From the
Department of Biochemistry and the
Microchemical and Proteomics Facility, Emory
University School of Medicine, Atlanta, Georgia 30322
Received for publication, April 9, 2003 , and in revised form, May 22, 2003.
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ABSTRACT |
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INTRODUCTION |
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Human MAO A and MAO B are encoded by separate genes and share a sequence
homology of 70% (3).
Activity of either enzyme is sensitive to thiol-modifying reagents
(4,
5), and cysteine residues have
been implicated in the catalytic mechanism of MAO based on the results of
site-directed mutagenesis (6,
7), thiol titration
(8), and inhibitor binding
(9) experiments. Human MAO A
and MAO B each contain 9 cysteine residues with 7 of them in highly conserved
positions (Scheme 1; A165/B156,
A201/B192, A306/B297, A321/B312, A374/B365, A398/B389, and A406/B397)
(3).2
In each enzyme, one conserved cysteine residue (Cys406 in MAO A and
Cys397 in MAO B) is linked in a thioether bond to the
8
-methylene of FAD (10,
11), therefore leaving 8
cysteine residues either as free thiols or in disulfide bonds. Gomes et
al. (8) reported that 2
cysteine residues in bovine liver MAO B are protected from reaction with thiol
probes by substrates, concluding that these 2 residues are located to the
active site and essential for enzyme catalytic activity. The proximity of
Cys365 to the catalytic site in bovine MAO B was proposed by Zhong
and Silverman (9) by
demonstrating this residue to be modified on inhibition of bovine liver MAO B
with N-cyclopropyl-
-methylbenzylamine. Mutagenesis experiments
by Wu et al. (6) have
shown Ser mutations of Cys374 and Cys406 to abolish MAO
A activity and Ser mutations of Cys156, Cys365, and
Cys397 to abolish MAO B activity. Mutations of the other cysteine
residues were shown not to alter catalytic activities of either enzyme
(6). Cesura et al.
(7) found that MAO B activity
is decreased significantly when Cys365 is mutated to Ala. The
recently solved crystal structure of MAO B
(12) reveals that
Cys365 and Cys156, previously suggested to be involved
in catalytic mechanism (6,
7,
9), are located on the surface
of the molecule.
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A recent study (13) on the stoichiometry of reduction of bovine MAO B or of recombinant human liver MAO A by sodium dithionite led to the conclusion that each enzyme contains a redox active disulfide group, which was suggested to function catalytically in shuttling reducing equivalent between the amine substrate and the FAD. This suggestion has been questioned because anaerobic titration of either enzyme requires stoichiometric levels of substrate to reduce the enzyme-bound flavin coenzyme (14, 15).
To address these uncertainties that exist in the literature regarding the role of thiol groups in MAO A and MAO B structure and function, a detailed study was conducted using purified enzyme preparations. The availability of large scale quantities of purified recombinant, fully functional human MAO A (14) and MAO B (15) in our laboratory facilitated this approach. Both enzymes were examined for the presence of disulfide bonds using mass spectrometry by analyzing the thiol-modified intact enzymes prior to and after any disulfide bond reduction. Cysteine reactivities in functional purified enzymes are also compared before and after inactivation by clorgyline or pargyline, their respective acetylenic inactivators (1618). The level of resolution achieved by electrospray ionization quadrupole mass spectrometry allows for monitoring of the distribution of reacted thiol groups in the enzymes (19). In addition, the positions of reactive cysteine residues were determined. The results reported in this manuscript demonstrate that neither MAO A nor MAO B contains any disulfide bonds and that despite the differences in cysteine reactivities between the two enzymes in their native states, cysteine reactivities of enzymes inactivated with clorgyline or pargyline are similar and also provide further documentation for the suggested binding mode of MAO B to the outer mitochondrial membrane.
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EXPERIMENTAL PROCEDURES |
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Enzyme PreparationBoth human recombinant MAO A and MAO B
were expressed in Pichia pastoris and purified as described
previously (14,
15); MAO B was kindly provided
by Dr. P. Newton-Vinson and MAO A by Min Li in our laboratory. Prior to each
experiment, the enzymes (12 mg) were desalted using a G-25 (fine)
Sephadex column (1 x 20 cm) in 50 mM potassium phosphate
buffer, pH 7.5, containing 0.8% (w/v) -octylglucopyranoside (Buffer
A).
Disulfide Bond AnalysisTo identify the number of cysteine residues reacting with NEM in the fully reduced form of the enzyme, MAO A (10 µM) was first incubated for 30 min at 25 °C in Buffer A containing 2 M guanidine HCl and 4 mM reduced dithiothreitol; NEM was then added to a final concentration of 11 mM. One hour following the addition of NEM, the reaction was quenched with 70 µl of formic acid/N,N-dimethylformamide/water (23%/70%/7% v/v/v, Solution B), desalted by microbore reversed-phase HPLC, and analyzed by ESI-MS (14, 15). The number of cysteine residues reacting with NEM in the native (unreduced, as isolated) form of the enzyme was identified by incubating MAO A (10 µM) with 100 µM NEM at 25 °C for 2 h in Buffer A containing 2 M guanidine HCl. The reaction was quenched with 70 µl of Solution B and analyzed by microbore HPLC and ESI-MS. The same experiments were performed with human recombinant MAO B.
N-Ethylmaleimide or Biotinyl-NEM ModificationAliquots of 8 mM solutions of NEM or Biotinyl-NEM were added to the enzyme (10 µM in Buffer A) to a final concentration of 800 µM (MAO-to-NEM ratio of 1:80), and the resulting solutions were incubated at either 0 °C or 25 °C. At specified times, 5 µl aliquots of the reaction mixtures were analyzed for catalytic activity using the kynuramine assay for MAO A (14) or the benzylamine assay for MAO B (15), and 100-µl aliquots of the reaction mixtures were quenched with 70 µl of Solution B and stored at 20 °C pending HPLC and MS analyses.
Tryptic DigestionDried HPLC-purified Biotinyl-NEM-modified
MAO was redissolved in 1 µl of 80% formic acid and immediately diluted with
10 µl of 50% aqueous isopropanol and 90 µl of 0.1 M ammonium
bicarbonate. The pH of this solution was adjusted to 8 by adding 5%
ammonium hydroxide. Aliquots of trypsin (1 µg; Promega, Madison, WI,
sequencing grade) were added to a final trypsin-to-MAO ratio of
1:30
(mol/mol), and the mixture was incubated in the dark at 37 °C for 24
h.
Streptavidin Affinity ChromatographyTo isolate Biotinyl-NEM modified peptides, the tryptic digests were applied to a streptavidin affinity cartridge (Applied Biosystems) according to the manufacturer's protocol. The resulting eluates were analyzed by ESI-LC-MS using a Zorbax SB-C18 (0.5 x 150 mm; Agilent Technologies) capillary HPLC column or by SELDI-MS. Samples for SELDI-MS analysis in 50% isopropanol, 30% formic acid were loaded onto protein chips (H4 protein chips; Ciphergen Biosystems, Fremont, CA) and washed with 0.1% aqueous formic acid prior to sinapinic acid (matrix) addition. (These conditions were found to increase the recovery of hydrophobic peptides.) In some experiments, an internal standard biotinylated peptide (ALSEGC(Biotinyl-NEM)TPYDIN; molecular mass = 1826.6 Da, maleamic acid form; molecular mass = 1808.6 Da, maleimide form) was added (100 pmol) to the MAO digest prior to streptavidin chromatography.
Biotinyl-NEM Modification of MAO A and MAO B in Intact
MitochondriaIntact mitochondria were isolated from P.
pastoris overexpressing either MAO A or MAO B as described earlier
(20). Intact mitochondria
containing 1 unit of either MAO A or MAO B in Hepes buffer (10 mM
Hepes, pH 7.4, containing 1 mM phenylmethylsulfonyl fluoride) were
incubated with 20-fold molar excess of clorgyline and pargyline, respectively,
on ice for 20 min, following which the suspensions were centrifuged at 20,000
x g for 10 min. After washing the pellet with Hepes buffer, the
mitochondria were resuspended in Hepes buffer and incubated with Biotinyl-NEM
(final concentration, 1 mM; 1/100 mol/mol MAO/Biotinyl-NEM)
for 15 h at 4 °C. The excess Biotinyl-NEM was removed by centrifugation
(20,000 x g for 10 min). The mitochondria were than resuspended
in SDS-PAGE sample buffer, and mitochondrial proteins were separated using
SDS-PAGE (7.5% gel) and stained with Coomassie Brilliant Blue. MAO-containing
bands were cut out of the gel and digested with trypsin
(21) prior to streptavidin
affinity chromatography and MS analyses (as described above). For Western blot
analysis of biotinylated proteins, the gels were electro-blotted onto a
nitrocellulose membrane, and the membrane was incubated with streptavidin
conjugated to alkaline phosphatase (Pierce) and developed using a
5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (Sigma).
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RESULTS |
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Kinetics of NEM ModificationMAO A and MAO B inactivation
with sulfhydryl-reactive reagents proceeds with different kinetic time courses
(Table I). MAO A is inactivated
by an 80-fold molar excess of NEM at 25 °C with a
of
2 min, whereas MAO B is inactivated with a
of
8 h under the same conditions. At 0 °C, MAO A is inactivated with a
of
3 h, and no inactivation of MAO B is observed
(
> 100 h). MAO B is inactivated at a faster rate
(
=
3 h) when an 800-fold excess of NEM was used
at 25 °C. All of the enzyme inactivations exhibit pseudo-first-order
kinetic behavior, with the exception of MAO A inactivation at 25 °C, which
is biphasic, in agreement with previous reports
(13).
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The ability of quadrupole ESI-MS to resolve mass differences corresponding
to the addition of single NEM (125 Da) allowed the monitoring of the time
course of the reaction. MAO A reacts with >7 NEM molecules during 1 h of
incubation at 25 °C (Fig.
2A), whereas clorgyline-inactivated MAO A reacts with 5
NEM molecules over a 2-h period (Fig.
2B). Two clusters of modified MAO B are observed after 24
h of incubation with NEM at 25 °C, a low stoichiometry cluster (14
NEM groups added) and a high stoichiometry cluster (810 NEM groups
added) (Fig. 2C).
These results suggest that once MAO B is modified with up to 4 NEM groups, it
is structurally altered to promote further modification, resulting in the
fully S-alkylated (8 NEM groups) and "overmodified"
enzyme (>8 NEM groups). Pargyline inactivation of MAO B prevents the
formation of the high stoichiometry cluster, suggesting that pargyline binding
to MAO B prevents the structural changes necessary for modification of all of
its thiol groups (Fig.
2D). Similar results to these reported above for MAO A
were observed when MAO B was incubated with an 800-fold excess of NEM at 25
°C for 5 h (data not shown).
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These results suggest that clorgyline and pargyline inactivations of MAO A and MAO B, respectively, provide additional structural stability for each enzyme. This is demonstrated by modification of three more cysteine residues in MAO A as compared with clorgyline-inactivated MAO A (Fig. 2, A and B) and, similarly, by modification of five more cysteine residues in MAO B as compared with pargyline-inactivated MAO B (Fig. 2, C and D). In some cases, residues other than cysteine are modified by NEM, resulting in instances where MAO B is modified by more than 8 NEM groups (Fig. 2C), especially when large excess of NEM over MAO B is used (data not shown). This finding is not surprising because NEM has previously been reported to react (albeit at slower rates) with residues such as histidine, lysine, and tyrosine when used in large excess (22). Under all experimental conditions used, cysteine residues in MAO A are more reactive than cysteine residues in MAO B.
Kinetics of Biotinyl-NEM ModificationAlthough NEM is
suitable for detailed kinetic studies, we found it difficult to directly
analyze NEM-modified cysteine-containing tryptic peptides of MAO A and MAO B
because of their hydrophobicity. Therefore we used water-soluble EZ-link
Biotinyl-NEM (Scheme 2) to aid
in the identification of specific cysteine reactive sites. Biotinyl-NEM was
used to modify reactive cysteine residues because it facilitates rapid and
selective purification of the labeled peptides using streptavidin
chromatography (see below). The inactivation half-times for MAO A are
=
5 min at 25 °C and
=
14 h at 0 °C when the enzyme was incubated
with an 80-fold excess of Biotinyl-NEM. Under the same conditions, MAO B is
very slow to inactivate at either temperature (
>
100 h). Multiple cysteine residues (up to 7) in MAO A react with Biotinyl-NEM
after 24 h of incubation at 0 °C, whereas only a single cysteine residue
in clorgyline-inactivated MAO A reacts with Biotinyl-NEM under the same
reaction conditions (Fig. 3). A
single cysteine residue in MAO B and pargyline-inactivated MAO B reacts with
Biotinyl-NEM under the same conditions
(Fig. 3). Despite the large
differences in cysteine reactivities between MAO A and MAO B, both the
clorgyline- and pargyline-inactivated enzymes react with only a single
Biotinyl-NEM group, suggesting that clorgyline binding to MAO A stabilizes the
MAO A structure. Biotinyl-NEM reacted with fewer cysteine residues than NEM
under identical conditions. This is most likely due to its size and
hydrophilic properties, which diminish its access to the protein core.
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Identification of Modified Cysteine ResiduesTo determine
the locations of the single Biotinyl-NEM-modified cysteine residues in
clorgyline-inactivated MAO A and pargyline-inactivated MAO B
(Fig. 3), biotinylated tryptic
peptides were enriched by streptavidin affinity chromatography and analyzed by
either LC-ESI-MS or SELDI-MS. The use of SELDI-MS, which allowed for the
deposition of large sample volumes onto the SELDI sample plate and subsequent
sample desalting, has proved essential for the analyses. Peptide
240267, containing Cys266, was identified as the only
peptide modified by Biotinyl-NEM in clorgyline-inactivated MAO A
(Fig. 4). A relatively high
background is observed in this spectrum, which is due to the presence of
unmodified MAO A tryptic peptides that were retained throughout the
purification, likely because of their hydrophobicity. Upon the first analysis
of peptides from a singly biotinylated MAO B by SELDI-MS, no biotinylated
peptide was identified, although the internal standard was readily observed.
Only after detailed analysis of minor species present in the mass spectrum,
peptide 521, containing Cys5, was identified as the only
peptide modified by Biotinyl-NEM in pargyline-inactivated MAO B
(Fig. 4). None of the other
minor species in the spectrum correspond to a biotinylated MAO B peptide. The
low recovery of this peptide is not surprising given its very nonpolar nature
(CDVVVVGGGISGMAAAK). In addition, because of hydrolysis of the maleimide ring
(23), two forms of this
peptide were detected resulting in even lower recovery (maleimide form,
molecular mass = 2060 Da, 60%; maleamic acid form, molecular mass = 2078
Da,
40%, Fig. 4). In
contrast, the biotinylated peptide of MAO A was almost completely recovered in
its maleamic acid
form.3
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Structural Equivalence of Cys5 (MAO B) and Cys266 (MAO A)Although Cys5 in MAO B and Cys266 in MAO A are not aligned in the amino acid sequences of the two MAOs, they show similar reactivities toward Biotinyl-NEM. The recently solved x-ray structure of MAO B (12) reveals that Cys5 (MAO B) is located on the surface of the molecule opposite to the membrane-binding region. Cys266 (MAO A) aligns with Ala257 (MAO B) when the MAO A and MAO B sequences are aligned using a Clustal X algorithm using the Blosum 30 matrix (24). Ala257 is located in the same region of the molecule as Cys5 (Fig. 5), suggesting that Cys5 in MAO B and Cys266 in MAO A are structurally equivalent. The slower rate of modification of Cys266 in MAO A relative to Cys5 in MAO B (Fig. 3) could be explained by steric hindrance of Cys266 by Phe14 in MAO A (Fig. 5). In contrast, the thiol group of Cys5 in MAO B is quite accessible to solvent.
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Analysis of MAO A and MAO B in Intact MitochondriaTo
determine whether the conclusions from the above experiments performed with
detergent-solubilized enzymes (in vitro) hold true for the
membrane-bound forms, we performed similar modification experiments using
intact mitochondria from P. pastoris overexpressing either enzyme. In
this expression system, >95% of either enzyme produced is bound to the
yeast mitochondrial membrane (data not shown). SDS-PAGE analysis of the intact
mitochondria shows that either overexpressed enzyme accounts for >80% of
the total mitochondrial protein (Fig.
6). Cysteine residues of either enzyme react with Biotinyl-NEM
when intact mitochondria are incubated with the reagent as demonstrated by
Western blots of mitochondrial proteins developed with streptavidin conjugated
to alkaline phosphatase (Fig.
6). In addition to MAO (that represented the most intense band),
as expected, several other proteins were also biotinylated, including a
90-kDa protein. Given the relative intensities of the protein and biotin
staining, the stoichiometry of Biotinyl-NEM modification of MAO (1:1) is low
relative to the 90-kDa protein. This is probably due to a larger number of
cysteine residues in the 90-kDa protein that are accessible to Biotinyl-NEM
modification. No attempts were made to identify the 90-kDa protein.
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If the MAO structure in the mitochondrial membrane is the same as the structure of purified enzyme in detergent solution, then the same cysteine residues should react with Biotinyl-NEM in either case. Indeed, a biotinylated MAO A peptide 240267 containing Cys266 was identified using SELDI MS analysis of a MAO A in-gel tryptic digest (Fig. 7). However, no biotinylated peptide could be identified by SELDI analysis of a MAO B in-gel tryptic digest (Fig. 7). The expected peptide 521 is likely to be extracted from the gel in very low yields because of its hydrophobicity (CDVVVVGGGISGMAAAK). However, evidence of this biotinylated MAO B peptide containing Cys5 is obtained by using selective ion monitoring LC-ESI-MS of the MAO B in-gel tryptic digest (Fig. 7). Peptides containing Cys266 and Cys5 were the only biotinylated peptides identified in mitochondrial membrane-bound clorgyline-inactivated MAO A and pargyline-inactivated MAO B, respectively, similar to the results obtained for detergent-solubilized purified enzymes. These results provide experimental evidence suggesting that MAO A and MAO B structures in outer mitochondrial membrane are similar to their structures in detergent solutions.
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DISCUSSION |
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To investigate thiol group reactivities in a 60-kDa protein at the
sequence level, an approach was required that would yield high resolution and
enable direct identification of modified cysteine residues. A second
consideration was the very hydrophobic nature of the resulting tryptic,
Cys-containing peptides. The first requirement was satisfied by modifying the
thiol groups with a reagent that exhibits a high level of specificity and
selectivity; N-ethylmaleimide was chosen because this reagent
exhibits a good selectivity for thiol groups. Although modification of
histidine, lysine, and tyrosine residues have been observed at high reagent
concentrations and long reaction times
(22), the selectivity of NEM
for cysteine residues is still superior as compared with the alternatives,
which include haloacyl- or acryloyl-based reagents. ESI quadrupole MS was
applied because it provides a high resolution allowing us to monitor additions
of 125 Da to a
60-kDa protein
(19). Using MAO A and MAO B,
we demonstrated that this approach is feasible and provides a better
description of the relative levels of different populations of reaction
products as compared with more traditional approaches, such as radiolabeling
or spectrophotometric monitoring of the reaction.
The findings of no detectable disulfide bonds in MAO A or MAO B are in
accord with x-ray data on MAO B
(12), which showed the
shortest distance between two thiol groups to be 7.5 Å,
i.e. much greater than a typical SS distance (
2.2
Å). No disulfide bonds were identified in MAO A despite the fact that
two cysteine residues, Cys321 and Cys323, are present in
close proximity in the primary sequence. The findings of no disulfide bonds in
either enzyme do not support the conclusions of Sablin and Ramsay
(13), which proposed the
presence of redox active disulfide bonds in both enzymes on the basis of
reducing equivalents required for flavin reduction upon dithionite titrations.
Stoichiometric amounts of substrates were previously found sufficient for full
reduction of either enzyme
(14,
15). The lack of disulfide
bonds in either enzyme is also consistent with the results of Nandigama and
Edmondson (25), which
demonstrated that apoMAO A can be inserted into the outer mitochondrial
membrane in the absence of flavin in the rib5
strain of Saccharomyces cerevisiae riboflavin auxotroph, and the
activity of this enzyme is restored upon the addition of exogenous FAD. It had
previously been demonstrated that yeast thiol/disulfide isomerase is a
flavoprotein (26) and would
not be active in the absence of exogenous flavin and therefore would not be
able to catalyze disulfide bond formation in newly synthesized proteins.
The reaction of MAO A with NEM proceeds rapidly to form a species with 7 or 8 modified thiol groups. These data suggest that after the initial alkylation of a few thiol groups on the enzyme with NEM, the enzyme structure unfolds to one in which all of the thiol groups are now reactive with the thiol reagent (Fig. 3). This behavior is also observed with the larger Biotinyl-NEM reagent where the major species observed with MAO A is either an unmodified enzyme or a species with 5 or 7 Biotinyl-NEM molecules added. The reactivity profile of MAO B with this reagent suggests a more stable structure where extensive reaction with NEM occurs only on prolonged incubation. These data are consistent with the relative stabilities of the two enzymes observed by investigators who have worked with both purified enzymes.
A covalent modification of the active site flavin by acetylenic amines
(16) leads to reduced
stoichiometry of NEM or Biotinyl-NEM modification of MAO A and MAO B (Figs.
3 and
4). These findings demonstrate
that binding of an inhibitor to the catalytic site of either enzyme results in
a pronounced stabilization of their respective structures. Thus, the
protective effect of substrate/inhibitor binding to thiol reagents observed by
others can be attributed to this stabilizing influence of the
three-dimensional fold of the protein(s) structure rather than steric
hindrance of the active site thiol group by the bound substrate/inhibitor.
There are no detectable changes in the -helical content of either MAO A
or MAO B upon acetylenic inhibitor binding as judged by UV circular dichroism
studies (27). The
-helical contents of both enzymes are similar (estimated at
45%)
and in excellent agreement with the
-helical content calculated based
on MAO B x-ray structure (44%)
(12,
27). The similar estimated
helical contents of MAO A and MAO B predict that both enzymes have similar
three-dimensional structures. This conclusion is consistent with their thiol
reactivity profiles (Fig.
5).
The observation that Biotinyl-NEM reacts with the same cysteinyl residues in the membrane-bound and detergent-solubilized forms of MAO A and MAO B supports the view that the C-terminal helix of MAO B serves as the major anchor of the protein in the outer mitochondrial membrane, and the N-terminal part of the enzyme is in a solvent accessible environment. Although our experiments were performed with overexpressed human enzymes in P. pastoris, we suggest that our findings are relevant to the natural environment of the enzyme because the extraction of the enzymes from yeast mitochondria requires conditions similar to those used when extracting the enzymes from either beef liver or human placental mitochondria (14, 15). The substantial permeability of the outer membrane precludes any conclusions regarding the orientation of the enzymes with respect to facing the cytoplasmic or the inner membrane compartments. It is worthwhile to note that although both Cys266 and Cys5 are conserved in all known MAO A and MAO B sequences, respectively (Scheme 1), Ser mutation of either Cys residue has no effect on enzyme activity (6).
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Biochemistry, Emory University School of Medicine, Rollins Research Bldg., 1510 Clifton Rd., Atlanta, GA 30322. Tel.: 404-727-5972; Fax: 404-727-3452; E-mail: dedmond{at}bimcore.emory.edu.
1 The abbreviation used are: MAO, monoamine oxidase(s); Biotinyl-NEM,
polyethylene oxide maleimide-activated biotin; ESI, electrospray ionization;
MS, mass spectrometry; NEM, N-ethylmaleimide; HPLC, high performance
liquid chromatography; SELDI, surface enhanced laser desorption ionization;
LC, liquid chromatography.
2 Numbering for both enzymes refers to the gene-predicted amino acid
sequences. On the protein level, the initial Met residue of MAO B is
removed.
3 This phenomenon could be explained by the presence of histidine residues
within the sequence of the MAO A peptide that could facilitate ring hydrolysis
by acting as a general base. Similar findings, when maleimide-modified
peptides from the same protein showed different degrees of the ring
hydrolysis, were observed earlier (F. Hubálek, unpublished data).
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ACKNOWLEDGMENTS |
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REFERENCES |
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