Bovine Liver Monoamine Oxidase Research Articles

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).

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.

Structure activity studies of the substrate binding site in monoamine oxidase B

The influence of para and meta substitution of benzylamine analogues on their interaction with bovine liver monoamine oxidase B has been investigated to provide insights into the nature of the substrate binding site. Binding data with para-substituted benzylamine analogues show the are of the binding site about the para position to be hydrophobic and exhibiting some steric constraints. Alkylation of the benzylamine nitrogen with methyl groups results in a dominance of steric constraints about the para-position as an influence on binding. meta-Substitution of the benzylamine ring results in a decreased binding affinity which exhibits a dependence on the van der Waals volume of the substituent indicating steric constraints also occur about this area of the bound substrate. The independence of the rate of enzyme reduction with the nature of the meta-substituent suggests these benzylamine analogues are bound in the substrate site in a manner which optimizes overlap of the pro-R benzyl CH bond with the lone pair orbital on the nitrogen. In contrast, the observed rates of enzyme reduction by para-substituted benzylamine analogues exhibit a dominant steric dependence which suggests the mode of binding of this class of analogues does not provide this optimal overlap for efficient CH bond cleavage. Support for this conclusion also comes from the observation that para-substituted N,N-dimethylbenzylamine analogues are competitive inhibitors and not substrates for monoamine oxidase B while the meta-substituted analogues are substrates. albeit poor ones. The demonstration of a tunneling contribution to the CH bond cleavage step demonstrates the absence of any motion or changes in solvation coupled with that catalytic event and the close proximity of the enzyme group accepting the H to the pro-R position of the bound substrate. Little or no influence of meta or para benzylamine substituent on the rate of O 2 reaction with the reduced flavin-protonated imine complex is observed which suggests alterations in the configuration of the bound substrate do not influence the reactivity of the reduced flavin.

Spectral and kinetic studies of imine product formation in the oxidation of p-(N,N-dimethylamino)benzylamine analogs by monoamine oxidase B

The oxidative deamination of p-(N,N-dimethylamino)benzylamine and N-methyl-p-(N,N-dimethylamino)benzylamine by bovine liver monoamine oxidase B has been investigated by absorption spectral, steady-state, and stopped-flow kinetic studies. An absorbing intermediate with a maximum at 390 nm is observed with either analogue in turnover experiments at neutral pH and is identified as due to the formation of protonated imine as the initial product. p-(N,N-Dimethylamino)benzaldehyde is the final product formed from either substrate analogue. Anaerobic stopped-flow measurements show N-methyl-p-(N,N-dimethylamino)benzylamine to reduce enzyme-bound flavin with a limiting rate of 1.8 s-1 concurrent with the appearance of a 390-nm absorption due to protonated imine product with a limiting rate of 1.7 s-1. Both observed rates are somewhat faster than catalytic turnover (1.5 s-1). Under anaerobic conditions, the decay of protonated N-methyl-p-(N,N-dimethylamino)benzenimine is much slower than turnover (k = 4.8 x 10(4) s-1). p-(N,N-Dimethylamino)benzylamine reduces the enzyme with a limiting rate of 2.1 s-1, which is faster than catalytic turnover (1.2 s-1). Protonated imine formation is also observed with this substrate with an apparent limiting rate of 1.3 s-1. The decay of the protonated p-(N,N-dimethylamino)benzenimine absorbance is slower than catalytic turnover but faster than the rate of aldehyde formation under anaerobic conditions. Deuterium kinetic isotope effect values of approximately 10 are observed both for flavin reduction and for protonated imine formation. No isotope effect is observed for the rate of imine decay.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of the Catharanthus alkaloids: from Streptomyces griseus to monoamine oxidase B.

More than three decades after their discovery and implementation in medicine, essentially nothing is known about the metabolism or the implications of metabolism in mechanism of action or toxicity of the Catharanthus alkaloids. The frustrating paucity of information about pathways of metabolism has limited a major source of structure-activity relationship information and has blocked a critical avenue necessary for the logical development of new and more useful Catharanthus alkaloids. Microbial transformations, peroxidases, copper oxidases, mouse and rat cytochrome P-450 systems, and mouse brain and bovine liver monoamine oxidase (MAO) preparations have been explored in the study of Catharanthus alkaloid metabolism. In this report, we present results which have clarified the involvement of enzymatic and chemically catalyzed one-electron oxidations that yield nitrogen-centered cation radicals, iminium, and carbinolamine intermediates, all of which explain how new carbon-carbon and carbon-oxygen bonds form, or break and rearrange. The dimeric Catharanthus alkaloids are recalcitrant to oxidations catalyzed by monoamine oxidases and to both normal and induced P-450 rat microsomal preparations. However, the Catharanthus alkaloids appear to be selective reversible inhibitors of MAO-B. Chemical and biochemical aspects of the metabolic transformations of dimeric Catharanthus alkaloids are reviewed together with the implications of our findings.

Partial amino acid sequence analysis of human placenta monoamine oxidase A and bovine liver monoamine oxidase B

We have prepared peptide maps from human placenta monoamine oxidase type A (MAO-A) and bovine monoamine oxidase type B (MAO-B) and determined the amino acid sequences of 21 of these peptides. These sequences have been compared to the cDNA deduced amino acid sequences of human MAO-A and -B. A result of special interest is the identification of two sets of MAO-A peptides which have sequences different from those deduced from cDNA sequences. This observation is consistent with the notion that MAO-A may be composed of at two subunits which are similar but not identical in primary amino acid sequence.

Interaction of spin-labeled tryptamine with monoamine oxidase: probing the microenvironment of the active site by spin probe-spin label techniques

The spin-labeled substrate, tryptamine, was used as a structural probe of the active site of bovine liver monoamine oxidase B (amine: oxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4). When the reaction was monitored by electron spin resonance (ESR), line broadening effects indicative of binding with an apparent relation to substrate specificity of the highly purified enzyme were observed. The spectrum indicated that the bound tryptamine was ‘partially immobilized’ with a dissociation constant of 39 μM and 2.2 mol bound per enzyme dimer. The correlation time, reflecting the environment of the tryptamine binding site, was determined to be 6.2 ns. The topology of the active site was investigated by using dual spin-label methodology in which the spin-labeled substrate, tryptamine, and the 15N-substituted and deuterated maleimide spin label covalently bound to the essential sulfhydryl groups were used. The ESR spectral data suggested that the essential sulfhydryl groups are at least 14 Å away from the tryptamine-binding site. The environment surrounding both spin-labeled substrates, tryptamine and [ 2H, 15N]MSL, and the motional properties of the enzyme are discussed.

Stopped-flow studies on the mechanism of oxidation of N-methyl-4-phenyltetrahydropyridine by bovine liver monoamine oxidase B.

The kinetic mechanism of monoamine oxidase B involves either a binary or a ternary complex, depending on the substrate. In this study, stopped-flow kinetic data provide direct evidence for ternary complexes not only of reduced enzyme, oxygen, and product but also of reduced enzyme, oxygen, and substrate, both for benzylamine and for the tertiary amine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, the mechanism for a given substrate is not exclusive but, rather, is determined by competition between the alternate pathways as a result of different rate constants for the oxidation of the reduced enzyme, the reduced enzyme-product complex, and the reduced enzyme-substrate complex, as well as the different dissociation constants for the complexes. Comparison of the rate constants obtained from the stopped-flow studies with steady-state data indicates that the overall rate of reaction for the oxidation of MPTP by monoamine oxidase is dominated by the reductive step, but for benzylamine the steady-state rate is determined by a complex function of the rates of both the reductive and oxidative half-reactions.

ESR analysis of the FAD in bovine liver monoamine oxidase

Two hydrazine spin labels, 1-oxyl-2,2,5,5-tetramethylpyrroline-3-carbonyl ethyl hydrazine and 1-oxyl-2,2,6,6-tetramethylpiperidino-4-hydrazine, were synthesized as probes of the FAD binding site of monoamine oxidase. The reporter nitroxide moiety showed an ESR spectrum classified as partially immobilized which is indicative of FAD near the surface of the enzyme. Attempts to pick up flavin semiquinone or free radical intermediates during substrate oxidation with the spin traps 5,5-dimethyl-1-pyrroline-1-oxidase and phenyl-t-butylnitrone were not successful.

Investigations of the mechanism of selective inhibition of type B mitochondrial monoamine oxidase by phosphatidylserine

Liposomes of phosphatidylserine (PS) were found to inhibit strongly the B-form of membrane bound monoamine oxidase (MAO) isolated from rat and bovine liver, while having no effect on the rat liver A-form. Use of 14C-liposomes demonstrated high levels of PS association with the membrane, which could not be removed by extensive washing with high ionic strength buffers. The inhibition of MAO-B was not reversed on further perturbation of the membrane by chaotropic agents, sonication, or treatment with additional liposome preparations of phosphatidylcholine or phosphatidylinositol. Partial reversal of the inhibition was found when the PS-treated bovine liver membrane was solubilizcd with the detergent octyl glucoside. PS, however, had no effect on a solubilized preparation of bovine liver MAO. These results suggest a specific interaction between MAO and PS rather than an indirect effect of bulk changes in membrane properties, but an intact membrane was. nevertheless, required to mediate the inhibition. Comparison of the decreases in apparent levels of MAO-B in rat liver mitochondrial membranes that were calculated from changes in relative catalytic activities with A and B specific substrates or changes in sensitivity to A-form specific reversible and irreversible inhibitors. all showed good quantitative correlation. Lineweaver-Burk plots of the effect of PS incorporation into bovine liver mitochondrial membranes on MAO oxidation of phenylethylamine exhibited the expected pattern for a noncompetitive inhibitor acting on a ping-pong mechanism bireactant enzyme. On the basis of these results, a possible in vivo role for the acidic phospholipids in regulating apparent levels of MAO from one tissue to another and/or in response to environmental effects is proposed.

Bovine liver mitochondrial monoamine oxidase is not an iron-dependent enzyme.

It has been reported that rat liver monoamine oxidase is an iron-dependent enzyme. Calculations from reported data suggest that rat liver monoamine oxidase contains 2 g atoms of iron/mol of enzyme. However, our data over a 16-year period show that our purified bovine liver monoamine oxidase does not contain sufficient iron to justify being considered an iron protein. In order to ensure the correctness of the iron content, two microchemical analyses and atomic absorption spectroscopy were used to determine the iron content of wet ashed samples. The iron content was also determined during successive steps of enzyme purification and was found to decrease rather than increase. Monoamine oxidase, being a hydrophobic outer membrane enzyme, is very difficult to purify and detailed tests for homogeneity are necessary. Our preparations show a single band when examined by sodium dodecyl sulfate-polyacrylamide disc electrophoresis and behave as a pure protein by end group analysis. Thus, bovine liver monoamine oxidase is not an iron-dependent enzyme.

Iron content and spectral properties of highly purified bovine liver monoamine oxidase

Centrifugation of monoamine oxidase preparations from bovine liver mitochondria in sucrose gradients enabled the removal of most of the iron containing impurities without loss of catalytic activity. The nonheme iron content of the enzyme purified with a gradient was found to be not more than 5%, and the total iron not more than 25% of the flavin content of the enzyme. Thus, iron is not a component of monoamine oxidase. Most of the iron present was in the form of cytochrome impurities, the presence of which, in less pure preparations, obscured the observation of a red semiquinone formed on reduction of the enzyme with sodium dithionite. This semiquinone was not found on substrate reduction.

Bovine liver monoamine oxidase: A modified purification procedure and preliminary evidence for two subunits and one FAD

Bovine liver mitochondrial monoamine oxidase was isolated in a more active state and in higher yields by an improved purification method which utilized β-mercaptoethanol and which contained several other important modifications. The subunit structure of the purified enzyme components was investigated by chemical and enzymatic methods. The subunit molecular weight of the three enzyme components isolated was estimated to be 52,000 by sodium dodecyl sulfate disc electrophoresis and by exclusion-diffusion chromatography on Biogel A-5m with 6 m guanidine HCl as the solvent. The number of peptides observed in the peptide map of the tryptic digest of the S-β-carboxymethylcysteine derivative of the enzyme also showed that the subunit molecular weight was about 52,000. Since it was previously reported that the monomer molecular weight of the enzyme was about 110,000, the active enzyme is made up of two subunits. The NH 2-terminus of the enzyme of both subunits is blocked since Edman degradation and aminopeptidase failed to release an NH 2-terminal amino acid. The COOH-terminal amino acid of both subunits was shown to be leucine by carboxypeptidase digestion of the enzyme since it was liberated quantitatively. From the FAD content of the enzyme and the subunit data, it is proposed that the enzyme probably consists of two subunits which differ possibly in that only one subunit contains 8-α-cysteinyl FAD.

The reaction of sulfhydryl reagents with bovine hepatic monoamine oxidase: Evidence for the presence of two cysteine residues essential for activity

The bovine liver monoamine oxidase (EC 1.4.3.4) was found to be inactivated by various well-known sulfhydryl reagents like p- mercuribenzoate , methylmercuric idodide and 5,5′-dithiobis-(2-nitro benzoic acid). The present investigation shows that the inactivation of the enzyme results from reactions of these reagents with 2 out of 8 titratable sulfhydryl groups per 10 5 g of the enzyme. The substrate, benzylamine, and competitive inhibitors like benzaldehyde, p- nitrobenzaldehyde , benzyl alcohol protected the enzyme from inactivation by the mercurials or the Ellman reagent. The inactivation experiments with these sulfhydryl reagents, the protection experiments, and the kinetics as well as physicochemical observations suggest that there are only two cysteine residues that are required for activity of the enzyme. It is possible that these two residues may be active-center residues.

Separation of two monoamine oxidases from bovine bran

1. Two forms of monoamine oxidase have been separated from bovine brain mitochondria. The two enzymes differ in substrate specificity, sensitivity to inhibitors and are immunologically distinct. 2. Monoamine oxidase A does not cross-react with an antibody against bovine liver monoamine oxidase. It catalyzes the oxidation of tyramine, tryptamine and the biogenic amines norepinephrine, serotonin and dopamine. Benzylamine is very slowly oxidized. Harmaline inhibits the enzyme at 10−7 M concentration, amphetamine at 10−4 M. 3. Monoamine oxidase B is precipitated by an antibody against bovine liver monoamine oxidase but the precipitate is enzymatically active. The enzyme catalyzes the oxidation of benzylamine as well as tyramine, tryptamine and dopamine, whereas little or no activity was found with norepinephrine, or serotonin, as substrates. The enzyme is inhibited by pargyline and is relatively insensitive to harmaline. 4. Some of the physiological implications of these findings are discussed.