO protein from turbid material containing no protein which was apparently of lipid character (not shown in table).

Platelet MAO was further purified by binding to a tyramine-butyl-Sepharose column which had been equilibrated with 0.01 M potassium phosphate buffer, pH 7.2, with 0.25% (v/v) Triton X-100. Platelet monoamine oxidase, unlike human or rat liver enzyme, did not bind to the column when it was equilibrated with medium containing glycerol (12). The reason for such differences in binding between liver and platelet monoamine oxidases onto this column are not known. MAO was eluted from the column with a continuous gradient of KCl. The most active fractions were eluted between 0.075 and 0.115 M KC1 and had an average specific activity of 113 nmole/min/mg of protein (Fig. 1) corresponding to 185- fold purification. These most active fractions were located between two major

O- 0

FRACTION NUMBER

FIG. 1. Elution profile of MAO from tyramine-butyl-Sepharose column by a linear gradient of O-O.25 M KCl: enzyme elution pattern (0); protein elution pattern (0). I, II, and III indicate pools of the fractions collected between 55-75, 75-l 15, and 115-155 mM KC1 concentrations, respectively.

PLATELET MONOAMINE OXIDASE 5

protein peaks eluting from the column at lower and higher KC1 concentrations (Fig. I). At this stage of purification the enzyme was relatively unstable and loss of its specific activity to 72 nmole/min/mg of protein was observed during concentration on the Micro-Con-Filt device. The cause of this loss of activity has not been established. After concentration to 0.5 mg of protein/ml, however, the enzyme remained stable up to 2 weeks which was the longest time tested.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme preparation indicates that at this stage of purification it still contains many con- taminating proteins (Fig. 2). One can, however, see that a distinct protein band corresponding to a molecular weight of 58,000 is present in the electrophoretogram (Fig. 2). Enzyme fractions which have been eluted from the tyramine-butyl- Sepharose column either before or after the most active fractions showed higher contamination either by low or high molecular weight proteins, respectively (Fig. 2). Positive identification of a protein band of 58,000 Da as monoamine oxidase is documented by the fact that a peak of [3H]Pargyline binding corresponded to this protein band (Fig. 3).

The K,,, value for the purified monoamine oxidase with tyramine as a substrate was equal to 0.165 5 0.006 mM. The K,,, for monoamine oxidase in the crude platelet membrane fraction was 0.124 ?I 0.008 mM (3 experiments). This finding is different from that reported by Oreland (17) who showed a decrease of K,,, for MAO purified from pig liver, or from Stadt et al. (18) who found no change

MW -3

XI0 200

116 92

66

45

FIG. 2. Sodium dodecyl sulfate-10% polyacrylamide slab gel electrophoresis of partially purified platelet MAO. Molecular weight standard (MW Std) line contained 2.5 pg of each protein standard. Remaining lines contained 25 pg of protein from the following specimens: TX-100 = 051.5% Triton X-100 extract; Tyr-B-S I, II, and III = concentrated eluates of fractions from the tyramine-butyl- Sepharose column (see Fig. 1): CLdB = eluate from Sepharose CLdB column.

6 SZUTOWICZ, ORSULAK, AND KOBES

C3 l-4) parsvhe binding

1 2 3 4 5

FIG. 3. Sodium dodecyl sulfate-7.5% polyacrylamide slab gel electrophoresis of r3H]Pargyline- labeled MAO. Lines 1 and 4 are molecular weight standards; lines 2 and 3 were 35 and 20 pg of protein from DE-52 and Sepharose CL&B column eluates, respectively. Line 5 indicates [‘H]Pargyline radioactivity in sliced gel (see Materials and Methods for details). Sample in this line was the same as that in line 3.

in rat liver enzyme-substrate interaction during its purification. These variable effects of purification on K, value may result from removal of different lipid components from the enzyme environment during various purification procedures. It is generally known that in liver and brain, MAO-A is much more sensitive to changes in the lipid environment than MAO-B (19). However, in human platelets marked loss of MAO-B activity has been observed during delipidation (20). Our finding is compatible with the former, and indicates that in human platelets, the membrane environment facilitates the interaction of MAO with its substrates.

SUMMARY

Monoamine oxidase B has been purified from human blood platelets 185fold to a specific activity of 113 nmole/min/mg protein by a combination of Triton X-100 solubilization and ion exchange chromatography. A protein fraction cor- responding to 58,000 Da on sodium dodecyl sulfate-polyacrylamide gel electro- phoresis was identified as monoamine oxidase by its ability to bind [3H]Pargyline.

ACKNOWLEDGMENTS This study was supported in part by Grant MH37810 from the National Institutes of Mental Health

and by administrative support from the Department of Psychiatry. We thank Dr. Paul A. Srere for making his facilities at the Veterans Administration Medical Center, Dallas, Texas, available for our research.

PLATELET MONOAMINE OXIDASE 7

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