scholarly journals Novel NADP-linked alcohol–aldehyde/ketone oxidoreductase in thermophilic ethanologenic bacteria

1981 ◽  
Vol 195 (1) ◽  
pp. 183-190 ◽  
Author(s):  
R J Lamed ◽  
J G Zeikus
Keyword(s):  
Metal Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Secondary Alcohol ◽  
Sodium Dodecyl ◽  
Secondary Alcohols ◽  
Metal Ion Binding ◽  
Primary Alcohols ◽  
Cell Extracts ◽  
Alcohol Dehydrogenase Activity ◽  
Clostridium Thermohydrosulfuricum

An NADP-specific alcohol--aldehyde/ketone oxidoreductase was detected in cell extracts of Thermoanaerobium brockii and Clostridium thermohydrosulfuricum, but not in Thermobacteroides acetoethylicus or Clostridium thermocellum. The enzyme was purified from Ta. brockii by differential procedures that included heat treatment and an affinity-chromatography step on Blue Dextran--Sepharose. The 44-fold-purified enzyme displayed one band (mol.wt. approx. 40000) after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme had a broad substrate specificity that included linear and branched primary alcohols, linear and cyclic secondary alcohols, linear and cyclic ketones, and acetaldehyde. The NADP-specific alcohol--aldehyde/ketone oxidoreductase was considerably more active towards secondary alcohols than towards other substrates. The enzyme had remarkable stability to heating at 86 degrees C for 70 min, but was rapidly denatured on boiling. Secondary-alcohol dehydrogenase activity displayed a noticeable inflexion point at 50 degrees C in Arrhenius plots and a high Q10 value (greater than 2.0). The enzyme was inactivated by the thiol-blocking reagent p-chloromercuribenzoate, but was not significantly inhibited by common metal-ion-binding agents. The NADP-linked alcohol--aldehyde/ketone oxidoreductase of Ta. brockii appears to have properties distinct from those of previously described primary- and secondary-alcohol dehydrogenases.

Regulation of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldDH) in Aspergillus nidulans

1983 ◽  
Vol 217 (1208) ◽  
pp. 243-264 ◽  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Aspergillus Nidulans ◽  
Structural Gene ◽  
Aldehyde Dehydrogenase ◽  
Gel Electrophoresis ◽  
Regulatory Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Cell Extracts

There is a single major alcohol dehydrogenase (ADH) and a single major aldehyde dehydrogenase (AldDH) in Aspergillus nidulans . Both ADH and AldDH are induced by ethanol and by acetaldehyde and both are subject to carbon catabolite repression. ADH and AldDH are necessary for the utilization of ethanol and of threonine, indicating that both compounds are utilized via acetaldehyde. ADH and AldDH each give a single major activity band on gel electrophoresis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell extracts shows at least two similar ADH polypeptides of approximate relative molecular mass (r. m. m.) 41000 and two similar AldDH polypeptides of approximate r. m. m. 57000. The in vitro translation of mRNA from induced, carbon derepressed wild-type cells gives up to three ADH polypeptides in the r. m. m. range 39000-43000 and an AldDH polypeptide of approximate r. m. m. 57000. The mRNA from uninduced, carbon repressed wild-type cells does not direct the synthesis of the ADH and AldDH polypeptides. This indicates that the regulation of ADH and AldDH is at the level of transcription and/or post-transcriptional modification. The probable explanation of the multiple ADH polypeptides is post-transcriptional modification of the mRNA. Allyl alcohol mutants were made by using diepoxyoctane and γ-rays as mutagens. There are two classes, alcA and alcR . Neither class can utilize ethanol or threonine as a carbon source. The alcA mutants lack normal ADH and are recessive. Of the 47 alcA mutants examined 39 do not make the ADH polypeptides while eight do so. Therefore alcA is the structural gene for ADH. The two alcA mutants tested do not make functional mRNA for ADH. The alcR mutants lack both ADH and AldDH and are recessive. No alcR mutants make the ADH or the AldDH polypeptides. The three alcR mutants tested do not make functional ADH or AldDH mRNA. The mutant alcR 125 is a nonsense mutant, which establishes that alcR codes for a protein. The alcA and alcR genes are adjacent on chromosome VII and a preliminary fine-structure map of the alcA gene has been made. Three mutants that cannot utilize ethanol or threonine and have ADH, but lack AldDH, define a gene AldA on chromosome VIII. The aldA 23 mutant makes the AldDH polypeptides, the other two aldA mutants do not. Therefore aldA is probably the structural gene for AldDH. Our current hypothesis is that alcA and aldA are the structural genes for ADH and AldDH respectively and alcR is a transacting regulatory gene coding for a protein whose function is necessary for the expression of the alcA and aldA genes.

Plant NAD-Dependent Glutamate Dehydrogenase. Purification, Molecular Properties and Metal Ion Activation of the Enzymes from Lemna minor and Pisum sativum

1980 ◽  
Vol 35 (3-4) ◽  
pp. 213-221 ◽  
Author(s):  
Heinz-Walter Scheid ◽  
Adelheid Ehmke ◽  
Thomas Hartmann
Keyword(s):  
Amino Acid ◽  
Pisum Sativum ◽  
Glutamate Dehydrogenase ◽  
Gel Electrophoresis ◽  
Metal Ion ◽  
Lemna Minor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Equilibrium ◽  
Electron Microscopic

Abstract Glutamate dehydrogenase (ʟ-glutamate: NAD+ oxidoreductase (deaminating) EC 1.4.1.2) has been purified to homogeneity from Lemna minor and seeds of Pisum sativum. As established by polyacrylamide gel electrophoresis the Pisum-enzyme constitutes a multiple pattern of seven char­ge isoenzymes whereas the Lemna enzyme shows one single protein band. Molecular weights of 230 000 were calculated for both enzymes by sedimentation equilibrium measurements (Pisum-enzyme) and comparative gel filtration (Lemna-enzyme). Sodium dodecyl sulfate gel electrophoresis and electron microscopic observations revealed that both enzymes are composed of four identical subunits (molecular weight 58 500) arranged in a tetraedric structure. The amino acid compositions of both enzymes are similar to those of various hexameric glutamate dehydrogenases. The N-terminal amino acid of the Pisum-enzyme is alanine. Both enzymes require Ca2+ for maximal catalytic activity. For the Lemna-enzyme the K0.5 values for Ca2+ are 22 µᴍ (NADH-dependent reaction) and 4 µᴍ (NAD+ -dependent reaction), respectively. Ca2+ which to some extent can be replaced by Zn2+ does not affect the enzyme aggregation but seems to govern a reversible equilibrium between catalytically active and inactive enzyme forms.

scholarly journals Chemically defined inducers of alkylsulphatases present in Pseudomonas C12B

1974 ◽  
Vol 138 (1) ◽  
pp. 53-62 ◽  
Author(s):  
Kenneth S. Dodgson ◽  
John W. Fitzgerald ◽  
William J. Payne
Keyword(s):  
Secondary Alcohol ◽  
Sodium Dodecyl ◽  
Nutrient Broth ◽  
Phase Cell ◽  
Alkyl Sulphate ◽  
Cell Extracts ◽  
Complex Process ◽  
Commercial Detergent ◽  
Process Studies ◽  
Long Chain

When Pseudomonas C12B is grown on nutrient broth to the stationary phase, cell extracts contain two secondary alkylsulphatases (S1 and S2) active towards potassium decan-5-yl sulphate but not towards potassium pentan-3-yl sulphate and one primary alkylsulphatase (P1) active towards sodium dodecan-1-yl sulphate (sodium dodecyl sulphate). When 10mm-sodium hexan-1-yl sulphate is included in the nutrient broth an additional primary alkylsulphatase (P2) is produced. The S1, S2, P1 and P2 enzymes are also present in extracts of cells grown on broth containing the commercial detergent Oronite, together with an additional secondary alkylsulphatase (S3) active towards pentan-3-yl sulphate as well as decan-5-yl sulphate. The P2 primary alkylsulphatase can be induced by a number of primary and secondary alkyl sulphate esters but the induction of the S3 enzyme appears to be a more specific and complex process. Studies on the ability of different fractions separated from Oronite to act as inducers suggest that the combination of a long-chain secondary alkyl sulphate(s) and a long-chain secondary alcohol(s) is responsible for the appearance of the S3 enzyme. Potassium hexadecan-2-yl sulphate or potassium tetradecan-2-yl sulphate, in combination with either hexadecan-2-ol or tetradecan-2-ol, can serve as inducers for the enzyme. Some characteristics of these specific inducer systems have been elucidated.

scholarly journals Alpha 2-macroglobulin binds and inhibits activated protein C

Blood ◽  
1991 ◽  
Vol 78 (9) ◽  
pp. 2283-2290 ◽  
Author(s):  
H Hoogendoorn ◽  
CH Toh ◽  
ME Nesheim ◽  
AR Giles
Keyword(s):  
Complex Formation ◽  
Active Site ◽  
Metal Ion ◽  
Protein C ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
High Molecular Weight Complex

In previous studies using a nonhuman primate model of Protein C (PC) activation in vivo, immunoblotting showed substantial amounts of activated PC (APC) in a high molecular weight complex with what was presumed to be a previously unrecognized APC binding protein. This APC complex can also be formed in citrated plasma in vitro. It is of low electrophoretic mobility, sodium dodecyl sulfate (SDS) stable, with an apparent Mr of 320 Kd. Its purification from human plasma was accomplished using barium citrate adsorption, sequential polyethylene glycol (PEG) precipitations, diethylaminoethyl sepharose chromatography, AcA-34 gel filtration, and zinc-chelate affinity chromatography. This was monitored by subjecting the fractions to nondenaturing polyacrylamide gel electrophoresis (PAGE), transfer to polyvinylidene-difluoride membranes, and probing with 125I-labeled human APC. The purified APC-binding protein was homogeneous by SDS-PAGE with an Mr of 275 Kd. Its identity as alpha 2-macroglobulin (alpha 2M) was demonstrated immunochemically. Complex formation between alpha 2M and APC was found to be almost completely inhibited by EDTA, but to a lesser extent by citrate. Complex formation could also be prevented by active site inhibition with D-Phenylalanyl-L-Prolyl-L-Arginine- Chloromethyl Ketone (PPACK) or pretreatment of alpha 2M with methylamine. Incubation of APC (33 nmol/L) with alpha 2M (1 mumol/L) resulted in time-dependent inhibition of APC anticoagulant activity when measured using an activated partial thromboplastin time based APC assay. These data show that alpha 2M binds and inhibits APC in vitro and the interaction is both metal-ion and active-site dependent, requiring functionally intact alpha 2M. As the complexes formed in vitro comigrate electrophoretically with those observed in vivo after PC activation, it is suggested that alpha 2M is a physiologically relevant inhibitor involved in the processing of APC in vivo.

scholarly journals Purification, Characterization, and Sequence Analysis of 2-Aminomuconic 6-Semialdehyde Dehydrogenase from Pseudomonas pseudoalcaligenes JS45

1998 ◽  
Vol 180 (17) ◽  
pp. 4591-4595 ◽  
Author(s):  
Zhongqi He ◽  
John K. Davis ◽  
Jim C. Spain
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Gene Encoding ◽  
Multiple Sequence ◽  
Pseudomonas Pseudoalcaligenes ◽  
Semialdehyde Dehydrogenase ◽  
Cell Extracts ◽  
High Degree

ABSTRACT 2-Aminonumconic 6-semialdehyde is an unstable intermediate in the biodegradation of nitrobenzene and 2-aminophenol by Pseudomonas pseudoalcaligenes JS45. Previous work has shown that enzymes in cell extracts convert 2-aminophenol to 2-aminomuconate in the presence of NAD+. In the present work, 2-aminomuconic semialdehyde dehydrogenase was purified and characterized. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 57 kDa. The molecular mass of the native enzyme was estimated to be 160 kDa by gel filtration chromatography. The optimal pH for the enzyme activity was 7.3. The enzyme is able to oxidize several aldehyde analogs, including 2-hydroxymuconic semialdehyde, hexaldehyde, and benzaldehyde. The gene encoding 2-aminomuconic semialdehyde dehydrogenase was identified by matching the deduced N-terminal amino acid sequence of the gene with the first 21 amino acids of the purified protein. Multiple sequence alignment of various semialdehyde dehydrogenase protein sequences indicates that 2-aminomuconic 6-semialdehyde dehydrogenase has a high degree of identity with 2-hydroxymuconic 6-semialdehyde dehydrogenases.

scholarly journals Cellobiose-6-Phosphate Hydrolase (CelF) ofEscherichia coli: Characterization and Assignment to the Unusual Family 4 of Glycosylhydrolases

1999 ◽  
Vol 181 (23) ◽  
pp. 7339-7345 ◽  
Author(s):  
John Thompson ◽  
Sergei B. Ruvinov ◽  
Darón I. Freedberg ◽  
Barry G. Hall
Keyword(s):  
Metal Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Divalent Metal ◽  
Oligomeric Structure ◽  
Sequence Alignments ◽  
Functional Roles ◽  
Catalytically Active ◽  
Divalent Metal Ion ◽  
Substrate Cleavage

ABSTRACT The gene celF of the cryptic cel operon ofEscherichia coli has been cloned, and the encoded 6-phospho-β-glucosidase (cellobiose-6-phosphate [6P] hydrolase; CelF [EC 3.2.1.86 ]) has been expressed and purified in a catalytically active state. Among phospho-β-glycosidases, CelF exhibits unique requirements for a divalent metal ion and NAD+ for activity and, by sequence alignment, is assigned to family 4 of the glycosylhydrolase superfamily. CelF hydrolyzed a variety of P-β-glucosides, including cellobiose-6P, salicin-6P, arbutin-6P, gentiobiose-6P, methyl-β-glucoside-6P, and the chromogenic analog,p-nitrophenyl-β-d-glucopyranoside-6P. In the absence of a metal ion and NAD+, purified CelF was rapidly and irreversibly inactivated. The functional roles of the cofactors have not been established, but NAD+ appears not to be a reactant and there is no evidence for reduction of the nucleotide during substrate cleavage. In solution, native CelF exists as a homotetramer (M w, ∼200,000) composed of noncovalently linked subunits, and this oligomeric structure is maintained independently of the presence or absence of a metal ion. The molecular weight of the CelF monomer (M r, ∼50,000), estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is in agreement with that calculated from the amino acid sequence of the polypeptide (450 residues;M r = 50,512). Comparative sequence alignments provide tentative identification of the NAD+-binding domain (residues 7 to 40) and catalytically important glutamyl residues (Glu112 and Glu356) of CelF.

scholarly journals Evidence for an Inducible Nucleotide-Dependent Acetone Carboxylase in Rhodococcus rhodochrousB276

1999 ◽  
Vol 181 (9) ◽  
pp. 2752-2758 ◽  
Author(s):  
Daniel D. Clark ◽  
Scott A. Ensign
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Time Dependent ◽  
Nucleoside Triphosphate ◽  
Chromatography Analysis ◽  
Cell Extracts ◽  
Nocardia Corallina ◽  
Common Strategy ◽  
Molecular Masses ◽  
Do So

ABSTRACT The metabolism of acetone was investigated in the actinomyceteRhodococcus rhodochrous (formerly Nocardia corallina) B276. Suspensions of acetone- and isopropanol-grownR. rhodochrous readily metabolized acetone. In contrast,R. rhodochrous cells cultured with glucose as the carbon source lacked the ability to metabolize acetone at the onset of the assay but gained the ability to do so in a time-dependent fashion. Chloramphenicol and rifampin prevented the time-dependent increase in this activity. Acetone metabolism by R. rhodochrous was CO2 dependent, and 14CO2 fixation occurred concomitant with this process. A nucleotide-dependent acetone carboxylase was partially purified from cell extracts of acetone-grownR. rhodochrous by DEAE-Sepharose chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the acetone carboxylase was composed of three subunits with apparent molecular masses of 85, 74, and 16 kDa. Acetone metabolism by the partially purified enzyme was dependent on the presence of a divalent metal and a nucleoside triphosphate. GTP and ITP supported the highest rates of acetone carboxylation, while CTP, UTP, and XTP supported carboxylation at 10 to 50% of these rates. ATP did not support acetone carboxylation. Acetoacetate was determined to be the stoichiometric product of acetone carboxylation. The longer-chain ketones butanone, 2-pentanone, 3-pentanone, and 2-hexanone were substrates. This work has identified an acetone carboxylase with a novel nucleotide usage and broader substrate specificity compared to other such enzymes studied to date. These results strengthen the proposal that carboxylation is a common strategy used for acetone catabolism in aerobic acetone-oxidizing bacteria.

scholarly journals Metal Ion-Chelated Tannic Acid Coating for Hemostatic Dressing

Materials ◽  
2019 ◽  
Vol 12 (11) ◽  
pp. 1803 ◽  
Author(s):  
Bing Song ◽  
Liwei Yang ◽  
Lulu Han ◽  
Lingyun Jia
Keyword(s):  
Tannic Acid ◽  
Metal Ion ◽  
Blood Clotting ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fluorescence Signal ◽  
Large Area ◽  
Sds Page ◽  
Hemostatic Dressing ◽  
Acid Coating

Tannic acid (TA), a high-molecular-weight polyphenol, is used as a hemostasis spray and unguent for trauma wound remedy in traditional medical treatment. However, the use of tannic acid on a large-area wound would lead to absorption poisoning. In this work, a TA coating was assembled on a quartz/silicon slide, or medical gauze, via chelation interaction between TA and Fe3+ ions and for further use as a hemostasis dressing. Protein adsorption on the TA coating was further investigated by fluorescence signal, ellipsometry analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The adsorbed bovine serum albumin (BSA), immunoglobulin G (IgG) and fibrinogen (Fgn) on the TA coating was in the manner of monolayer saturation adsorption, and fibrinogen showed the largest adsorption. Furthermore, we found the slight hemolysis of the TA coating caused by the lysed red blood cells and adsorption of protein, especially the clotting-related fibrinogen, resulted in excellent hemostasis performance of the TA coating in the blood clotting of an animal wound. Thus, this economic, environmentally friendly, flexible TA coating has potential in medical applications as a means of preparing novel hemostasis materials.

Biological characteristics of recombinant enhancin ofTrichoplusia nigranulovirus expressed inEscherichia coli

2006 ◽  
Vol 3 (3) ◽  
pp. 171-176
Author(s):  
Yuan Zhe-Ming ◽  
Meng Xiao-Lin ◽  
Liu Shu-Sheng
Keyword(s):  
Trichoplusia Ni ◽  
Metal Ion ◽  
Inclusion Bodies ◽  
Ethylenediaminetetraacetic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Biological Characteristics ◽  
Cotton Bollworm ◽  
Peritrophic Membrane

AbstractA recombinant enhancin fromTrichoplusia nigranulovirus, expressed inEscherichia coliand named P96, had been shown in a previous study to increase significantly the efficacy of bio-insecticides applied to insect pests. Here, several biological characteristics were clarified to provide further proof for the activities of P96. The inclusion bodies formed by P96 were quite stable when expressed inE. coli; however, they could be effectively dissolved in sodium carbonate (Na2CO3) solution or in the midgut digestive secretion of the fifth-instar larvae of the cotton bollworm,Helicoverpa armigera,in vitro. According to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), the peritrophic membrane of the cotton bollworm was clearly decomposed by P96. With the increase of P96 concentration, the mortality of the cotton bollworm caused by nuclear polyhedrosis virus increased up to its saturation concentration, 6.76×105inclusion bodies/ml. The bioactivity of P96 was strongly inhibited by ethylenediaminetetraacetic acid (EDTA), which formed a complex compound with the metal ion (Zn2+) in the protein.

scholarly journals Rhizobium etli Genetically Engineered for the Heterologous Expression of Vitreoscilla sp. Hemoglobin: Effects on Free-Living and Symbiosis

1999 ◽  
Vol 12 (11) ◽  
pp. 1008-1015 ◽  
Author(s):  
Mario Ramírez ◽  
Brenda Valderrama ◽  
Raúl Arredondo-Peter ◽  
Mario Soberón ◽  
Jaime Mora ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Heterologous Expression ◽  
Energy Content ◽  
Nitrogenase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rhizobium Etli ◽  
Free Living ◽  
Cell Extracts ◽  
Bean Plants

Oxygen concentration is an environmental signal that regulates nitrogen fixation in the Rhizobium-legume symbiosis. We investigated the effect of the heterologous expression of Vitreoscilla sp. hemoglobin (VHb), which is an oxygen-binding protein, in Rhizobium etli. The vhb gene and its native promoter were subcloned in the plasmid pMR4 and transformed into the R. etli strain CE3. Free-living cultures of engineered R. etli CE3 expressed the vhb gene, as shown by the CO-difference spectral and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of cell extracts. The expression of vhb in free-living R. etli grown under most limiting oxygen concentrations resulted in an increase in respiratory activity, chemical energy content, and expression of the nitrogen-fixation gene nifHc. Bacteroids isolated from nodules of bean plants inoculated with the engineered R. etli CE3 expressed the vhb gene, as shown by RNA slot-blot analysis. Bean plants inoculated with the engineered strain exhibited higher nitrogenase activity and total nitrogen content (68% and 14 to 53%, respectively) than bean plants inoculated with the R. etli wild type. These results suggest that the synthesis of VHb in engineered R. etli stimulated the respiratory efficiency of free-living rhizobia, and also probably of symbiotic bacteroids, thus leading to higher levels of symbiotic nitrogen fixation.

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