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dc.contributor.authorElias, DA
dc.contributor.authorJuck, DF
dc.contributor.authorBerry, KA
dc.contributor.authorSparling, R
dc.date.accessioned2007-12-13T12:54:58Z
dc.date.available2007-12-13T12:54:58Z
dc.date.issued2000-11-30
dc.identifier.citation0008-4166; CAN J MICROBIOL, NOV 2000, vol. 46, no. 11, p.998 to 1003.en
dc.identifier.urihttp://hdl.handle.net/1993/2969
dc.description.abstractMethanosphaera stadtmanae (DSM 3091) is a methanogen that requires H-2 and CH3OH for methanogenesis. The organism does not possess an F-420-dependent hydrogenase and only low levels of F-420. It does however possess NADP(+):F-420 oxidoreductase activity. The NADP(+):F-420 oxidoreductase, the enzyme which catalyses the electron transfer between NADP(+) and F-420 in this organism, was purified and characterized. NAD(+), NADH, FMN, and FAD could not be used as electron acceptors. Optimal pH for F-420 reduction was 6.0, and 8.5 for NADP(+) reduction. During the purification process, it was noted that precipitation with (NH4)(2)SO4 increased total activity 16-fold but reduced the stability of the enzyme. However, recombination of cell-free extracts with resuspended 65-90% (NH4)(2)SO4 pellet returned activity to near cell-free extract levels. Neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme. The purified enzyme from M. stadtmanae possessed a molecular weight of 148 kDa as determined by gel filtration chromatography and native-PAGE, consisting of alpha, beta, and gamma subunits of 60, 50, and 45 kDa, respectively, using SDS-PAGE. The K-m values were 370 muM for NADP(+), 142 muM for NADPH, 62.5 muM for F-420, and 7.7 muM for F420H2. These values were different from the K-m values observed in the cell-free extract.en
dc.format.extent151973 bytes
dc.format.mimetypeapplication/pdf
dc.language.isoengen_US
dc.rightsNo part of the NRC Research Press electronic journals may be reproduced, stored, or transmitted in any form or by any means, without the written permission of the publisher, except as stated below. Under the Canadian Copyright Act, individuals may download or print single copies of articles for personal research or study. Any person may reproduce short excerpts from articles in the journals for any purpose that respects the moral rights of authors, provided that the source is fully acknowledged. As a courtesy, the consent of authors of such material should be obtained directly from the author. Authorization to reproduce items for other than personal research or study, as stated above, may be obtained via Access © upon payment of the copyright fee of $10.00 per copy. NRC Research Press also extends certain additional rights to authors. The above rights do not extend to copying or reproduction for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. For such copying or reproduction, arrangements must be made with NRC Research Press.en
dc.rightsinfo:eu-repo/semantics/openAccess
dc.subjectmethanogenen
dc.subjectNADP : F-420 oxidoreductaseen
dc.subjectNADP reductaseen
dc.subjectF-420en
dc.subjectNADP(+)en
dc.subjectMETHANOBACTERIUM-THERMOAUTOTROPHICUMen
dc.subjectSTREPTOMYCES-GRISEUSen
dc.subjectREDUCTASEen
dc.subject8-HYDROXY-5-DEAZAFLAVINen
dc.subjectMEMBRANEen
dc.subjectHYDROGENen
dc.subjectCARBONen
dc.titlePurification of the NADP(+): F-420 oxidoreductase of Methanosphaera stadtmanaeen
dc.typejournal articleen_US
dc.typeinfo:eu-repo/semantics/article
dc.statusPeer revieweden
dc.identifier.doihttp://dx.doi.org/10.1139/w00-090


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