[3H]Ro 19-6327: A reversible ligand and affinity labelling probe for monoamine oxidase-B

Cesura, Andrea M.; Galva, M. D.; Imhof, R.; Kyburz, E.; Picotti, G. B.; Prada, M. Da

doi:10.1016/0014-2999(89)90336-1

Cited by 44 publications

(25 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thereafter, in analogy to the inactivation pathway proposed by Silverman and co-workers [13, 391 for other mechanism-based M A 0 inactivators, the activated inhibitor may undergo combination with an active-site nucleophile or, alternatively, as suggested in the case of some benzylamine derivatives [38], the imine product of the inhibitor may form a stable complex with the reduced enzyme. In both cases, the amide group of this class of inhibitors is likely to plsiy an important role in stalsilising the enzyme-inhibitor adduct [21,24,251. The affinity-labeling reaction of MAO-B with iV-(2-aminoethy1)arylcarboxamide inhibitors may involve the addition of the inhibitor enzyme-activated intermediate to an active-site nucleophile followed by proton-catalyzed reduction to give a stable adduct [24-261.…”

Section: Discussionmentioning

confidence: 99%

“…1 for position of tritium labeling) was synthesized by Dr H. Harder (Isotope Synthesis Department, F. Hoffmann-La Roche, Basel, Switzerland) as previously described [24]. [ Enzyme activity determination.…”

Section: Methodsmentioning

confidence: 99%

“…Reversible binding of ['Hllazabemide was performed essentially as previously described [24,271 by incubating the enzyme preparation at 25°C in the presence of various concentrations of ['Hllazabemide. Bound radioligand was then separated from free radioactivity by filtering the samples through polyethylenimine-treated GF/B glass-fiber filters (Whatmann) [28].…”

Section: Methodsmentioning

confidence: 99%

“…The concentration of MAO-B in this preparation was 35 pmol . mg protein-', as determined by ['Hllazabemide binding [24]. The enzyme was solubilised with digitonin at a proteiddetergent ratio of 1 : 8 and subsequently purified by affinity chromatography as previously described [30].…”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Investigation on the Structure of the Active Site of Monoamine Oxidase‐B by Affinity Labeling with the Selective Inhibitor Lazabemide and by Site‐Directed Mutagenesis

Cesura

Gottowik

Lahm

et al. 1996

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

The structural features of the active site of human monoamine oxidase El (MAO-B) were investigated by affinity labeling and site-directed mutagenesis. The pseudosubstrate inhibitor N-[2-aminoethyl1-5-chloro-2-pyridine carboxamide HCI (lazabemide) can be irreversibly linked to MAO-B by reduction of the enzyme-inhibitor complex with NaBH,CN. Analysis of the flavin spectrum of ['Hllazabemide-labeled human MAO-B indicated that insertion of the inhibitor did not occur into the isoalloxazine ring of FAD. After trypsin digestion and HPLC peptide mapping of the radiolabeled enzyme, two labeled peptides were observed. Sequence analysis showed that both peptides started at Val371 of human MAO-B. These results indicate that ['Hllazabemide is incorporated into the MAO-B peptide stretch containing the FADmodified Cys397. The function of putative active-site residues contained in this region was investigated by site-directed mutagenesis and expression of the mutant proteins in HEK-293 cells. Substitution of His382 of MAO-B with an Arg greatly reduced the enzymic activity, suggesting that this residue may represent a nucleophile relevant for the MAO-B catalytic mechanism. Whereas it has been shown that MAO-A and MAO-B are the products of separate genes [5-71 encoding proteins with a high degree of amino acid identity (72.6%) [5]. In both M A 0 forms, the co-factor-binding site appears to be constituted by at least the following two regions: a domain near the N-terminus constituting the non-covalent FAD co-factor-binding site which contains an ADP-binding sequence

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Investigation on the Structure of the Active Site of Monoamine Oxidase‐B by Affinity Labeling with the Selective Inhibitor Lazabemide and by Site‐Directed Mutagenesis

Cesura

Gottowik

Lahm

et al. 1996

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…HCl (Ro 41-2064) were synthesised at Hoffmann-La Roche, Basel. ['HIRo 41-1049 (30.8 Ci/mmol) and ['Hllazabemide (17.0 Cil mmol) were synthesised by Dr H. Harder (Isotope Synthesis Department, Hoffmann-La Roche, Basel) as previously described (Cesura et al, 1989 Engineering of MAO-A and MAO-B chimeras. A 1670-bp MAO-B EcoRI-Hind111 fragment and a 1680-bp MAO-A fragment were separately subcloned into the vector pCMV.…”

Section: Materials N-(2-aminoethyl)-5-(rn-fluorophenyl)-4-thiazolementioning

confidence: 99%

Structure/Function Relationships of Mitochondrial Monoamine Oxidase A and B Chimeric Forms

Gottowik

Malherbe

Lang

et al. 1995

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

Monoamine oxidases (MAO) A and B show a high degree of amino acid similarity. Apart from the NH,-terminus, which contains an ADP-binding consensus sequence, little is known about their structural features or the sequences involved in the binding of substrates. In the present paper, we have studied the structure/function relationships of MAOs by constructing 18 different chimeric forms of MAO, engineered by moving progressively the junction between the NH,-terminus of one M A 0 form with the COOH-terminus of its isoenzyme. After transient expression in HEK-293 cells, the properties of these chimeric enzymes were investigated using both selective and nonselective substrates and inhibitors. Whereas exchange of the ADP-binding sequence did not modify the catalytic properties of either M A 0 isoforms, chimeras with increasing length of the NH,-terminus of MAO-A (up to residue 256) showed a marked decrease in affinity towards the MAO-B substrate phenylethylamine and the inhibitor N(2-aminoethyl)-5-chloro-2-pyridine carboxamide . HC1 (lazabemide) when compared to wild-type MAO-B. No major changes were observed in the k,,, values of these chimeras. From the data obtained, two sequences, i.e. 62-103 and 146-220, appeared of particular importance in constituting the binding site of MAO-B. On the other hand, the catalytic properties and specificity of MAO-A appeared to be relatively insensitive to substitution of both the NH,-(up to position 112) and COOH-termini (from residue 395) of MAO-A with the corresponding MAO-B sequences. However, further modification of the central 283-residue sequence of MAO-A did not appear compatible with enzymic activity. None of the engineered chimeras showed a shift in specificity from one isoform to the other.

show abstract

Neuroprotection in Parkinson Disease

Simpkins

Jankovic²

2003

Arch Intern Med

View full text Add to dashboard Cite

Treatment of Parkinson disease has improved dramatically over the past quarter of a century and promising therapies are emerging. Although treatment with levodopa results in marked symptomatic improvement, mortality rates of the disease have remained relatively unchanged. Recent findings of abnormal protein folding, coupled with oxidative stress, provide scientific rationale for novel therapeutic strategies designed to slow disease progression. To be effective, these disease-modifying and neuroprotective therapies must be instituted early in the course of the disease and early diagnosis therefore is critical. Consequently, primary care physicians will play an increasingly important role in early institution of such neuroprotective strategies. This review is designed to highlight some of the recent advances in our understanding of the mechanisms of neurodegeneration and to draw attention to the importance of early recognition and implementation of the new therapeutic interventions.

show abstract

[3H]Ro 19-6327: A reversible ligand and affinity labelling probe for monoamine oxidase-B

Cited by 44 publications

References 16 publications

Investigation on the Structure of the Active Site of Monoamine Oxidase‐B by Affinity Labeling with the Selective Inhibitor Lazabemide and by Site‐Directed Mutagenesis

Investigation on the Structure of the Active Site of Monoamine Oxidase‐B by Affinity Labeling with the Selective Inhibitor Lazabemide and by Site‐Directed Mutagenesis

Structure/Function Relationships of Mitochondrial Monoamine Oxidase A and B Chimeric Forms

Neuroprotection in Parkinson Disease

Contact Info

Product

Resources

About