Liver MAO Research Articles

Sensitivity of the mink (Mustela vison) liver monoamine oxidase to isoquinoline alkaloids

MAO. Note that the inhibitory specificity of mink MAO is virtually unstudied. Only one [11] of the available scarce papers on the catalytic properties of this enzyme reports its sensitivity to two acridine derivatives. Analysis of the effects of benzo[c]phenanthridine alkaloids (sanguinarine and chelerythrine), the diisoquinoline alkaloid berberine, and the drugs Ukrain and Sangviritrin on the mink ( Mustela vison ) liver MAO activity, conducted for the first time, is of doubtless interest for expanding the biochemical studies into the properties of enzymes of domesticated fur-bearing animals. Mitochondrial fragments of the American mink ( M. vison Schreb.) liver were the source of MAO. The liver was excised from mature 2-year-old male minks with a weight of 1.5‐1.7 kg kept under standard conditions in the Kondopoga State Fur Plant [12]. The animals were euthanized by decapitation. The mitochondrial membranes were isolated from tissue homogenates partially purified from the ballast protein by extraction in 0.0075 M potassium phosphate buffer pH 7.4 [13]. The mitochondrial fragments were obtained by freezing at ‐20 ° C with subsequent centrifugation and stored frozen until used. The protein content, determined by biuret method, amounted to 17 ± 2 mg/ml. MAO activity was determined spectrophotometrically (at 420 nm) according to the amount of ammonia formed over 30 min in the enzymatic reaction of oxidative deamination of the monoamines— tyramine hydrochloride, benzylamine hydrochloride, and β -phenylethylamine hydrochloride (Sigma, United States)—with serotonin creatinine sulfate (Reanal, Hungary) according to the modified Conway method with subsequent nesslerization [13]. The samples (final volume, 2.5 ml) contained fragments of mitochondrial membranes in an amount corresponding to approximately 1 mg protein, 0.5 ml of inhibitor (tested preparation) at a specified concentration or 0.5 ml of water (in control experiments), 0.5 ml of 0.01 M phosphate buffer pH 7.4, and 0.5 ml of a substrate at a specified concentration (1 mM tyramine, tryptamine, benzy

Structure and mechanism of monoamine oxidase.

Monoamine oxidases A and B (MAO A and MAO B) are mitochondrial outer membrane-bound flavoproteins that catalyze the oxidative deamination of neurotransmitters and biogenic amines. A number of mechanism-based inhibitors (MAOI's) have been developed for clinical use as antidepressants and as neuroprotective drugs. To facilitate the development of more effective and specific inhibitors, a detailed understanding of the structures and catalytic mechanisms of these enzymes is required. The recent development of high level expression systems for producing recombinant human liver MAO A and MAO B in Pichia pastoris has facilitated the determination of the three dimensional crystal structures of MAO B (up to 1.7 angstroms resolution) in complex with different reversible (isatin, 1,4-diphenyl-2-butene) and irreversible inhibitors (pargyline, N-(2-aminoethyl)-p-chlorobenzamide, and trans-2-phenylcyclopropylamine). The binding of substrates or inhibitors to MAO B involves an initial negotiation of a protein loop occurring near the surface of the membrane and two hydrophobic cavities; an "entrance" cavity and an "active site" cavity. These two cavities can either be separate or in a fused state depending on the conformation of the Ile199 side chain, which appears to function as a gate. The amine function of the bound substrate approaches the re face of the bent and "puckered" covalent FAD through an "aromatic cage" formed by two tyrosine residues that are perpendicular to the plane of the flavin ring. No amino acid residues that could function as acids or bases are found near the catalytic site. The existing structural data on MAO B support previous QSAR results and are also supportive of a proposed polar nucleophilic mechanism for MAO A and B catalysis rather than the alternatively proposed single electron transfer mechanism.

Loss of serotonin oxidation as a component of the altered substrate specificity in the Y444F mutant of recombinant human liver MAO A.

To investigate the roles of tyrosyl residues located near the covalent 8alpha-S-cysteinyl FAD in monoamine oxidase A (MAO A) and to test the suggestion that MAO A and plant polyamine oxidase may have structural homology, tyrosyl to phenylalanyl mutants of MAO A at positions 377, 402, 407, 410, 419, and 444 were constructed and expressed in Saccharomyces cerevisiae. All mutant enzymes were expressed and exhibited lower specific activities as compared to WT MAO A using kynuramine as substrate. The lowest specific activities in this assay are exhibited by the Y407F and Y444F mutant enzymes. On purification and further characterization, these two mutants were found to each contain covalent FAD. Both mutant enzymes are irreversibly inhibited by the MAO A inhibitor clorgyline and exhibit binding stoichiometries of 0.54 (Y407F) and 0.95 (Y444F) as compared to 1.05 for WT MAO A. Y444F MAO A oxidizes kynuramine with a k(cat) <2% of WT enzyme and is greater than 100-fold slower in catalyzing the oxidation of phenylethylamine or of serotonin. In contrast, Y444F MAO A oxidizes p-CF(3)-benzylamine at a rate 25% that of WT enzyme. Steady state and reductive half-reaction stopped-flow data using a series of para-substituted benzylamine analogues show Y444F MAO A exhibits quantitative structure activity relationships (QSAR) properties on analogue binding and rates of substrate oxidation very similar to that exhibited by the WT enzyme (Miller and Edmondson (1999) Biochemistry 38, 13670): log K(d) = -(0.37 +/- ()()0.07)V(W)(x0.1) - 4.5 +/- 0.1; log k(red) = +(2.43 +/- 0.19)sigma + 0.17 +/- 0.05. The Y444F MAO A mutant also exhibits similar QSAR properties on the binding of phenylalkyl side chain amine analogues as WT enzyme: log K(i) = (4.37 +/- 0.51)E(S) + 1.21 +/- 0.77. These data show that mutation of Y444F in MAO A results in a mutant that has lost its ability to efficiently oxidize serotonin (its physiological substrate) but, however, exhibits unaltered quantitative structure-activity parameters in the binding and rate of benzylamine analogues. The mechanism of C-H abstraction is therefore unaltered. The suggestion that polyamine oxidase and monoamine oxidase may have structural homology appears to be valid as regards Y444 in MAO A and Y439 in plant polyamine oxidase.

Inactivation of monoamine oxidase B by 1-phenylcyclopropylamine: mass spectral evidence for the flavin adduct

Incubation of 1-phenylcyclopropylamine with bovine liver MAO (MAO B), followed by complete enzymatic digestion to single amino acid residues and subsequent analysis by on-line liquid chromatography–electrospray ionization mass spectrometry, was used to investigate the resulting flavin adduct structure.

High-level expression of human liver monoamine oxidase B in Pichia pastoris.

The high-level heterologous expression, purification, and characterization of the mitochondrial outer membrane enzyme human liver monoamine oxidase B (MAO B) using the methylotrophic yeast Pichia pastoris expression system are described. A 2-L culture of P. pastoris expresses approximately 1700 U of MAO B activity, with the recombinant enzyme associated tightly with the membrane fraction of the cell lysate. By a modification of the published procedure for purification of bovine liver MAO B [Salach, J. I. (1979) Arch. Biochem. Biophys. 192, 128-137], recombinant human liver MAO B is purified in a 34% yield ( approximately 200 mg from 2 L of cell culture). The isolated enzyme exhibits an M(r) of approximately 60, 000 on SDS-PAGE and 59,474 from electrospray mass spectrometry measurements, which is in good agreement with the mass predicted from the gene sequence and inclusion of the covalent FAD. One mole of covalent FAD per mole of MAO B is present in the purified enzyme and is bound by an 8alpha-S-cysteinyl(397) linkage, as identified by electrospray mass spectrometry of the isolated tryptic/chymotryptic flavin peptide. Recombinant human liver MAO B and bovine liver MAO B are shown to be acetylated at the seryl residues at their respective amino termini. The benzylamine oxidase activity of recombinant MAO B ranges from 3.0 to 3.4 U/mg and steady-state kinetic parameters for this enzyme preparation compare well with those published for the bovine liver enzyme: k(cat) = 600 min(-1), K(m)(benzylamine) = 0.50 mM, and K(m)(O(2)) = 0.33 mM. Kinetic isotope effect parameters using [alpha,alpha-(2)H(2)]benzylamine are also similar to those found for the bovine enzyme. Recombinant MAO B exhibits a (D)k(cat) = 4.7, a (D)[k(cat)/K(m)(benzylamine)] = 4.5, and a (D)[k(cat)/K(m)(O(2))] = 1.0. In contrast to bovine liver MAO B, no evidence was found for the presence of any anionic flavin radical either by UV-vis or by EPR spectroscopy in the resting form of the enzyme. These data demonstrate the successful heterologous expression of a functional, membrane-bound MAO B, which will permit a number of mutagenesis studies as structural and mechanistic probes not previously possible.

Evidence for alternative binding modes in the interaction of benzylamine analogues with bovine liver monoamine oxidase B

The interaction of purified bovine liver MAO B with the benzylamine analogues N, N-dimethylbenzylamine and α-methylbenzylamine has been investigated. Both classes of analogues are competitive inhibitors of benzylamine oxidase activity. The K i values were determined for nine different para-substituted N, N-dimethylbenzylamine analogues. Analysis of the binding affinities demonstrate the deprotonated forms of the tertiary amines are preferentially bound to MAO B and the affinity decreases with increasing van der Waals volume of the para-substituent. The correlation for this relation is: Log K i =−0.97±(0.28)σ+(0.75±0.11)(0.1×V w )−4.24±(0.16) α-Methyl benzylamine analogues are also found to be competitive inhibitors of MAO B-catalyzed benzylamine oxidation. Similar K i values were determined using either the S or R stereoisomers. Analysis of the binding affinities of five para-substituted α-methylbenzylamine analogues to MAO B shows the deprotonated form also to be preferentially bound and the affinity is marginally increased with increasing van der Waals volume of the para-substituent: Log K i =−0.71σ−(0.32)(0.1×V w )−3.50 Comparison of these data with that previously published for para-substituted benzylamine binding to MAO B (Walker and Edmondson, Biochemistry 33 (1994) 7088–7098) demonstrates that these benzylamine analogues exhibit differing modes of binding to the active site of MAO B. The presence of an electronic substituent effect in the binding of these two classes of analogues compared with the lack of an observable electronic effect in the binding of benzylamine to MAO B is consistent with the proposal that orientation of the benzyl ring of the bound substrate is responsible for the absence of an electronic substituent effect on the rate of the reductive half reaction (Miller and Edmondson, Biochemistry 38 (1999) 13670–13683).

Structure-activity relationships in the oxidation of para-substituted benzylamine analogues by recombinant human liver monoamine oxidase A.

Monoamine oxidase A (MAO A) plays a central role in the oxidation of amine neurotransmitters. To investigate the structure and mechanism of this enzyme, recombinant human liver MAO A was expressed and purified from Saccharomyces cerevisiae. Anaerobic titrations of the enzyme require only 1 mol of substrate per mole of enzyme-bound flavin for complete reduction. This demonstrates that only one redox-active group (i.e., the covalent FAD cofactor) is involved in catalysis. The reaction rates and binding affinities of 17 para-substituted benzylamine analogues with purified MAO A were determined by steady state and stopped flow kinetic experiments. For each substrate analogue that was tested, the rates of steady state turnover (k(cat)) and anaerobic flavin reduction (k(red)) are similar in value. Deuterium kinetic isotope effects on k(cat), k(red), k(cat)/K(m), and k(red)/K(s) with alpha, alpha-[(2)H]benzylamines are similar for each substrate analogue that was tested and range in value from 6 to 13, indicating that alpha-C-H bond cleavage is rate-limiting in catalysis. Substrate analogue dissociation constants determined from reductive half-reaction experiments as well as from steady state kinetic isotope effect data [Klinman, J. P., and Matthews, R. G. (1985) J. Am. Chem. Soc. 107, 1058-1060] are in excellent agreement. Quantitative structure-activity relationship (QSAR) analysis of dissociation constants shows that the binding of para-substituted benzylamine analogues to MAO A is best correlated with the van der Waals volume of the substituent, with larger substituents binding most tightly. The rate of para-substituted benzylamine analogue oxidation and/or substrate analogue-dependent flavin reduction is best correlated with substituent electronic effects (sigma). Separation of the electronic substituent parameter (sigma) into field-inductive and resonance effects provides a more comprehensive treatment of the electronic correlations. The positive correlation of rate with sigma (rho approximately 2.0) suggests negative charge development at the benzyl carbon position occurs and supports proton abstraction as the mode of alpha-C-H bond cleavage. These results are discussed in terms of several mechanisms proposed for MAO catalysis and with previous structure-activity studies published with bovine liver MAO B [Walker, M. C., and Edmondson, D. E. (1994) Biochemistry 33, 7088-7098].

Influence of age on stress responses to metabolic cage housing in rats.

1. We studied the effect of isolation stress in 3- and 12-month-old rats individually housed in metabolic cages for 7 days. Urine (24 hr) was collected daily from one group of animals of each age. The other group was tested in an open field and on a hot plate on days 1 and 7. 2. Total deambulation in the open-field test was lower in young than in older rats both on day 1 (54.7 +/- 9.9 vs 80 +/- 8.9 crossings/session; P < 0.04) and on day 7 (21 +/- 9 vs 48 +/- 7 crossings per session; P < 0.04) and decreased significantly in the two groups when tested on day 7 (P < 0.03). Latency to paw-licking in the hot-plate test was longer in young than in older animals on day 1 (14 +/- 2 vs 8 +/- 4 sec; P < 0.05) but was similar in the two groups on day 7. 3. Urinary excretions of norepinephrine (NE) and epinephrine (E) were determined by HPLC with electrochemical detection. Urinary NE in day 1 was similar in young and older animals (2627 +/- 828 vs 3069 +/- 598 ng/24 hr). In young animals NE excretion decreased along the study and was significantly (P < 0.02) lower than on day 1 during the last 3 days of the study. Conversely, in older animals urinary excretion of NE remained similar throughout the study. On day 7 urinary excretion of NE in older animals was about two fold that in young rats. Urinary E was similar in young and older rats (341 +/- 127 vs 532 +/- 256 ng/24 hr) on day 1 and showed a tendency to increase throughout the study. 4. Urinary monoamine oxidase inhibitory (IMAO) activity was determined by testing the ability of urine extracts to inhibit rat liver MAO activity in vitro and was higher in young than in older animals throughout the study (day 1, 54.8 +/- 4.2 vs 25.1 +/- 5.1%; P < 0.02). In young rats excretion of IMAO was significantly higher during the last 3 days of the study than on day 1 (P < 0.05). In older animals urinary IMAO showed a tendency to increase at the end of the study. 5. Isolation stress caused by housing rats in metabolic cages results in different behavioral and metabolic responses in young and older animals. Young animals exhibit a lower locomotor and analgesic response and excrete lower amounts of NE and higher IMAO activity in the urine than older rats. The metabolic and behavioral responses to isolation stress are highly dependent on the age of the animals tested. These results should be taken into consideration when designing experiments requiring the use of metabolic cages.

Selective inhibitors of monoamine oxidase. 4. SAR of tricyclic N-methylcarboxamides and congeners binding at the tricyclics' hydrophilic binding site.

Linear [6.6.6] tricyclic moieties whose center ring is made of two atoms of differing size (here primarily thioxanth-9-ones and phenoxathiins) monosubstituted meta to the sulfur by C(O)NHMe include potent and selective inhibitors of monoamine oxidase A. Similarities with effects on SAR of acylamide and of diazapentacyclic substitution on such rings, including positional variables, the requirement for monomethylation (primary and dialkylated amides are inactive and higher monoalkylated amides show little or no potency), and that sulfur is optimally in sulfone form, suggest that binding to the enzyme occurs similarly in each series. No significantly greater rise in blood pressure was found in rats given sufficient 8 to inhibit most brain and liver MAO A and then followed by oral tyramine than was found on administration of tyramine to controls. This is in contrast to a large blood pressure rise in rats pretreated with phenelzine followed by tyramine, and in accord with the belief that an inhibitor selective for MAO A which is reversibly bound to the enzyme and therefore displaced by any ingested tyramine will not lead to the "cheese effect" (hypertension during treatment with MAO inhibitors usually caused by ingestion of foods containing tyramine).

Monoamine oxidase B isolated from bovine liver exists as large oligomeric complexes in vitro.

The quaternary structure and subunit composition of bovine liver monoamine oxidase B (MAO B) was investigated using size-exclusion chromatography, sucrose gradient centrifugation and electron microscopy. Purified enzyme was subjected to Superdex gel filtration column chromatography in the presence of the non-ionic detergents, n-octyl beta-D-glucopyranoside (octyl glucoside) and Triton X-100R-PC (Triton). The specific activity and elution profiles indicate that the enzyme exists as a dimer and preferentially functions as larger oligomeric complexes. Distribution of the oligomeric forms of MAO B was found to be dependent upon protein concentration. Dilution of the enzyme, however, had little or no effect upon the specific activity profiles. In Triton and octyl glucoside, plots of specific activity versus molecular mass displayed a sigmoidal shape. The chromatographic data suggest that detergent-solubilized bovine liver MAO B exists as cooperative oligomeric enzyme complexes. Similarly, sucrose density gradient centrifugation of purified MAO B exhibited a direct correlation between enzyme activity and molecular mass of the MAO complexes. MAO B activity was found to be widely distributed throughout the sucrose gradient and the highest enzyme activity was contained in the high-density sucrose layers. MAO B specific activity is dependent upon the size of the protein complexes and, therefore, oligomerization of the enzyme may play a role in the regulation of MAO B. Transmission electron microscopy of purified MAO B was performed using protein prepared by octyl glucoside extraction. Purified enzyme was applied to Formvar-coated copper grids and negatively stained with methylamine tungstate. MAO-B-specific monoclonal antibody (MAO B-1C2) conjugated to colloidal gold was used as a probe. Contrast enhancement of the electron microscopy data showed that detergent-depleted enzyme tends to aggregate in a linear arrangement of oligomeric complexes. Our data suggest that the MAO B oligomeric complexes are hexamers which contain threefold rotational symmetry. The individual complexes have globular morphology and the hexamers appear to be composed of a trimer of MAO B homodimers.

Regions of the molecule responsible for substrate specificity of monoamine oxidase A and B: a chimeric enzyme analysis.

To examine regions of the monoamine oxidase (MAO, EC 1.4.3.4) molecule responsible for substrate recognition, a series of these enzymes, in which discrete regions in one molecule were substituted by corresponding sequences of the other, were constructed from the cDNAs of rat liver MAO A and MAO B and were expressed in yeast, Saccharomyces cerevisiae. Substrate specificities of the original and chimeric enzymes were examined in terms of the maximum activity (Vmax) and the affinity (Km) for serotonin, beta-phenylethylamine (PEA), and benzylamine. Chimeric enzymes with the amino-terminal portion (about 220 residues) and the amino-terminal and middle portions (about 400 residues) of MAO A and MAO B, respectively, exhibited substantially the same Km values as those of the parent enzymes. Extension of the substitution in the middle portion of a chimeric enzyme to the second half of the amino-terminal portion resulted in conversion of the Km values for serotonin to those of the counterpart. Data on relative Vmax values of the chimeric enzymes for the three substrates revealed that the relative catalytic activities were mainly determined by the presence of the middle portion. We conclude from these observations that the region between about residues 120-220 and about residues 50-400 is responsible for determination of the substrate specificity of MAO A and MAO B, respectively, while the middle portion, of about residues 220-400, may relate to the relative catalytic activity towards substrates.

Dramatic Species Differences in the Susceptibility of Monoamine Oxidase B to a Group of Powerful Inhibitors

Oxadiazolones and oxadiazolethiones are potent, reversible competitive inhibitors of MAO B in rat brain mitochondria. We have compared the Ki values of six of these inhibitors toward MAO B from rat, human and beef liver mitochondria, using benzylamine as substrate. Unexpectedly, their inhibitory potency varies by 3 to 4 orders of magnitude between rat and beef liver MAO B, whereas the inhibition of the rat and human liver enzymes is quite similar. Examples are 5-(4-benzyl-oxyphenyl)-3-(2-cyano-ethyl)-1,3,4-oxadiazole-2(3H)- thione, with Ki at 30° of 0.5 nM for rat, 0.8 nM for human, and 1,830 nM for beef liver mitochondria and 5-(4-benzyloxyphenyl)-3-(2-hydroxyethyl)-1,3,4-oxadiazol-2(3H)- one with Ki values of 1.2, 1.1 and 1,320 nM for MAO B from these three sources. Since solubilized and membrane-bound enzymes had similar sensitivities to the inhibitors, the differences seen must arise from differences in the amino acid sequences of the three enzymes.

CDNA cloning and sequencing of rat monoamine oxidase A: Comparison with the human and bovine enzymes

1. 1. The nucleotide and deduced amino acid sequences of rat liver MAO A were determined, and sequence identities among MAO A and B from rat, human and bovine were compared. 2. 2. MAO A from rat exhibited greater than 85% sequence identity with bovine and human MAO A, and 70% identity with rat MAO B. 3. 3. Rat adrenal cDNAs were restriction mapped, partially sequenced and found to be identical to rat liver MAO A, suggesting that these two tissues express the same polypeptide.

The effect of debrisoquin on MAO A and MAO B activities

To examine the mode of action of debrisoquin (DEB), we studied the effect of this drug in vitro on MAO A and MAO B enzyme activities. DEB was shown to be a competitive inhibitor of highly purified human MAO A and MAO B enzyme activities. DEB inhibited placental MAO A with a Ki value of 0.5 μM and liver MAO B with a Ki value of 8.8 μM, 18-fold greater effect on the A form. Kynuramine was used as substrate for both enzymes. Additional studies using a dilution technique showed that DEB was a reversible inhibitor of both forms of the enzyme. The results of this study show that DEB is a potent competitive and reversible inhibitor of both MAO A and MAO B enzymes.

Molecular basis of mao A and B: cDNA cloning of human liver MAO A and B

Molecular basis of mao A and B: cDNA cloning of human liver MAO A and B

4-Fluoro-3-nitrophenyl azide binding sites on purified beef liver monoamine oxidase B

Previous work from this laboratory has shown that 4-fluoro-3-nitrophenyl azide (FNPA) is an effective photoaffinity labeling probe for MAO-B (Chen et al., Biochem. Pharmac.34, 781-785, 1985). The FNPA binding sites have been further studied by using [(3)H]FNPA. When [(3)H]FNPA was photolyzed with purified beef liver MAO, then subjected to tryptic and chymotryptic digestion, three radioactive peaks were observed after Sephadex G-25 column chromatography procedure. The extent of [(3)H]FNPA incorporation varied directly with [(3)H]FNPA concentration. They could be protected by the presence of the substrate (phenylethylamine) or inhibitors (pargyline and trans-phenylcyclopropylamine) of MAO-B during photolysis. These protections were concentration dependent. Furthermore, the decrease in [(3)H]FNPA labeling in the presence of inhibitors paralleled the decrease in MAO catalytic activity. These results suggest that the FNPA binding sites were related to the active site of MAO-B. Under the same conditions, the separation profiles of [(3)H]FNPA labeled and [(3)H]pargyline labeled tryptic-chymotryptic peptides after Sephadex G-25 column chromatography are distinctly different. This result suggests that FNPA labeling sites may be different from the pargyline binding site. Since pargyline binds to the prosthetic group(-FAD) of MAO, [(3)H]FNPA may label different domains of the active site. This probe may be useful for the characterization of the active site of MAO-B.

Studies on the mechanism of MPTP oxidation by human liver monoamine oxidase B

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its deuterated analogues were oxidized to their corresponding dihydropyridinium species (MPDP +) by preparations of pure human liver MAO B : monoclonal antibody complex to investigate the mechanism of MPTP activation. Lineweaver-Burk plots of initial reaction rates revealed that the K m,app values for the various deuterated MPTP analogues were similar to those of MPTP. In contrast, V max,app values were substantially decreased by substitution of deuterium for hydrogen on the tetrahydropyridinium ring, especially at C-6. Deuterium substitution on the N-methyl group alone did not significantly reduce V max,app. These studies support the interpretation that oxidation of MPTP at the C-6 position on the tetrahydropyridine ring is a major rate-determining step in its biotransformation by MAO B.

Metabolism of the neurotoxin in MPTP by human liver monoamine oxidase B

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was oxidized to dihydropyridine MPDP + and pyridine MPP + by preparations of monoamine oxidase B (MAO B), including pure human liver MAO B:monoclonal antibody complex. K m, app values for MPTP and benzylamine, a preferred MAO B substrate, were 316 and 64 μ M, respectively. 4-Phenyl-1,2,3,6-tetrahydropyridine (PTP), the nor derivative of MPTP, was also a substrate ( K m,app = 221μM). MPDP + MPTP, and MPP +, but not PTP, were found tobe irreversible inhibitors of MAO B. Our studies support the hypothesis that MPTP is oxidized in primate brain by MAO B to MPDP + which is then converted to MPP + a major metabolite found in the substantia nigra.

1240 PLATELET MONOAMINE OXIDASE (MAO) ACTIVITY IN REYE'S SYNDROME (RS)

In patients with RS we have shown that MAO activity was significantly reduced in liver and tyramine, dopamine, norepinephrine, epinephrine, and octopamine were increased in plasma, urine, and cerebrospinal fluid. The purpose of the present study was to determine whether generalized MAO dysfunction is a metabolic abnormality associated with RS. Platelet and liver MAO activities were evaluated by radioenzymatic technique in RS and control patients. Platelet MAO activity was significantly decreased (p<0.025) in RS patients (n=13) [3.3 ± 2.4 nmoles of [3H]4-hydroxy-phenylacetic acid formed x (mg protein)−1 × hr−1] when compared to control patients without liver disease (n=8) [9.8 ± 2.5 nmoles] and in non-RS liver disease patients (n=9) [9.1 ± 2.0 nmoles]. Following recovery platelet MAO levels in RS patients were not significantly different from controls. In contrast, there were no significant differences between hepatic MAO activities in RS patients and those without liver disease or non-RS liver disease. Conclusion: 1) reduced platelet MAO activity is a specific abnormality in RS and represents generalized mitochondrial dysfunction; 2) the pattern of MAO activities in platelet and liver may differentiate RS from other hepatopathologic states; 3) the disturbance of MAO activity may be responsible for hypercatecholaminemia and neurologic dysfunction in RS patients.

Molecular characteristics of a new type, distinct from type A and type B, of monoamine oxidase

Based on identical inhibitor sensitivities bo clorgyline and deprenyl, we previously reported the existence of a new type of MAO in carp liver. Now we compared enzymic properties of MAO in carp brain and liver. MAO activities in both preparations were highly inhibited by reversible inhibitors, in agreement with previous findings on inhibition by irreversible inhibitors, but extents of inhibition by compounds tested were similar regardless of whether 5-HT, tyramine or PEA was used as substrate. Substrate specificities of liver and brain MAO were also similar, and the Km values for these three substrates were respectively similar, indicating the similarity of both enzymes. Inhibitors for pyridoxal phosphate-containing amine oxidases were not effective in inhibiting either liver or brain MAO activities, but when activities were assayed at various tyramine and O2 concentrations,results showed parallel lines. This was also found when O2 tension was varied, indicating that both tissue MAOs react according to the ping-pong mechanism, as shown for mammalian MAO. SDS gel electrophoresis showed 60,000 dalton MW’s for both enzymes. These findings indicate that MAO in carp brain and liver seem to be identical to one another and similar to MAO in mammals, except for inhibitor sensitivity.