Anti-flavin antibodies

Barber, M.J.; Eichler, Duane C.; Solomonson, L P; Ackrell, Brian A.C.

doi:10.1042/bj2420089

Cited by 32 publications

(19 citation statements)

References 22 publications

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“…1), which demonstrates that the nature of the flavin analogue or even its absence has no influence on the level of C406A MAO A expression. As expected, immunoblots using the flavin antisera (22) showed no evidence for the presence of covalent flavin co-migrating with C406A MAO A (data not shown).…”

mentioning

confidence: 72%

Influence of FAD Structure on Its Binding and Activity with the C406A Mutant of Recombinant Human Liver Monoamine Oxidase A

Nandigama¹,

Edmondson

2000

Journal of Biological Chemistry

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2 The molecular basis for the requirement of a covalent flavin linkage has been addressed in MAO as well as other enzyme systems containing covalent flavin coenzymes. Mutagenesis studies by Shih and co-workers (5) demonstrate that replacement of Cys-397 in MAO B with a seryl residue resulted in an inactive enzyme in a COS cell expression system. More recent work from Ito and co-workers shows that recombinant rat liver MAO A in which Cys 406 is mutated to an alanyl residue does exhibit catalytic activity at a level estimated to be 40 -60% that of the wild-type enzyme (depending on the nature of the substrate) when expressed in S. cerevisiae (6).Mutagenesis studies on other flavoenzymes containing 8␣-substituted covalent flavin coenzymes have shown that mutations of the linking amino acid result in enzymes still able to bind FAD non-covalently and to exhibit some catalytic activity. Replacement of His-44 with Cys in fumarate reductase results in a mutant enzyme that contains non-covalent FAD that can be reversibly resolved and reconstituted with FAD to form an active enzyme (7). Recent studies on vanillyl-alcohol oxidase demonstrate that replacement of His-422 with Ala results in an enzyme that binds FAD non-covalently, exhibits ϳ10% the activity of the native enzyme, and has essentially the same structure of the native enzyme as determined by x-ray crystallography (8). Expression of rat L-gulono-␥-lactone oxidase (containing an N(1)-histidyl-FAD (9, 10)) in a baculovirus insect cell system where the medium is deficient in riboflavin results in the formation of an apoprotein that is reactivated by the addition of FAD (11). The binding of FAD to this apoenzyme is apparently non-covalent even though the liganding histidyl residue is present.These studies with enzymes containing 8␣-histidyl-FAD demonstrate that covalent linkage of the flavin is not necessary for protein folding to a form capable of binding the flavin coenzyme by non-covalent interactions. Although the catalytic activities of these mutated forms are lower than those of the native wild-type enzymes, covalent binding of the flavin does not appear to be a requirement for functionality. In the cases tested, however, catalytic turnover is more efficient when the flavin cofactor is covalently bound.This manuscript describes experiments on the C406A mutant form of human liver MAO A expressed in a riboflavinrequiring strain of S. cerevisiae described in a previous publication from this laboratory (3). The results demonstrate that the apo form of the enzyme is incorporated into the outer mitochondrial membrane in a folded form that can bind FAD and a number of FAD analogues. Catalytic activity is reconstituted upon binding of FAD and several of the FAD analogues tested, which allow the determination of binding constants.

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mentioning

confidence: 72%

Influence of FAD Structure on Its Binding and Activity with the C406A Mutant of Recombinant Human Liver Monoamine Oxidase A

Nandigama¹,

Edmondson

2000

Journal of Biological Chemistry

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“…Polyclonal antibodies against PchF were raised in rabbit and purified by standard procedures (24). Anti-flavin antibodies (25) Pseudomonas stutzerii cytochrome c 551 , Pseudomonas aeruginosa cytochrome c 551 , and halophilic Paracoccus (ATCC 12084) cytochrome c 554 .…”

Section: Methodsmentioning

confidence: 99%

The Cytochrome Subunit Is Necessary for Covalent FAD Attachment to the Flavoprotein Subunit of p-Cresol Methylhydroxylase

Kim

Fuller

Kuusk

et al. 1995

Journal of Biological Chemistry

100

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No completely satisfying explanation has been provided for the first question. The attachment of an aminoacyl group to the 8␣-or 6-position of riboflavin does not confer any unusual properties on the flavin, either free in solution or in an enzyme, with the exception of the ultraviolet-visible spectrum of 6-Scysteinylriboflavin, which is quite different from that of other forms of free and bound aminoacyl flavins (2, 3). While the oxidation-reduction potentials (E m 7 ) are about 50 -60 mV more positive for aminoacyl flavins than for unmodified forms (2), this increase in potential can also be achieved by noncovalent interactions with protein. Additionally, enzymes with covalently bound flavins do not catalyze a unique or specific set of reactions.As to the second question, it is known that 2-electron reduction of protein-free 8␣-O-tyrosylriboflavin or 8␣-S-cysteinylsulfonylriboflavin cause expulsion of the aminoacyl groups, thus producing unmodified, oxidized riboflavin (2, 4). The principle of microscopic reversibility suggests that the reverse reaction could occur within an enzyme, with or without intervention of an external enzyme. A mechanism for covalent flavinylation, which has long been in the literature, is shown in Fig. 2 (5, 6). An analogous mechanism was proposed for nonenzymic basecatalyzed nucleophilic attack at the 8␣-carbon of riboflavin derivatives in organic solvents (7).It has been suggested that covalent tethering might require prior activation of the flavin or proteins, e.g. a high energy phosphate bond (8). Of several enzymes studied to date, no specific enzyme has been implicated in the covalent modification process, which contrasts with examples of nonflavin-cofactor covalent attachment to apoenzymes (9 -13).The structural genes of several bacterial enzymes containing covalently bound flavin have been cloned into vectors for expression in new hosts: succinate dehydrogenase, fumarate reductase (14, 15), sarcosine oxidase (16), 6-hydroxy-D-nicotine oxidase (6-HDNO) 1 from Arthrobacter aerogenes (15) (all containing 8␣-N 3 -histidyl-FAD), trimethylamine dehydrogenase

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“…Western blot analyses using antiseras raised against human MAOA (hMAOA) or to BSA-conjugated FAD [22] show positive immunochemical reactivities with the purified recombinant rMAOB ( Figure 1A, lanes 3 and 4). This data shows the presence of a covalently bound flavin cofactor ( Figure 1A, lane 4) in the rMAOB sequence.…”

Section: Characterization Of Purified Rmaobmentioning

confidence: 99%

“…The purified protein was characterized by SDS-polyacrylamide gel electrophoresis followed by either Coomassie blue staining or by Western blot analyses using anti-MAO antisera raised in rabbits against hMAOA, and anti-covalent flavin antisera raised in rabbits against FAD conjugated bovine serum albumin [22].…”

Section: Immunochemical Analysesmentioning

confidence: 99%

Characterization of detergent purified recombinant rat liver monoamine oxidase B expressed in Pichia pastoris

Upadhyay

Edmondson

2008

Protein Expression and Purification

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The high level expression and purification of rat monoamine oxidase B (rMAOB) in the methylotrophic yeast Pichia pastoris is reported. Nearly 100 mg of purified rMAOB is obtained from 130 grams (wet weight) of cells (0.5 liter of culture). The MALDI-TOF mass spectrum of the purified protein shows a single species with a molecular mass of 59.228 ± 0.064 kDa, which agrees with the calculated molecular weight of 59.172 kDa for the rMAOB protein sequence assuming one mole of covalent FAD per mole of the enzyme. Consistent with the MALDI-MS data, purified rMAOB shows a single band near 60 kDa in Coomassie stained SDS/PAGE gel as well as on Western blot analyses performed using antiseras raised against human MAOA and BSA conjugated FAD. A partial amino acid sequence of the purified protein is confirmed to be that of the wild type rMAOB by in-gel trypsin digestion and MALDI-TOF-MS analyses of the liberated peptide fragments. Steady state kinetic data show that purified rMAOB exhibits a K m (amine) of 176 ± 15 µM and a k cat of 497 ± 83 min −1 for benzylamine oxidation, and a K m (O 2 ) of 170 ± 10 µM. Kinetic parameters obtained for purified rMAOB are compared with those reported earlier for recombinant human liver MAOB expressed in Pichia pastoris. KeywordsMonoamine Oxidase B; Rat Liver MAOB; Pichia pastoris Monoamine oxidase B (MAOB) 1 is an outer mitochondrial membrane bound flavoenzyme responsible for oxidative deamination and subsequent degradation of dopamine and dietary amines in our body [1]. These oxidation processes are important in maintaining physiological levels of dopamine and free dietary amines in our body and proper functioning of the central nervous system. Age related increases in the expression levels of MAOB in neural tissues [2,3] are implicated in several neurological disorders like, Parkinson's and Alzheimer's diseases and selective MAOB inhibitors are clinically proven drug adjuvants for treating these disorders [4,5].Owing to their therapeutic applications, developments of MAOB inhibitors with greater clinical potency have gained momentum in recent years. Rat, being one of the most common 1 This work was supported by National Institute of Health Grant GM-29433 (DEE).2To whom correspondence should be addressed: 1510 Clifton Road NE, Atlanta, GA 30322. E-mail: deedmon@emory.edu, Phone: 404-727-5972, Fax: 404-727-2738. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. animal models, is generally used for the screening of potential drug candidates. However, recent quantitative studies have shown differences in inhibitor specificities and properties b...

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Anti-flavin antibodies

Cited by 32 publications

References 22 publications

Influence of FAD Structure on Its Binding and Activity with the C406A Mutant of Recombinant Human Liver Monoamine Oxidase A

Influence of FAD Structure on Its Binding and Activity with the C406A Mutant of Recombinant Human Liver Monoamine Oxidase A

The Cytochrome Subunit Is Necessary for Covalent FAD Attachment to the Flavoprotein Subunit of p-Cresol Methylhydroxylase

Characterization of detergent purified recombinant rat liver monoamine oxidase B expressed in Pichia pastoris

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