scholarly journals Characterization of Energy-Conserving Hydrogenase B in Methanococcus maripaludis

2010 ◽  
Vol 192 (15) ◽  
pp. 4022-4030 ◽  
Author(s):  
Tiffany A. Major ◽  
Yuchen Liu ◽  
William B. Whitman
Keyword(s):  
Gene Replacement ◽  
Methanococcus Maripaludis ◽  
Gene Encoding ◽  
Western Blots ◽  
Growth Defects ◽  
Energy Conserving ◽  
Membrane Bound ◽  
Membrane Spanning ◽  
And Function

ABSTRACT The Methanococcus maripaludis energy-conserving hydrogenase B (Ehb) generates low potential electrons required for autotrophic CO2 assimilation. To analyze the importance of individual subunits in Ehb structure and function, markerless in-frame deletions were constructed in a number of M. maripaludis ehb genes. These genes encode the large and small hydrogenase subunits (ehbN and ehbM, respectively), a polyferredoxin and ferredoxin (ehbK and ehbL, respectively), and an ion translocator (ehbF). In addition, a gene replacement mutation was constructed for a gene encoding a putative membrane-spanning subunit (ehbO). When grown in minimal medium plus acetate (McA), all ehb mutants had severe growth deficiencies except the ΔehbO::pac strain. The membrane-spanning ion translocator (ΔehbF) and the large hydrogenase subunit (ΔehbN) deletion strains displayed the severest growth defects. Deletion of the ehbN gene was of particular interest because this gene was not contiguous to the ehb operon. In-gel activity assays and Western blots confirmed that EhbN was part of the membrane-bound Ehb hydrogenase complex. The ΔehbN strain was also sensitive to growth inhibition by aryl acids, indicating that Ehb was coupled to the indolepyruvate oxidoreductase (Ior), further supporting the hypothesis that Ehb provides low potential reductants for the anabolic oxidoreductases in M. maripaludis.

scholarly journals Characterization of two monoclonal antibodies against cytochrome b558 of human neutrophils

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1686-1694 ◽  
Author(s):  
AJ Verhoeven ◽  
BG Bolscher ◽  
LJ Meerhof ◽  
R van Zwieten ◽  
J Keijer ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transmembrane Protein ◽  
Autosomal Gene ◽  
Human Neutrophils ◽  
Western Blots ◽  
Binding Characteristics ◽  
Cytochrome B558 ◽  
Spectrophotometric Methods ◽  
Membrane Bound

Abstract Monoclonal antibodies (MoAbs) were raised against cytochrome b558, a membrane-bound component of the NADPH:O2 oxidoreductase in human neutrophils. This cytochrome consists of a low-molecular-weight (low- mol-wt) subunit of 22 to 23 Kd, probably encoded by an autosomal gene, and a high-mol-wt subunit of 75 to 90 Kd, encoded on the X-chromosome. MoAb 449 reacts with the low-mol-wt subunit and MoAb 48 with the high- mol-wt subunit on Western blots of purified cytochrome b558 and on blots of whole neutrophil extracts. In extracts of neutrophils from patients with chronic granulomatous disease (CGD) in which cytochrome b558 is not detectable by spectrophotometric methods, the low-mol-wt subunit is present, albeit in a much smaller amount. The high-mol-wt subunit is not detected by MoAb 48 in neutrophils of patients with X- linked CGD and in neutrophils of patients with the autosomal, cytochrome-b558-negative form of the disease. These results can be explained by a marked instability of these subunits when the synthesis of either of the two is disturbed. In differentiated HL-60 cells, the high-mol-wt subunit appears to be present in a different form. Cloning of the low-mol-wt subunit with the help of MoAb 449 suggests the presence of a heme-binding site on this subunit. By comparison of the binding characteristics of MoAb 449 to intact and permeabilized neutrophils with those of MoAb 7D5, recently isolated by Nakamura et al (Blood 69:1404, 1987), the low-mol-wt subunit was established as a transmembrane protein.

A single external enzyme confers alternative NADH:ubiquinone oxidoreductase activity in Yarrowia lipolytica

1999 ◽  
Vol 112 (14) ◽  
pp. 2347-2354 ◽  
Author(s):  
S.J. Kerscher ◽  
J.G. Okun ◽  
U. Brandt
Keyword(s):  
Yarrowia Lipolytica ◽  
Complex I ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Inner Mitochondrial Membrane ◽  
Gene Encoding ◽  
Oxidoreductase Activity ◽  
Mitochondrial Inner Membrane ◽  
Energy Conserving ◽  
And Function ◽  
Complex I Activity

NADH:ubiquinone oxidoreductases catalyse the first step within the diverse pathways of mitochondrial NADH oxidation. In addition to the energy-conserving form commonly called complex I, fungi and plants contain much simpler alternative NADH:ubiquinone oxido-reductases that catalyze the same reaction but do not translocate protons across the inner mitochondrial membrane. Little is known about the distribution and function of these enzymes. We have identified YLNDH2 as the only gene encoding an alternative NADH:ubiquinone oxidoreductase (NDH2) in the obligate aerobic yeast Yarrowia lipolytica. Cells carrying a deletion of YLNDH2 were fully viable; full inhibition by piericidin A indicated that complex I activity was the sole NADH:ubiquinone oxidoreductase activity left in the deletion strains. Studies with intact mitochondria revealed that NDH2 in Y. lipolytica is oriented towards the external face of the mitochondrial inner membrane. This is in contrast to the situation seen in Saccharomyces cerevisiae, Neurospora crassa and in green plants, where internal alternative NADH:ubiquinone oxidoreductases have been reported. Phylogenetic analysis of known NADH:ubiquinone oxidoreductases suggests that during evolution conversion of an ancestral external alternative NADH:ubiquinone oxidoreductase to an internal enzyme may have paved the way for the loss of complex I in fermenting yeasts like S. cerevisiae.

scholarly journals Identification and characterization of the 2D6 and Mr 23000 antigens on the plasma membrane of rat spermatozoa

1987 ◽  
Vol 241 (2) ◽  
pp. 353-360 ◽  
Author(s):  
R Jones ◽  
C R Brown
Keyword(s):  
Plasma Membrane ◽  
Antigenic Determinant ◽  
Egg Recognition ◽  
Western Blots ◽  
One Dimensional ◽  
Antigen Present ◽  
Membrane Bound ◽  
Species Specific ◽  
Identification And Characterization

Previous investigations [Jones, Brown, von Glos & Gaunt (1985) Exp. Cell Res. 156, 31-44] have demonstrated the appearance of a new antigenic determinant (recognized by monoclonal antibody 2D6) on the plasma membrane of rat spermatozoa during post-testicular maturation in the epididymis. Identification of the 2D6 antigen on Western blots from one-dimensional SDS/polyacrylamide gels revealed that it co-migrated with a membrane protein (designated Mr 23,000 antigen) present on testicular and immature germ cells, suggesting that one antigen might be a modified version of the other. In the present work, however, we demonstrate that, although they have similar Mr and are present in soluble and membrane-bound forms, the 2D6 and Mr 23,000 antigens are biochemically and immunologically distinct molecules. The properties of the antigens are described and compared. The Mr 23,000 antigen is present on both testicular and cauda epididymidal spermatozoa, has a pI of 6.1, contains no detectable carbohydrate, is not tissue-specific and is degraded by V8 protease. By contrast, the 2D6 antigen is glycosylated, has a broad pI from 4.5 to 6.1, is tissue- and species-specific and is resistant to digestion with V8 protease. Its role in sperm-egg recognition is discussed.

scholarly journals Existence and functions of hypothalamic kisspeptin neuropeptide signaling system in a non-chordate deuterostome species

2019 ◽  
Author(s):  
Tianming Wang ◽  
Zheng Cao ◽  
Zhangfei Shen ◽  
Jingwen Yang ◽  
Xu Chen ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Apostichopus Japonicus ◽  
Receptor Internalization ◽  
Neurosecretory System ◽  
Vertebrate Lineage ◽  
Gene Encoding ◽  
And Function ◽  
First Time ◽  
New Evidence

AbstractThe kisspeptin (Kp) system is a central modulator of the hypothalamic-pituitary-gonadal axis in vertebrates. Its existence outside the vertebrate lineage remains largely unknown. Here we report the identification and characterization of Kp system in the sea cucumber Apostichopus japonicus. The gene encoding the Kp precursor, generates two mature neuropeptides, AjKiss1a and AjKiss1b. The Kp receptors, AjKissR1 and AjKissR2, are strongly activated by synthetic A. japonicus and vertebrate Kps, triggering a rapid intracellular mobilization of Ca2+, followed by receptor internalization. AjKissR1 and AjKissR2 share similar intracellular signaling pathways via Gαq/PLC/PKC/MAPK cascade, when activated by C-terminal decapeptide (AjKiss1b-10). The A. japonicus Kp system functions in mutiple tissues which are closely related to reproduction and metabolism. Overall, our findings uncover for the first time, to our knowledge, the existence and function of the Kp system in a non-chordate species and provide new evidence to support the ancient origin of the hypothalamic neurosecretory system.

scholarly journals Menaquinone (Vitamin K2) Biosynthesis: Localization and Characterization of the menA Gene fromEscherichia coli

1998 ◽  
Vol 180 (10) ◽  
pp. 2782-2787 ◽  
Author(s):  
K. Suvarna ◽  
D. Stevenson ◽  
R. Meganathan ◽  
M. E. S. Hudspeth
Keyword(s):  
High Performance ◽  
Promoter Sequence ◽  
Anaerobic Growth ◽  
Gene Encoding ◽  
Reading Frame ◽  
Chromatography Analysis ◽  
Naphthoic Acid ◽  
Membrane Bound ◽  
Carbon Side Chain

ABSTRACT A key reaction in the biosynthesis of menaquinone involves the conversion of the soluble bicyclic naphthalenoid compound 1,4-dihydroxy-2-naphthoic acid (DHNA) to the membrane-bound demethylmenaquinone. The enzyme catalyzing this reaction, DHNA-octaprenyltransferase, attaches a 40-carbon side chain to DHNA. The menA gene encoding this enzyme has been cloned and localized to a 2.0-kb region of the Escherichia coli genome between cytR and glpK. DNA sequence analysis of the cloned insert revealed a 308-codon open reading frame (ORF), which by deletion analyses was shown to restore anaerobic growth of amenA mutant. Reverse-phase high-performance liquid chromatography analysis of quinones extracted from theorf-complemented cells independently confirmed the restoration of menaquinone biosynthesis, and similarly, analyses of isolated cell membranes for DHNA octaprenyltransferase activity confirmed the introduction of the menA product into theorf-complemented menA mutant. The validity of an ORF-associated putative promoter sequence was confirmed by primer extension analyses.

Disruption of the gene encoding the ChiB1 chitinase of Aspergillus fumigatus and characterization of a recombinant gene product

Microbiology ◽  
2003 ◽  
Vol 149 (10) ◽  
pp. 2931-2939 ◽  
Author(s):  
Alex K. Jaques ◽  
Tamo Fukamizo ◽  
Diana Hall ◽  
Richard C. Barton ◽  
Gemma M. Escott ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Wild Type Strain ◽  
Chitinase Activity ◽  
Gene Replacement ◽  
Wild Type ◽  
Gene Encoding ◽  
Batch Cultures ◽  
Putative Active Site ◽  
Hydrolysis Of

The gene encoding a major, inducible 45 kDa chitinase of Aspergillus fumigatus was cloned and analysis of the deduced amino acid sequence identified a chitinase of the fungal/bacterial class which was designated ChiB1. Recombinant ChiB1, expressed in Pichia pastoris, was shown to function by a retaining mechanism of action. That is, the β-conformation of the chitin substrate linkage was preserved in the product in a manner typical of family 18 chitinases. Cleavage patterns with the N-acetylglucosamine (GlcNAc) oligosaccharide substrates GlcNAc4, GlcNAc5 and GlcNAc6 indicated that the predominant reaction involved hydrolysis of GlcNAc2 from the non-reducing end of each substrate. Products of transglycosylation were also identified in each incubation. Following disruption of chiB1 by gene replacement, growth and morphology of disruptants and of the wild-type strain were essentially identical. However, during the autolytic phase of batch cultures the level of chitinase activity in culture filtrate from a disruptant was much lower than the activity from the wild-type. The search for chitinases with morphogenetic roles in filamentous fungi should perhaps focus on chitinases of the fungal/plant class although such an investigation will be complicated by the identification of at least 11 putative active site domains for family 18 chitinases in the A. fumigatus TIGR database (http://www.tigr.org/).

scholarly journals Structure and function of an unusual flavodoxin from the domainArchaea

2019 ◽  
Vol 116 (51) ◽  
pp. 25917-25922 ◽  
Author(s):  
Divya Prakash ◽  
Prashanti R. Iyer ◽  
Suharti Suharti ◽  
Karim A. Walters ◽  
Michel Geovanni Santiago-Martinez ◽  
...  
Keyword(s):  
Atp Synthesis ◽  
Flavin Mononucleotide ◽  
Methanosarcina Acetivorans ◽  
Physiological Characterization ◽  
Electron Transfer Proteins ◽  
Reduction Potentials ◽  
Transport Chain ◽  
Membrane Bound ◽  
And Function

Flavodoxins, electron transfer proteins essential for diverse metabolisms in microbes from the domainBacteria, are extensively characterized. Remarkably, although genomic annotations of flavodoxins are widespread in microbes from the domainArchaea, none have been isolated and characterized. Herein is described the structural, biochemical, and physiological characterization of an unusual flavodoxin (FldA) fromMethanosarcina acetivorans, an acetate-utilizing methane-producing microbe of the domainArchaea. In contrast to all flavodoxins, FldA is homodimeric, markedly less acidic, and stabilizes an anionic semiquinone. The crystal structure reveals an flavin mononucleotide (FMN) binding site unique from all other flavodoxins that provides a rationale for stabilization of the anionic semiquinone and a remarkably low reduction potentials for both the oxidized/semiquinone (−301 mV) and semiquinone/hydroquinone couples (−464 mV). FldA is up-regulated in acetate-grown versus methanol-grown cells and shown here to substitute for ferredoxin in mediating the transfer of low potential electrons from the carbonyl of acetate to the membrane-bound electron transport chain that generates ion gradients driving ATP synthesis. FldA offers potential advantages over ferredoxin by (i) sparing iron for abundant iron-sulfur proteins essential for acetotrophic growth and (ii) resilience to oxidative damage.

scholarly journals Characterization of two monoclonal antibodies against cytochrome b558 of human neutrophils

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1686-1694 ◽  
Author(s):  
AJ Verhoeven ◽  
BG Bolscher ◽  
LJ Meerhof ◽  
R van Zwieten ◽  
J Keijer ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Transmembrane Protein ◽  
Autosomal Gene ◽  
Human Neutrophils ◽  
Western Blots ◽  
Binding Characteristics ◽  
Cytochrome B558 ◽  
Spectrophotometric Methods ◽  
Membrane Bound

Monoclonal antibodies (MoAbs) were raised against cytochrome b558, a membrane-bound component of the NADPH:O2 oxidoreductase in human neutrophils. This cytochrome consists of a low-molecular-weight (low- mol-wt) subunit of 22 to 23 Kd, probably encoded by an autosomal gene, and a high-mol-wt subunit of 75 to 90 Kd, encoded on the X-chromosome. MoAb 449 reacts with the low-mol-wt subunit and MoAb 48 with the high- mol-wt subunit on Western blots of purified cytochrome b558 and on blots of whole neutrophil extracts. In extracts of neutrophils from patients with chronic granulomatous disease (CGD) in which cytochrome b558 is not detectable by spectrophotometric methods, the low-mol-wt subunit is present, albeit in a much smaller amount. The high-mol-wt subunit is not detected by MoAb 48 in neutrophils of patients with X- linked CGD and in neutrophils of patients with the autosomal, cytochrome-b558-negative form of the disease. These results can be explained by a marked instability of these subunits when the synthesis of either of the two is disturbed. In differentiated HL-60 cells, the high-mol-wt subunit appears to be present in a different form. Cloning of the low-mol-wt subunit with the help of MoAb 449 suggests the presence of a heme-binding site on this subunit. By comparison of the binding characteristics of MoAb 449 to intact and permeabilized neutrophils with those of MoAb 7D5, recently isolated by Nakamura et al (Blood 69:1404, 1987), the low-mol-wt subunit was established as a transmembrane protein.

Sign in / Sign up

Export Citation Format

Share Document