scholarly journals Cloning and Characterization of the Pseudomonas aeruginosa zwf Gene Encoding Glucose-6-Phosphate Dehydrogenase, an Enzyme Important in Resistance to Methyl Viologen (Paraquat)

1998 ◽  
Vol 180 (7) ◽  
pp. 1741-1749 ◽  
Author(s):  
Ju-Fang Ma ◽  
Paul W. Hager ◽  
Michael L. Howell ◽  
Paul V. Phibbs ◽  
Daniel J. Hassett
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mass ◽  
Methyl Viologen ◽  
Size Exclusion ◽  
Logarithmic Phase ◽  
G6pdh Activity ◽  
Gene Encoding ◽  
Exclusion Chromatography ◽  
Increased Sensitivity ◽  
Glucose 6 Phosphate Dehydrogenase

ABSTRACT In this study, we cloned the Pseudomonas aeruginosa zwfgene, encoding glucose-6-phosphate dehydrogenase (G6PDH), an enzyme that catalyzes the NAD+- or NADP+-dependent conversion of glucose-6-phosphate to 6-phosphogluconate. The predictedzwf gene product is 490 residues, which could form a tetramer with a molecular mass of ∼220 kDa. G6PDH activity andzwf transcription were maximal in early logarithmic phase when inducing substrates such as glycerol, glucose, or gluconate were abundant. In contrast, both G6PDH activity and zwftranscription plummeted dramatically when bacteria approached stationary phase, when inducing substrate was limiting, or when the organisms were grown in a citrate-, succinate-, or acetate-containing basal salts medium. G6PDH was purified to homogeneity, and its molecular mass was estimated to be ∼220 kDa by size exclusion chromatography. Estimated Km values of purified G6PDH acting on glucose-6-phosphate, NADP+, and NAD+ were 530, 57, and 333 μM, respectively. The specific activities with NAD+ and NADP+ were calculated to be 176 and 69 μmol/min/mg. An isogenic zwf mutant was unable to grow on minimal medium supplemented with mannitol. The mutant also demonstrated increased sensitivity to the redox-active superoxide-generating agent methyl viologen (paraquat). Since one by-product of G6PDH activity is NADPH, the latter data suggest that this cofactor is essential for the activity of enzymes critical in defense against paraquat toxicity.

Molecular and biochemical characteristics of inulosucrase InuBK from Alkalihalobacillus krulwichiae JCM 11 691

2021 ◽  
Author(s):  
Ken-ji Yokoi ◽  
Sosyu Tsutsui ◽  
Gen-ya Arakawa ◽  
Masakazu Takaba ◽  
Koichi Fujii ◽  
...  
Keyword(s):  
High Performance ◽  
Culture Medium ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Resonance Spectroscopy ◽  
Gene Encoding ◽  
Reading Frame ◽  
Exclusion Chromatography ◽  
Chain Lengths

Abstract Information about the inulosucrase of non-lactic acid bacteria is scarce. We found a gene encoding inulosucrase (inuBK) in the genome of the gram-positive bacterium Alkalihalobacillus krulwichiae JCM 11691. The inuBK open reading frame encoded a protein comprising 456 amino acids. We expressed His-tagged InuBK in culture medium using a Brevibacillus system. The optimal pH and temperature of purified InuBK were 7.0–9.0 and 50 °C–55 °C, respectively. The findings of high-performance anion-exchange chromatography, nuclear magnetic resonance spectroscopy, and high-performance size-exclusion chromatography with multi-angle laser light scattering showed that the polysaccharide produced by InuBK was an inulin with a molecular weight of 3,806, a polydispersity index (PI) of 1.047, and fructosyl chain lengths with 3–27 degrees of polymerization. The size of InuBK was smaller than commercial inulins, and the PI of the inulin that it produced was lower.

scholarly journals Bifidobacterium longum Endogalactanase Liberates Galactotriose from Type I Galactans

2005 ◽  
Vol 71 (9) ◽  
pp. 5501-5510 ◽  
Author(s):  
Sandra W. A. Hinz ◽  
Marieke I. Pastink ◽  
Lambertus A. M. van den Broek ◽  
Jean-Paul Vincken ◽  
Alphons G. J. Voragen
Keyword(s):  
Molecular Mass ◽  
Transmembrane Domain ◽  
Bifidobacterium Longum ◽  
Cell Extract ◽  
Size Exclusion ◽  
Glycoside Hydrolase Family ◽  
Type I ◽  
Native Molecular Mass ◽  
Exclusion Chromatography ◽  
Hydrolase Family

ABSTRACT A putative endogalactanase gene classified into glycoside hydrolase family 53 was revealed from the genome sequence of Bifidobacterium longum strain NCC2705 (Schell et al., Proc. Natl. Acad. Sci. USA 99:14422-14427, 2002). Since only a few endo-acting enzymes from bifidobacteria have been described, we have cloned this gene and characterized the enzyme in detail. The deduced amino acid sequence suggested that this enzyme was located extracellularly and anchored to the cell membrane. galA was cloned without the transmembrane domain into the pBluescript SK(−) vector and expressed in Escherichia coli. The enzyme was purified from the cell extract by anion-exchange and size exclusion chromatography. The purified enzyme had a native molecular mass of 329 kDa, and the subunits had a molecular mass of 94 kDa, which indicated that the enzyme occurred as a tetramer. The optimal pH of endogalactanase activity was 5.0, and the optimal temperature was 37°C, using azurine-cross-linked galactan (AZCL-galactan) as a substrate. The Km and V max for AZCL-galactan were 1.62 mM and 99 U/mg, respectively. The enzyme was able to liberate galactotrisaccharides from (β1→4)galactans and (β1→4)galactooligosaccharides, probably by a processive mechanism, moving toward the reducing end of the galactan chain after an initial midchain cleavage. GalA's mode of action was found to be different from that of an endogalactanase from Aspergillus aculeatus. The enzyme seemed to be able to cleave (β1→3) linkages. Arabinosyl side chains in, for example, potato galactan hindered GalA.

scholarly journals VirB8 homolog TraE from plasmid pKM101 forms a hexameric ring structure and interacts with the VirB6 homolog TraD

2018 ◽  
Vol 115 (23) ◽  
pp. 5950-5955 ◽  
Author(s):  
Bastien Casu ◽  
Charline Mary ◽  
Aleksandr Sverzhinsky ◽  
Aurélien Fouillen ◽  
Antonio Nanci ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Secretion System ◽  
High Molecular Mass ◽  
Full Length ◽  
Size Exclusion ◽  
Cross Linking ◽  
X Ray ◽  
Exclusion Chromatography ◽  
Plasmid Pkm101 ◽  
Membrane Bound

Type IV secretion systems (T4SSs) are multiprotein assemblies that translocate macromolecules across the cell envelope of bacteria. X-ray crystallographic and electron microscopy (EM) analyses have increasingly provided structural information on individual T4SS components and on the entire complex. As of now, relatively little information has been available on the exact localization of the inner membrane-bound T4SS components, notably the mostly periplasmic VirB8 protein and the very hydrophobic VirB6 protein. We show here that the membrane-bound, full-length version of the VirB8 homolog TraE from the plasmid pKM101 secretion system forms a high-molecular-mass complex that is distinct from the previously characterized periplasmic portion of the protein that forms dimers. Full-length TraE was extracted from the membranes with detergents, and analysis by size-exclusion chromatography, cross-linking, and size exclusion chromatography (SEC) multiangle light scattering (MALS) shows that it forms a high-molecular-mass complex. EM and small-angle X-ray scattering (SAXS) analysis demonstrate that full-length TraE forms a hexameric complex with a central pore. We also overproduced and purified the VirB6 homolog TraD and show by cross-linking, SEC, and EM that it binds to TraE. Our results suggest that TraE and TraD interact at the substrate translocation pore of the secretion system.

scholarly journals Trefoil Factor Family (TFF) Modules Are Characteristic Constituents of Separate Mucin Complexes in the Xenopus laevis Integumentary Mucus: In Vitro Binding Studies with FIM-A.1

2020 ◽  
Vol 21 (7) ◽  
pp. 2400 ◽  
Author(s):  
René Stürmer ◽  
Jana Reising ◽  
Werner Hoffmann
Keyword(s):  
Molecular Mass ◽  
Complex I ◽  
Size Exclusion ◽  
Trefoil Factor ◽  
Binding Studies ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Exclusion Chromatography ◽  
Trefoil Factor Family

The skin of the frog Xenopus laeevis is protected from microbial infections by a mucus barrier that contains frog integumentary mucins (FIM)-A.1, FIM-B.1, and FIM-C.1. These gel-forming mucins are synthesized in mucous glands consisting of ordinary mucous cells and one or more cone cells at the gland base. FIM-A.1 and FIM-C.1 are unique because their cysteine-rich domains belong to the trefoil factor family (TFF). Furthermore, FIM-A.1 is unusually short (about 400 amino acid residues). In contrast, FIM-B.1 contains cysteine-rich von Willebrand D (vWD) domains. Here, we separate skin extracts by the use of size exclusion chromatography and analyze the distribution of FIM-A.1 and FIM-C.1. Two mucin complexes were detected, i.e., a high-molecular-mass Complex I, which contains FIM-C.1 and little FIM-A.1, whereas Complex II is of lower molecular mass and contains the bulk of FIM-A.1. We purified FIM-A.1 by a combination of size-exclusion chromatography (SEC) and anion-exchange chromatography and performed first in vitro binding studies with radioactively labeled FIM-A.1. Binding of 125I-labeled FIM-A.1 to the high-molecular-mass Complex I was observed. We hypothesize that the presence of FIM-A.1 in Complex I is likely due to lectin interactions, e.g., with FIM-C.1, creating a complex mucus network.

Molecular mass determinations in coal-derived liquids by MALDI mass spectrometry and size-exclusion chromatography

Fuel ◽  
1997 ◽  
Vol 76 (13) ◽  
pp. 1225-1233 ◽  
Author(s):  
Maria-Jesus Lázaro ◽  
Alan A. Herod ◽  
Mike Cocksedge ◽  
Mark Domin ◽  
Rafael Kandiyoti
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mass ◽  
Size Exclusion Chromatography ◽  
Maldi Mass Spectrometry ◽  
Size Exclusion ◽  
Exclusion Chromatography

Oat spelts xylan molecular mass estimation by size exclusion chromatography

2003 ◽  
Vol 53 (3) ◽  
pp. 297-304 ◽  
Author(s):  
Andrei Sarbu ◽  
Fernando Gonçalves ◽  
Maria Norberta de Pinho
Keyword(s):  
Molecular Mass ◽  
Size Exclusion Chromatography ◽  
Size Exclusion ◽  
Mass Estimation ◽  
Exclusion Chromatography

scholarly journals Immunoassay for Sex Hormone-Binding Globulin in Undiluted Serum Is Influenced by High-Molecular-Mass Aggregates

2005 ◽  
Vol 51 (2) ◽  
pp. 401-407 ◽  
Author(s):  
Markus Thaler ◽  
Jochen Metzger ◽  
Anita Schreiegg ◽  
Barbara Denk ◽  
Andreas Gleixner ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Size Exclusion Chromatography ◽  
High Molecular Mass ◽  
Sex Hormone ◽  
Size Exclusion ◽  
Sex Hormone Binding Globulin ◽  
Hormone Binding ◽  
Spr Biosensor ◽  
Undiluted Serum ◽  
Exclusion Chromatography

Abstract Background: The new Elecsys® chemiluminescence assay for measurement of homodimeric sex hormone-binding globulin (SHBG) was designed for use with undiluted serum, in contrast to other methods that require predilution. During assay development, unexpected calibration difficulties were observed that were attributable to particular biochemical properties of the highly concentrated SHBG in solution. Methods: We used a surface plasmon resonance (SPR) biosensor, which enables biomolecular interaction analysis of SHBG, and size-exclusion chromatography for this investigation. The immunoassay was evaluated for imprecision, linearity, and suitability of the dilution medium, and the method was compared with an IRMA for SHBG. Results: The SPR biosensor characterized the special protein properties of SHBG in various concentrations. Above 200 nmol/L there was a strong tendency toward formation of high-molecular-mass aggregates. This was also detectable by size-exclusion chromatography and could be reversed by simple dilution of the sample. On the basis of these results, the dynamic measuring range of the SHBG assay is restricted to 0.350–200 nmol/L. Assay evaluation on a 2010 analyzer revealed excellent precision (CV ≤2.5%). Mean recoveries were 84.2–98.8%. Intermethod comparison with an IRMA yielded a satisfactory concordance of the two assays with a Spearman correlation coefficient of 0.8807. Conclusions: Aggregates of human SHBG may have a detrimental impact on the accurate measurement of the protein if measurements are performed with undiluted serum samples. Further work is needed to clarify whether these high-molecular-mass aggregates influence the free fraction of steroid hormones in vivo.

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