Distinct Cellular Localization of Membrane-Bound and Soluble Forms of Catechol-O-Methyltransferase in Brain

Abstract: The cellular localization of the two forms of catechol- O -methyltransferase (COMT) was investigated by measuring their activities in rat striatum following unilateral stereotaxic injection of kainic acid, which causes degeneration of striatal neurons followed by proliferation of astroglial cells. Membrane-bound COMT activity was decreased in the lesioned striatum, while soluble COMT activity was increased. There was a statistically significant correlation between the ratio of lesioned to control activity for membrane-bound COMT and the neuronal marker enzyme glutamate decarboxylase. Similarly the increase in soluble COMT activity paralleled that of the astroglial marker enzyme, glutamine synthetase. These results indicate that the K _m membrane-bound catechol- O -methyltransferase may be localized predominantly in neurons, whereas the high-K_m soluble enzyme is found in glial cells.     

Purification and Characterization of a Membrane-Bound Enkephalin-Forming Carboxypeptidase, "Enkephalin Convertase"

Enkephalin convertase, the enkephalin-synthesizing carboxypeptidase B-like enzyme, has been purified to apparent homogeneity from bovine pituitary and adrenal chromaffin granule membranes. The membrane-bound enkephalin convertase can be solubilized in high yield with 0.5% Triton X-100 in the presence of 1 M NaCl. Extensive purification is achieved by affinity chromatography with p-aminobenzoyl-L-arginine linked to Sepharose 6B. Enzyme purified from both pituitary and adrenal chromaffin granule membranes shows a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis with an apparent molecular weight of 52,500, whereas enkephalin convertase purified from soluble extracts of these tissues has an apparent molecular weight of 50,000. The regional distribution of the membrane-bound enzyme in the rat brain differs from that of the soluble enzyme. While the soluble enzyme shows 10-fold variations, resembling somewhat the enkephalin peptides, membrane-bound enkephalin convertase is more homogeneously distributed throughout the brain. In rat pituitary glands, membrane-bound enzyme activity is similar in the anterior and posterior lobes, whereas the soluble enzyme is enriched in the anterior lobe. Membrane-bound and soluble forms of enkephalin convertase isolated from either bovine pituitary glands or adrenal chromaffin granules show identical substrate and inhibitor specificities. As with the soluble enzyme, membrane-bound enkephalin convertase hydrolyzes [Met]- and [Leu]enkephalin-Arg6 and -Lys6 to enkephalin, with no further degradation of the pentapeptide.     

Association of Ala72Ser polymorphism with COMT enzyme activity and the risk of schizophrenia in Koreans

Catechol-O-methyltransferase (COMT) inactivates circulating catechol hormones, catechol neurotransmitters, and xenobiotic catecholamines by methylating their catechol moieties. The COMT gene has been suggested as a candidate gene for schizophrenia through linkage analyses and molecular studies of velo-cardio-facial syndrome. A coding polymorphism of the COMT gene at codon 108/158 (soluble/membrane-bound form) causing a valine to methionine substitution has been shown to influence enzyme activity, but its association with schizophrenia is inconclusive. We have screened 17 known polymorphisms of the COMT gene in 320 Korean schizophrenic patients and 379 controls to determine whether there is a positive association with a nonsynonymous single-nucleotide polymorphism (rs6267) at codon 22/72 (soluble/membrane-bound form) causing an alanine-to-serine (Ala/Ser) substitution. With the Ala/Ala genotype as a reference group, the combined genotype (Ala/Ser and Ser/Ser)-specific adjusted odds ratio was 1.82 (95% CI=1.19–2.76; P=0.005), suggesting the Ser allele as a risk allele for schizophrenia. However, the Val/Met polymorphism was not associated with an increased risk of schizophrenia in Koreans (OR=0.88, 95% CI=0.64–1.21; P=0.43). The Ala72Ser substitution was correlated with reduced COMT enzyme activity. Our results support previous reports that the COMT haplotype implicated in schizophrenia is associated with low COMT expression.     

Determination of differential activities of soluble and membrane-bound catechol-O-methyltransferase in tissues and erythrocytes

Catechol-O-methyltransferase (COMT) exists as two isoenzymes, a membrane-bound form (MB–COMT) and a soluble form (S–COMT), with different roles in the metabolism of catecholamines and other catechol compounds. This report documents an HPLC assay for separate estimation of S–COMT and MB–COMT activity and examines activities of the two isoezymes among different rat tissues and in human and rat erythrocytes. Activities of MB–COMT and S–COMT varied widely among tissues. There were higher activities of S–COMT than MB–COMT in all tissues except the adrenal medulla where MB–COMT was the predominant isoenzyme, consistent with the importance of this tissue and MB–COMT for the O-methylation of catecholamines. MB–COMT and S–COMT in rat and human erythrocytes showed divergent levels and patterns of activity. The assay represents a rapid and accurate method for quantifying MB–COMT and S–COMT in various tissues and examining the relative roles of COMT isoenzymes in the metabolism of catechol compounds in health and disease.     

O-methylation of (+)-(R)- and (−)-(S)-6,7-dihydroxy-1-methyl-1,2,3,4-tetra-hydroisoquinoline (salsolinol) in the presence of pig brain catechol-o-methyltransferase

(+)-(R)- and (−)-(S)-salsolinol, dopamine-derived tetrahydroisoquinolines, were tested as substrates of pig brain soluble and membrane-bound catechol-O-methyltransferase (COMT) and as inhibitors of O-methylation of dopamine by soluble COMT in vitro. Methylation products were separated by high-performance liquid chromatography with electrochemical detection. Quantification of the products showed that O-methylation of (+)-(R)-salsolinol by soluble COMT afforded the 7-O-methylated product salsoline preferentially, whereas (−)-(S)-salsolinol yielded almost equivalent amounts of the 6- and 7-methyl ethers. Unlike O-methylation by soluble COMT, 7-O/6-O-methylation ratio produced by membrane-bound COMT varied with (+)-(R)-salsolinol concentration. As to the O-methylation of dopamine by soluble COMT, comparable competitive inhibition was observed with both (+)-(R)- and (−)-(S)-salsolinol. Chirality 9:367–372, 1997. © 1997 Wiley-Liss, Inc.     

THE REGIONAL AND SUBCELLULAR DISTRIBUTION OF CATECHOL-O-METHYL TRANSFERASE IN THE RAT BRAIN

Catechol-O-methyl transferase (COMT) activities determined in different regions of rat brain showed small variations. Highest activities were found in the hypothalamus and corpora quadrigemina, and lowest activities in the hippocampus and corpus striatum. The regional distribution of COMT was thus at variance with the distribution of DOPA decar- boxylase in this study and with the distribution of catecholamines and tyrosine hydroxylase reported in the literature. Determinations of the subcellular distribution of COMT in rat forebrain showed that 50 per cent of the activity was recovered in the high speed supernatant fluid and about 33 per cent in the crude mitochondrial fraction. Further separation of the latter by discontinuous sucrose gradients showed that the particulate COMT was found in the synaptosomal fraction in an occluded form. Full enzyme activity was only obtained after treatment with a detergent or after resuspension in water. After hypo-osmotic rupture of the crude mitochondrial fraction, COMT was recovered in the cytoplasmic fraction. The subcellular distribution of COMT was very similar to the ones of lactate dehydrogenase and DOPA decarboxylase. The proportions of soluble COMT obtained from homogenates of various regions of the brain differed from that of choline acetyl transferase and DOPA decarboxylase but were similar to that of lactate dehydrogenase. In conclusion, COMT is a cytoplasmic enzyme almost evenly distributed in the CNS. Its distribution does not resemble the distributions of the catecholamines or of the enzymes participating in the synthesis of catecholamines.     

Changes of Acetylcholinesterase Activity in Brain Areas and Liver of Sucrose- and Ethanol-Fed Rats

The effects of chronic ethanol or sucrose administration to rats on acetylcholinesterase from brain and liver were investigated. Membrane-bound and soluble acetylcholinesterase activities were determined in fractions prepared by centrifugation. The thermal stability and the effects of temperature and different types of alcohols on acetylcholinesterase activity were also studied. Membrane-bound acetylcholinesterase activity increased (p < 0.01) in the liver after chronic ethanol administration, whereas no differences among groups in the encephalic areas, except in the brain stem soluble form, were found. Membrane-bound acetylcholinesterase from the ethanol- and sucrose-treated groups was more stable at the different temperatures assayed between 10 and 50°C than that corresponding to the control group. Non-linear Arrhenius plots were obtained with preparations of membrane-bound acetylcholinesterase from rat liver, with discontinuities at 30°C (control or sucrose groups) or 34–35°C (alcohol group). Assays made with membrane-bound or soluble enzyme from brain showed linear Arrhenius plots in all groups studied. The inhibitory effects of increasing concentrations of ethanol, n-propanol and n-butanol on acetylcholinesterase preparations from forebrain, cerebellum, brain stem and liver of the three experimental groups (control, sucrose-fed and ethanol-fed) were very similar. However, n-butanol displayed a biphasic action on particulate or soluble preparations of rat forebrain. n-butanol inhibited (competitive inhibition) at higher concentrations (250–500 mM), while at lower concentrations (10–25 mM), the alcohol inhibited at low substrate concentrations but activated at high substrate concentration. These results suggest that the liver is more affected by ethanol than the brain. Moreover, the lipid composition of membranes is probably modified by ethanol or sucrose ingestion and this would affect membrane fluidity and consecuently the behaviour of acetylcholinesterase.     

Anti-catechol-O-methyltransferase: Demonstration of specificity and immunological cross-reactivity with the enzyme from rat liver,kidney, brain,and choroid plexuses

An antiserum to rat liver catechol-O-methyltransferase (COMT) was utilized in the immunological characterization of COMT from rat kidney, brain, and choroid plexuses, in addition to rat liver. The presence of anti-COMT activity was confirmed by the direct inhibition of the activity of the enzyme from rat liver by small quantities of the antiserum and by the inhibition of the activity of the enzyme from rat brain. The specificity of the antiserum was demonstrated both by immunoelectrophoresis of rat liver COMT, and by a partial purification of rat liver COMT in which changes in COMT specific activity were correlated with the appearance of a precipitin line in double-immunodiffusion experiments. The antigenic similarity of the enzyme derived from rat liver, kidney, brain, and choroid plexuses was demonstrated by the formation of a precipitin line of identity when preparations from these four tissues were diffused against the antiserum.     

Catechol-o-methyltransferase in rat erythrocyte and three other tissues: comparison of biochemical properties after removal of inhibitory calcium

The biochemical characteristics of soluble catechol-O-methyltransferase (COMT) activity in rat erythrocytes were compared with the properties of the soluble enzyme in rat liver, heart, and brain. COMT was measured by a procedure that avoided artifacts of some other assay procedures including inhibition of the enzyme by endogenous calcium. After the removal of calcium from the reaction mixture the apparent Michaelis-Menten constants for the two cosubstrates of the COMT reaction, S-adenosyl-1-methionine (SAM) and 3,4-dihydroxybenzoic acid (DBA), were similar in tissue preparations of rat liver, brain, heart and blood. The apparent Km values for the four tissues ranged from 5.7 to 6.7 x 10(-6) M and from 0.9-1.4 x 10(-4) M for SAM and DBA, respectively. The optimal pH and the optimal concentration of magnesium for the assay of red blood cell COMT were also similar to those for the enzyme in the three other rat tissues. After the removal of endogenous calcium, COMT activity in all four tissues was inhibited by the addition of calcium, and the [CaCl2] necessary to inhibit the enzyme activity 50% was 3-5 x 10(-4) M in all cases. The relative activities of COMT in the rat heart, brain, erythrocyte, and liver when expressed per g tissue or per ml of packed red blood cells were 1 to 1.15 to 1.58 to 140, respectively.     

Ultracytochemistry as a tool for the study of the cellular and subcellular localization of membrane-bound guanylate cyclase (GC) activity. Applicability to both receptor-activated and receptor-independent GC activity

Membrane-bound guanylate cyclase activity was detected by ultracytochemistry at the electron microscope level in several mammalian tissues. The technique used in these studies allows the detection of active enzyme at the membrane site where it is located. In a few cases, such as normal and regenerating peripheral nerves and placenta, membrane-bound guanylate cyclase could be detected in the absence of stimulators of enzyme activity. However, in the majority of these studies membrane-bound guanylate cyclase was investigated following stimulation with natriuretic peptides, guanylin, or the Ca²⁺ sensor proteins, S100B and S100A1. In general, membrane-bound guanylate cyclase was localized to plasma membranes, in accordance with the functional role of this enzyme. Yet, in secretory cells the enzyme activity was localized on intracellular membranes, suggesting a role of membrane-bound guanylate cyclase in secretory processes. Finally, S100B and S100A1 were found to colocalize with membrane-bound guanylate cyclase on photoreceptor disc membranes and to stimulate enzyme activity at these sites in dark-adapted retinas in a Ca²⁺-dependent manner. The results of these analyses are discussed in relation to the proposed functional role(s) of this enzyme.     

Purification and characterization of membrane-bound inositolpolyphosphate 5-phosphatase

Membrane-bound inositolpolyphosphate 5-phosphatase was solubilized and highly purified from a microsomal fraction of rat liver. Its physiochemical and enzymological properties were compared with those of highly purified preparations of two types of soluble enzyme (soluble Type I and Type II) from rat brain. The molecular masses of the membrane-bound and soluble Type I enzymes were 32 kDa, while that of soluble Type II enzyme was 69 kDa, as determined by molecular sieve chromatography. The membrane-bound and soluble Type I enzymes showed similar broad peaks on isoelectric focusing (pI 5.8-6.4), while soluble Type II enzyme showed multiple peaks in the region between pI 4.0-5.8. All three enzymes required divalent cation for activity. Mg2+ was the most effective for both the membrane-bound and soluble Type I enzymes, while Co2+ enhanced soluble Type II enzyme activity about 1.5-fold relative to Mg2+ at 1 mM. The optimal pH of both the membrane-bound and soluble Type I enzymes was 7.8, while that of soluble Type II was 6.8. The Km values for inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] of all three enzymes were similar (5-8 microM), but those for inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] were quite different, the Km values of membrane-bound and soluble Type I enzymes being 0.8 microM, while that of soluble Type II was 130 microM. These similarities between the membrane-bound and soluble Type I enzymes suggest that these two molecules may be the same protein, and that concentrations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, both of which are considered to play critical roles in the regulation of intracellular Ca2+-concentration, may be differently regulated by two functionally distinct enzymes.     

Steady state kinetics of soluble and membrane-bound mitochondrial ATPase

Steady state kinetic measurements of the rate of hydrolysis of ATP to ADP and inorganic phosphate by beef heart mitochondrial ATPase have been performed with both the solubilized enzyme and with the enzyme attached to a mitochondrial membrane fraction at 25° in 0.1 M NaCl with Mg²⁺ as the metal ion activator. These studies indicate the ATP Michaelis constants are somewhat larger for the soluble enzyme and the turnover numbers are considerably larger. In addition, the steady state parameters are essentially independent of pH over the range 7–9 for the membrane-bound enzyme, while the turnover number for the soluble enzyme varies considerably with pH. The product, ADP, is a competitive inhibitor of ATP and inhibits the soluble enzyme much more strongly than the membrane-bound enzyme. Oligomycin inhibits the membrane-bound enzyme very strongly, but has no effect on the activity of the soluble enzyme. The oligomycin inhibition is noncompetitive in nature.     

Steady state kinetics of soluble and membrane-bound mitochondrial ATPase

Steady state kinetic measurements of the rate of hydrolysis of ATP to ADP and inorganic phosphate by beef heart mitochondrial ATPase have been performed with both the solubilized enzyme and with the enzyme attached to a mitochondrial membrane fraction at 25° in 0.1 M NaCl with Mg²⁺ as the metal ion activator. These studies indicate the ATP Michaelis constants are somewhat larger for the soluble enzyme and the turnover numbers are considerably larger. In addition, the steady state parameters are essentially independent of pH over the range 7–9 for the membrane-bound enzyme, while the turnover number for the soluble enzyme varies considerably with pH. The product, ADP, is a competitive inhibitor of ATP and inhibits the soluble enzyme much more strongly than the membrane-bound enzyme. Oligomycin inhibits the membrane-bound enzyme very strongly, but has no effect on the activity of the soluble enzyme. The oligomycin inhibition is noncompetitive in nature.     

Contribution of Sulfate Conjugation, Deamination, and O-Methylation to Metabolism of Dopamine and Norepinephrine in Human Brain

Abstract: The kinetic constants were determined for dopamine (DA) and norepinephrine (NE) metabolism by phenolsulfotransferase (PST), type A and B monoamine oxidase (MAO), and membrane-bound and soluble catechol- O - methyltransferase (COMT) in frontal lobe preparations of human brain. PST and membrane-bound COMT were found to have the lowest K _m, values for both catecholamines. By means of the appropriate rate equations and the calculated kinetic constants for each enzyme, the activity of each enzymatic pathway was determined at varying concentrations of DA and NE. Results indicate that deamination by MAO is the principal pathway for the enzymatic inactivation of DA whereas NE is largely metabolized by MAO type A and membrane-bound COMT under the in vitro assay conditions used. At concentrations less than 100 μ M , soluble COMT'contributes less than 5% to the total catabolism of either catecholamine. PST can contribute up to 15% of the total DA metabolism and 7% of NE metabolism.     

Selective Effects of Proteases and Phospholipase A2 on Monoamine Oxidases A and B of Human Brain and Liver

Human brain and liver mitochondria contain membrane-bound monoamine oxidase of both A and B types. Monamine oxidase-A (MAO-A), either membrane-bound or in detergent-solubilized extracts from these tissues, was selectively inhibited during incubations with trypsin, chymotrypsin, thermolysin, or papain. MAO-A in solubilized, but not in membrane-bound, preparations was also very sensitive to the action of phospholipase A2, while MAO-B was unaffected. Membrane-bound MAO-A of rat brain mitochondria was more sensitive to phospholipases and less sensitive to proteases than was human brain enzyme, indicating that these agents may reveal species differences in MAO properties. Human brain and liver MAO-A, either solubilized or bound in mitochondrial membranes, apparently contains basic and aromatic peptide moieties that are available to proteases. Hydrolysis of these peptide bonds leads to rapid denaturation unless substrate molecules stabilize the active site. Phospholipase A2 may disrupt the phospholipid microenvironment of MAO-A, the integrity of which is essential for MAO-A activity, but not for MAO-B. No interconversion of the two activities was observed. After phospholipase A2 treatment, remaining MAO-A activity was recovered in low-molecular-weight regions of a gel filtration gradient, suggesting that MAO-A subunits were released. Although these experiments argue against the proposal that phospholipids may regulate the ratio of A/B activities of a single enzyme molecule, it is conceivable that endogenous phospholipases or proteases in mitochondrial membranes may influence MAO-A activity independently of MAO-B activity.     

Purification and Kinetic Mechanism of Human Brain Soluble Catechol-O-Methyltransferase

The soluble form of human brain catechol-O-methyltransferase (EC 2.1.1.6, COMT) has been purified approximately 4,000-fold from a 250,000 X g supernatant solution. The purified enzyme exhibits a molecular weight near 27,500 and a pI value equal to approximately pH 5.0. Initial velocity and product inhibition studies are consistent with an ordered reaction mechanism for soluble COMT. Tropolone, a dead-end inhibitor, exhibited a competitive pattern of inhibition when dopamine (DA) was the varied substrate and an uncompetitive pattern when S-adenosyl-L-methionine (SAM) was the varied substrate. These observations strongly suggest that the soluble form of COMT from human brain catalyzes the O-methylation of catecholamines via an ordered reaction mechanism in which SAM is the leading substrate. Since the membrane-bound form of COMT catalyzes the O-methylation of catecholamines through an identical reaction mechanism, these data provide further evidence that two forms of COMT, while being localized in distinct subcellular compartments, are quite similar in their molecular structure.     

Kinetic properties of gamma-glutamyltranspeptidase and leucine-amino-peptidase in developing rat intestine: effect of hormones

Administration of cortisone and thyroxine produced adult-type increase in the activities of soluble and membrane-bound gamma-glutamyltranspeptidase (gamma-GTP) in suckling rat intestine. Membrane-bound enzyme activity remained unaltered while the soluble enzyme activity was reduced (27%) in insulin-injected pups. Kinetic analysis revealed that the observed changes in the enzyme levels were a consequence of altered Vmax with no change in apparent Km. A 2-fold increase in the Km value was observed in adult gamma-GTP activity compared to that of suckling animals. Membrane-bound and soluble gamma-GTP yielded similar values of the Ea (9.7-13.1 kcal/mole) but exhibited apparent differences in heat stability in the control and hormone-injected groups. Leucine-amino peptidase(LAP) activity was reduced to adult levels in insulin-treated suckling animals. Thyroxine- and cortisone-treatment did not affect soluble activity but significantly (P less than 0.001) augmented the membrane-bound LAP levels. This increase was due to enhanced (54-82%) Vmax with no change in Km. The observed decrease in LAP activity in response to insulin was due to reduced Vmax. There was no change in Ea (8-11.6 kcal/mole) except the value was raised to 19.1 kcal/mole in cortisone-injected pups. Both the soluble and membrane-bound LAP activities were quite resistant to heat inactivation upto 30 min at 60 degrees C except in weanling rats. Thus, the kinetic behaviour of normally developed and precociously induced gamma-GTP and LAP is essentially similar but there are apparent differences in the mode of action of insulin, cortisone and thyroxine in affecting the development of these enzymes.     

Identity of `acid'' β-glucosidase and glucocerebrosidase in human spleen

1. Glucocerebrosidase, in association with a membrane-bound ;acid' beta-glucosidase, was separated from a soluble ;neutral' beta-glucosidase that had no activity towards glucocerebroside as substrate. 2. Glucocerebrosidase, as well as ;acid' beta-glucosidase activity depended upon the association of factor P (a heat-stable, soluble, acidic glycoprotein) with factor C (a heat-labile membrane-bound protein). 3. Factor C was solubilized under certain conditions. 4. Solubilized factor C, as well as membrane-bound factor C, could be alternatively stimulated by sodium taurocholate to give both glucocerebrosidase and ;acid' beta-glucosidase activities. 5. Membrane-bound factor C reacted optimally with factor P whereas solubilized factor C was preferentially stimulated by taurocholate. 6. Factor P-dependent glucocerebrosidase activity differed in kinetic properties from the taurocholate-stimulated enzyme activity. 7. The results are discussed in the light of (a) identity of glucocerebrosidase and ;acid' beta-glucosidase, (b) application in clinical diagnosis, (c) physiological significance of the enzyme system, and (d) polygenic inheritance in adult Gaucher's disease.     

Metabolic relationship between invertase and acid phosphatase isoenzymes in Saccharomyces cerevisiae

Repressed cells of Saccharomyces cerevisiae, subjected to inhibition of both RNA and protein synthesis, showed a pattern of membrane-bound and cytosol acid phosphatase to the external enzyme which seemed to be linked through a precursor-product relationship.Gel exclusion chromatography did not indicate clear differences between the isoenzymes. Moreover, centrifugation experiments in CsCl and precipitation with concanavalin A suggested that there were no acid phosphatase molecules devoid of carbohydrate. Membrane-bound invertase displayed a molecular weight and a carbohydrate to protein ratio smaller than those of the exocellular enzyme. The values of molecular weight and buoyant density of the membrane-bound enzyme were closer to those found for the cytosol invertase. The stability of the level of the soluble invertase detected in the cytoplasm under derepression conditions, or after RNA or protein synthesis inhibition was found to be only apparent and represented the result of an equilibrium between synthesis and degradation.     

Orientation and cellular distribution of membrane-bound catechol-O-methyltransferase in cortical neurons: implications for drug development

Catechol-O-methyltransferase (COMT) is a key enzyme for inactivation and metabolism of catechols, including dopamine, norepinephrine, caffeine, and estrogens. It plays an important role in cognition, arousal, pain sensitivity, and stress reactivity in humans and in animal models. The human COMT gene is associated with a diverse spectrum of human behaviors and diseases from cognition and psychiatric disorders to chronic pain and cancer. There are two major forms of COMT proteins, membrane-bound (MB) COMT and soluble (S) COMT. MB-COMT is the main form in the brain. The cellular distribution of MB-COMT in cortical neurons remains unclear and the orientation of MB-COMT on the cellular membrane is controversial. In this study, we demonstrate that MB-COMT is located in the cell body and in axons and dendrites of rat cortical neurons. Analyses of MB-COMT orientation with computer simulation, flow cytometry and a cell surface enzyme assay reveal that the C-terminal catalytic domain of MB-COMT is in the extracellular space, which suggests that MB-COMT can inactivate synaptic and extrasynaptic dopamine on the surface of presynaptic and postsynaptic neurons. Finally, we show that the COMT inhibitor tolcapone induces cell death via the mechanism of apoptosis, and its cytotoxicity is dependent on dosage and correlated with COMT Val/Met genotypes in human lymphoblastoid cells. These results suggest that MB-COMT specific inhibitors can be developed and that tolcapone may be less hazardous at low doses and in specific genetic backgrounds.