Immunocytochemical localization of two key enzymes of the 2-hydroxyglutarate pathway of glutamate fermentation in Acidaminococcus fermentans

Rohde, Manfred; Mayer, Frank; Dutscho, Roland; Wohlfarth, Gert

doi:10.1007/bf00422295

Cited by 12 publications

(7 citation statements)

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“…In addition, the resolution limits of the immunogold technique due to gold marker scattering around the antigenic epitope (33) do not allow any reliable conclusion with respect to the sidedness of the hydrogenase from the results of our labeling experiments ( Fig. 3A and 4).…”

Section: Methodsmentioning

confidence: 37%

“…3A, small circles). This diffuse label within the cell wall could be due to gold marker scattering around the antigenic epitope (33), surface relief formation during ultrathin sectioning of the plastic-embed- ded cells (data not shown [13]), or a small-range distortion of the cellular border line by pressing and shearing during the sectioning process. Since treatment of the cells with osmium tetroxide during fixation has been omitted so as not to destroy antigenicity of the hydrogenase molecules, the typical appearance of the cytoplasmic membrane as a blackwhite-black double track is not found.…”

Section: Methodsmentioning

confidence: 99%

“…At this temperature the cells were embedded in Lowicryl K4M as described by Roth et al (35), and the samples were polymerized in the cooling unit for 24 h at -35°C and 6-W UV light immersed in 100% ethanol as coolant. Ultrathin sections (120 to 150 nm) were picked up on nickel grids (300 mesh) and, before labeling with protein A-goldlo nm' prepared by the method of Frens (18), sections were treated with 0.05% (vol/vol) Tween 20 and 0.25% (wt/vol) aqueous skim milk powder to prevent unspecific antibody binding (33). Immunogold labeling was done with a specific rabbit antiserum raised against the F420-hydrogenase from M. barkeri for 1 to 2 h at room temperature, followed by a 30-min incubation with protein A-gold.…”

Section: Methodsmentioning

confidence: 99%

“…The hydrophobic properties of the protein indicated a possible membrane association in whole cells which could have been lost in the course of cell rupture (34). Immunoelectron microscopy, a powerful method for localizing bacterial and eucaryotic proteins in situ (33)(34)(35), revealed that the F420-hydrogenase of M. barkeri was predominantly located in the vicinity of the cytoplasmic membrane during -exponential growth on methanol as the sole methanogenic substrate, suggesting that the enzyme is membrane associated in intact cells. This result with M. barkeri is in agreement with recent findings reported for Methanococcus voltae (27) and Methanobacterium formicicum (3) and raises the question of whether the deazaflavin-dependent hydrogenase could be involved in the generation of an electrochemical proton gradient across the cytoplasmic membrane.…”

Section: Ch3-s-com + Htp-sh -> Ch4 + Com-s-s-htp (Reaction 1)mentioning

confidence: 99%

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Immunocytochemical localization of the coenzyme F420-reducing hydrogenase in Methanosarcina barkeri Fusaro

1991

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The cytological localization of the 8-hydroxy-5-deazaflavin (coenzyme F420)-reducing hydrogenase of Methanosarcina barkenr Fusaro was determined by immunoelectron microscopy, using a specific polyclonal rabbit antiserum raised against the homogeneous deazaflavin-dependent enzyme. In Western blot (immunoblot) experiments this antiserum reacted specifically with the native coenzyme F420-reducing hydrogenase, but did not cross-react with the coenzyme F420-nonreducing hydrogenase activity also detectable in crude extracts prepared from methanol-grown Methanosarcina cells. Immunogold labeling of ultrathin sections of anaerobically fixed methanol-grown cells from the exponential growth phase revealed that the coenzyme F420-reducing hydrogenase was predominantly located in the vicinity of the cytoplasmic membrane. From this result we concluded that the deazaflavin-dependent hydrogenase is associated with the cytoplasmic membrane in intact cells of M. barkeri during growth on methanol as the sole methanogenic substrate, and a possible role of this enzyme in the generation of the electrochemical proton gradient is discussed.During methanogenesis from methanol and H2 by restingcell suspensions of methanol-grown Methanosarcina barkeri, ATP synthesis is stringently coupled to an electrochemical proton gradient and a functional DCCD (N,N'-dicyclohexylcarbodiimide)-sensitive ATP synthase, indicating a chemiosmotic mechanism for energy conservation (8, 10, 11). The central intermediate of methane formation from methanol is 2-(methylthio)ethanesulfonic acid (CH3-S-coenzyme M [CoM]) (19,39,43) which is reductively demrethylated with H2 to yield CH4 and 2-mercaptoethanesulfonic acid (CoM-SH) by the multicomponent methyl-coenzyme M methylreductase system (36, 37). Since the free energy change of this reaction (AG0' = -85 kJ/reaction) is large enough to drive ATP synthesis (9, 19), it has been hypothesized that one or several components of the methylreductase system are membrane associated and involved in proton translocation across the cytoplasmic membrane.It has been demonstrated recently (16,22) that the hydrogen-dependent reductive demethylation of CH3-S-CoM to CH4, a reaction common to all methanogenic bacteria irrespective of their growth substrate (19, 37), proceeds in two steps:CoM-S-S-HTP + H2 -_ CoM-SH + HTP-SH (reaction 2)In the first step (reaction 1) the methylreductase catalyzes the reduction of CH3-S-CoM to CH4 with L-7-mercaptoheptanoylthreonine phosphate (HTP-SH) as the electron donor (29). The heterodisulfide CoM-S-S-HTP formed is then reduced in the second step to CoM-SH and HTP-SH by a hydrogenase-linked heterodisulfide reductase system (reaction 2), and it has been proposed that the reduction of the mixed disulfide rather than methane formation is linked to proton translocation and thus coupled to energy conservation (16, 21).Therefore, we decided to determine the ultrastructural location of the coenzyme F420-reducing hydrogenase (F420-hydrogenase) in M. barkeri by immunoelectron microscopy, using a specific po...

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Section: Methodsmentioning

confidence: 37%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Ch3-s-com + Htp-sh -> Ch4 + Com-s-s-htp (Reaction 1)mentioning

confidence: 99%

See 2 more Smart Citations

Immunocytochemical localization of the coenzyme F420-reducing hydrogenase in Methanosarcina barkeri Fusaro

1991

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“…Like 2,4-dinitrophenol, FCCP did not act as uncoupler since a 200000×g supernatant retained the activity [31]. The location of (R)-2-hydroxyglutaryl-CoA dehydratase in the cytoplasm was confirmed later using the antibody-gold technique [46]. The activity of the debydratase was stimulated, however, by boiled membranes due to the RNA content.…”

Section: (R)-2-hydroxyglutaryl-coa Dehydratasesmentioning

confidence: 89%

Unusual dehydrations in anaerobic bacteria

Buckel

1992

FEMS Microbiology Letters

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2-Hydroxyglutaryl-CoA dehydratase from Fusobacterium nucleatum (subsp. nucleatum): an iron-sulfur flavoprotein

Klees

Linder

1992

Arch. Microbiol.

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Anaerobically prepared cell-free extracts from Fusobacterium nucleatum contain 2-hydroxyglutaryl-CoA dehydratase with a specific activity of 20 nkat mg-1. The enzyme was purified 24-fold to a specific activity of 480 nkat mg-1 by anion exchange chromatography, gel filtration and chromatography on Blue-Sepharose. The activity of the purified enzyme was strictly dependent on the reductant Ti(III)citrate and stimulated 25-fold by 0.15 mM ATP and 5 mM MgCl2. ATP is hydrolysed to ADP during incubation with 2-hydroxyglutaryl-CoA dehydratase in the presence or absence of the substrate. The enzyme is extremely sensitive towards oxygen and is inhibited by 10 microM chloramphenicol, 10 microM 2,4-dinitrophenol or 0.15 mM hydroxylamine. The pure enzyme consists of three subunits alpha (49 kDa), beta (39 kDa) and gamma (24 kDa) in approximately equal amounts. In this respect the enzyme differs from the related 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans and lactyl-CoA dehydratase from Clostridium propionicum both of which are composed of only two subunits with sizes comparable to those of alpha and beta but require an additional protein for activity. The relative molecular mass of the native enzyme of about 100 kDa suggests a trimeric alpha beta gamma-structure. The homogeneous enzyme contains riboflavin (0.5 mol/112 kDa), iron and sulfur (3.5 mol/112 kDa each). Polyclonal antibodies directed against the 2-hydroxyglutaryl-CoA dehydratase from A. fermentans did not crossreact with cell free extracts or purified dehydratase from F. nucleatum. A comparison of the N-terminal amino acid sequences of the dehydratase subunits from A. fermentans and F. nucleatum, however, showed some similarities in the beta-subunits.

show abstract

Immunocytochemical localization of two key enzymes of the 2-hydroxyglutarate pathway of glutamate fermentation in Acidaminococcus fermentans

Cited by 12 publications

References 14 publications

Immunocytochemical localization of the coenzyme F420-reducing hydrogenase in Methanosarcina barkeri Fusaro

Immunocytochemical localization of the coenzyme F420-reducing hydrogenase in Methanosarcina barkeri Fusaro

Unusual dehydrations in anaerobic bacteria

2-Hydroxyglutaryl-CoA dehydratase from Fusobacterium nucleatum (subsp. nucleatum): an iron-sulfur flavoprotein

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