MSpace - DSpace at UofM >
University of Manitoba Researchers (Research Publications) >
Research Publications (UofM Student, Faculty and Staff only access) >

Please use this identifier to cite or link to this item: http://hdl.handle.net/1993/2969

Title: Purification of the NADP(+): F-420 oxidoreductase of Methanosphaera stadtmanae
Authors: Elias, DA
Juck, DF
Berry, KA
Sparling, R
Keywords: methanogen
NADP : F-420 oxidoreductase
NADP reductase
F-420
NADP(+)
METHANOBACTERIUM-THERMOAUTOTROPHICUM
STREPTOMYCES-GRISEUS
REDUCTASE
8-HYDROXY-5-DEAZAFLAVIN
MEMBRANE
HYDROGEN
CARBON
Issue Date: 30-Nov-2000
Citation: 0008-4166; CAN J MICROBIOL, NOV 2000, vol. 46, no. 11, p.998 to 1003.
Abstract: Methanosphaera 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.
URI: http://hdl.handle.net/1993/2969
DOI: http://dx.doi.org/10.1139/w00-090
Appears in Collection(s):Research Publications (UofM Student, Faculty and Staff only access)

Files in This Item:

File Description SizeFormat
Elias_Purification_of_the_NADP.pdf148.41 kBAdobe PDFView/Open
View Statistics

Items in MSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

 

Valid XHTML 1.0! MSpace Software Copyright © 2002-2010  Duraspace - Feedback