Print Email Facebook Twitter Synthesis of a novel fluorescent ceramide analogue and its use in the characterization of recombinant ceramidase from Pseudomonas aeruginosa PA01 Title Synthesis of a novel fluorescent ceramide analogue and its use in the characterization of recombinant ceramidase from Pseudomonas aeruginosa PA01 Author Nieuwenhuizen, W.F. van Leeuwen, S. Götz, F. Egmond, M.R. Centraal Instituut voor Voedingsonderzoek TNO Publication year 2002 Abstract Ceramidase (CDase) hydrolyses the N-acyl linkage of the sphingolipid ceramide. We synthesized the non-fluorescent ceramide analogue (4E,2S,3R)-2-N-(10-pyrenedecanoyl)-1,3,17-trihydroxy-17- (3,5-dinitrobenzoyl)-4-heptadecene (10) that becomes fluorescent upon hydrolysis of its N-acyl bond. This novel substrate was used to study several kinetic aspects of the recombinant CDase from the pathogenic bacterium Pseudomonas aeruginosa PA01. Maximum CDase activity was observed above 1.5 μM substrate, with an apparent Km of 0.5±0.1 μM and a turnover of 5.5 min-1. CDase activity depends on divalent cations without a strong specificity. CDase is inhibited by sphingosine and by several sphingosine analogues. The lack of inhibition by several mammalian CDase inhibitors such as D-erythro-MAPP, L-erythro-MAPP or N-oleoylethanolamine points to a novel active site and/or substrate binding region. The CDase assay described here offers the opportunity to develop and screen for specific bacterial CDase inhibitors of pharmaceutical interest. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Chemicals/CAS: 13-(PLPC-OOH); Amidohydrolases, EC 3.5.-; ceramidase, EC 184.108.40.206; Ceramides; Enzyme Inhibitors; Fluorescent Dyes; Phosphatidylcholines; Recombinant Proteins Subject NutritionAmidohydrolasesBinding SitesCeramidesEnzyme InhibitorsFluorescent DyesHydrogen-Ion ConcentrationKineticsMagnetic Resonance SpectroscopyMolecular StructurePhosphatidylcholinesPseudomonas aeruginosaRecombinant ProteinsBacteria (microorganisms)MammaliaPseudomonasPseudomonas aeruginosa To reference this document use: http://resolver.tudelft.nl/uuid:e37b5521-7788-4244-b064-868b62abefaa DOI https://doi.org/10.1016/s0009-3084(01)00206-7 TNO identifier 57470 ISSN 0009-3084 Source Chemistry and Physics of Lipids, 114 (2), 181-191 Document type article Files To receive the publication files, please send an e-mail request to TNO Library.