scholarly journals Identity and Substrate Specificity of Reductive Dehalogenases Expressed in Dehalococcoides-Containing Enrichment Cultures Maintained on Different Chlorinated Ethenes

2015 ◽  
Vol 81 (14) ◽  
pp. 4626-4633 ◽  
Author(s):  
Xiaoming Liang ◽  
Olivia Molenda ◽  
Shuiquan Tang ◽  
Elizabeth A. Edwards
Keyword(s):  
Specific Activity ◽  
Enrichment Culture ◽  
Polyacrylamide Gel Electrophoresis ◽  
Peptide Sequencing ◽  
Enzyme Assays ◽  
Content Type ◽  
Substrate Preferences ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
History Of ◽  
Different Strains

ABSTRACTMany reductive dehalogenases (RDases) have been identified in organohalide-respiring microorganisms, and yet their substrates, specific activities, and conditions for expression are not well understood. We tested whether RDase expression varied depending on the substrate-exposure history of reductive dechlorinating communities. For this purpose, we used the enrichment culture KB-1 maintained on trichloroethene (TCE), as well as subcultures maintained on the intermediatescis-dichloroethene (cDCE) and vinyl chloride (VC). KB-1 contains a TCE-to-cDCE dechlorinatingGeobacterand severalDehalococcoidesstrains that together harbor many of the known chloroethene reductases. Expressed RDases were identified using blue native polyacrylamide gel electrophoresis, enzyme assays in gel slices, and peptide sequencing. As anticipated but never previously quantified, the RDase fromGeobacterwas only detected transiently at the beginning of TCE dechlorination. TheDehalococcoidesRDase VcrA and smaller amounts of TceA were expressed in the parent KB-1 culture during complete dechlorination of TCE to ethene regardless of time point or amended substrate. TheDehalococcoidesRDase BvcA was only detected in enrichments maintained on cDCE as growth substrates, in roughly equal abundance to VcrA. Only VcrA was detected in subcultures enriched on VC. Enzyme assays revealed that 1,1-DCE, a substrate not used for culture enrichment, afforded the highest specific activity.trans-DCE was substantially dechlorinated only by extracts from cDCE enrichments expressing BvcA. RDase gene distribution indicated enrichment of different strains ofDehalococcoidesas a function of electron acceptor TCE, cDCE, or VC. Each chloroethene reductase has distinct substrate preferences leading to strain selection in mixed communities.

scholarly journals Dehalogenimonas sp. Strain WBC-2 Genome and Identification of Itstrans-Dichloroethene Reductive Dehalogenase, TdrA

2015 ◽  
Vol 82 (1) ◽  
pp. 40-50 ◽  
Author(s):  
Olivia Molenda ◽  
Andrew T. Quaile ◽  
Elizabeth A. Edwards
Keyword(s):  
Protein Identification ◽  
Enrichment Culture ◽  
Polyacrylamide Gel Electrophoresis ◽  
Functional Characterization ◽  
Genomic Islands ◽  
Pairwise Identity ◽  
Enzymatic Assays ◽  
Content Type ◽  
Reductive Dehalogenase ◽  
Native Polyacrylamide Gel Electrophoresis

ABSTRACTTheDehalogenimonaspopulation in a dechlorinating enrichment culture referred to as WBC-2 was previously shown to be responsible fortrans-dichloroethene (tDCE) hydrogenolysis to vinyl chloride (VC). In this study, blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzymatic assays and protein identification using liquid chromatography coupled with mass spectrometry (LC-MS/MS) led to the functional characterization of a novel dehalogenase, TdrA. This new reductive dehalogenase (RDase) catalyzes the dechlorination of tDCE to VC. A metagenome of the WBC-2 culture was sequenced, and a completeDehalogenimonasgenome, only the secondDehalogenimonasgenome to become publicly available, was closed. ThetdrAdehalogenase found within theDehalogenimonasgenome appears to be on a genomic island similar to genomic islands found inDehalococcoides. TdrA itself is most similar to TceA fromDehalococcoidessp. strain FL2 with 76.4% amino acid pairwise identity. It is likely that the horizontal transfer ofrdhAgenes is not only a feature ofDehalococcoidesbut also a feature of otherDehalococcoidia, includingDehalogenimonas.A set of primers was developed to tracktdrAin WBC-2 subcultures maintained on different electron acceptors. This newest dehalogenase is an addition to the short list of functionally defined RDases sharing the usual characteristic motifs (including an AB operon, a TAT export sequence, two iron-sulfur clusters, and a corrinoid binding domain), substrate flexibility, and evidence for horizontal gene transfer within theDehalococcoidia.

scholarly journals Functional Characterization of Reductive Dehalogenases by Using Blue Native Polyacrylamide Gel Electrophoresis

2012 ◽  
Vol 79 (3) ◽  
pp. 974-981 ◽  
Author(s):  
Shuiquan Tang ◽  
Winnie W. M. Chan ◽  
Kelly E. Fletcher ◽  
Jana Seifert ◽  
Xiaoming Liang ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Functional Characterization ◽  
Polyacrylamide Gel ◽  
Content Type ◽  
Reductive Dehalogenase ◽  
Dehalococcoides Mccartyi ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Rdase Genes ◽  
Blue Native

ABSTRACTDehalococcoides mccartyistrains are obligate organohalide-respiring bacteria harboring multiple distinct reductive dehalogenase (RDase) genes within their genomes. A major challenge is to identify substrates for the enzymes encoded by these RDase genes. We demonstrate an approach that involves blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzyme activity assays with gel slices and subsequent identification of proteins in gel slices using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RDase expression was investigated in cultures ofDehalococcoides mccartyistrain BAV1 and in the KB-1 consortium growing on chlorinated ethenes and 1,2-dichloroethane. In cultures of strain BAV1, BvcA was the only RDase detected, revealing that this enzyme catalyzes the dechlorination not only of vinyl chloride, but also of all dichloroethene isomers and 1,2-dichloroethane. In cultures of consortium KB-1, five distinctDehalococcoidesRDases and oneGeobacterRDase were expressed under the conditions tested. Three of the five RDases included orthologs to the previously identified chlorinated ethene-dechlorinating enzymes VcrA, BvcA, and TceA. This study revealed substrate promiscuity for these three enzymes and provides a path forward to further explore the largely unknown RDase protein family.

scholarly journals Isolation of a human hepatic 60 kDa aspartylglucosaminidase consisting of three non-identical polypeptides

1989 ◽  
Vol 262 (1) ◽  
pp. 189-194 ◽  
Author(s):  
M Baumann ◽  
L Peltonen ◽  
P Aula ◽  
N Kalkkinen
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Inherited Disease ◽  
Ph Optimum ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Molecular Masses ◽  
Polypeptide Chains

We have characterized the properties of human aspartylglucosaminidase (EC 3.5.1.26), the lysosomal enzyme which is deficient in the human inherited disease aspartylglucosaminuria. The purification procedure from human liver included affinity chromatography, gel filtration, strong-anion- and strong-cation-exchange h.p.l.c., chromatofocusing and reverse-phase h.p.l.c. In a denaturing SDS/polyacrylamide-gel electrophoresis, the 6600-fold purified enzyme was shown to be composed of three non-identical inactive polypeptide chains of molecular masses 24, 18 and 17 kDa. In a native polyacrylamide-gel electrophoresis, these polypeptide chains ran as one active enzyme complex. As judged from the elution position of the native enzyme in a Biogel P-100 gel filtration, the approximate molecular mass of this complex was 60 kDa. The enzyme had a pI of 5.7, a pH optimum at 6, of 0.48 mM and a specific activity of 200,000 nkat for the substrate 2-acetamido-1-beta-(L-aspartamido)-1,2-dideoxy-D-glucose. The enzyme showed a 57% loss of activity at 60 degrees C after 45 h but was practically inactive after incubation at 72 degrees C for a few minutes. The molecular structure, Km and specific activity as well as the thermostability of the enzyme described here are different from those reported previously for human aspartylglucosaminidase.

Actinobacterial chitinase-like enzymes: profiles of rhizosphere versus non-rhizosphere isolates

2003 ◽  
Vol 49 (11) ◽  
pp. 683-698 ◽  
Author(s):  
Ana C Gonzalez-Franco ◽  
Lee A Deobald ◽  
Aaron Spivak ◽  
Don L Crawford
Keyword(s):  
Cell Wall ◽  
Gel Electrophoresis ◽  
Rhizosphere Soil ◽  
Polyacrylamide Gel Electrophoresis ◽  
Hydrolytic Enzymes ◽  
Fungal Cell ◽  
Native Page ◽  
Substrate Preferences ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Over Time

The objective of this study was to determine if antifungal actinomycetes isolated from rhizosphere and non-rhizosphere soils exhibit different chitinase-like production and (or) induction patterns. Selected isolates from both habitats were compared. Chitinase-like levels and isoform characteristic patterns were evaluated over time in culture fluids of isolates grown on media containing different combinations of colloidal chitin and fungal cell wall (FCW) preparation. Supernatants were also subjected to native and non-native polyacrylamide gel electrophoresis (PAGE), using glycol chitin amended gels. For non-native PAGE, protein samples were denatured by two different approaches. Multiple active bands, ranging from 20 to 53 kDa and present in varying amounts, were detected in gels for most strains. Different substrate preferences were observed among strains, and different chitinase-like enzymes were produced, depending upon the substrate combinations used. The presence of FCW in the medium induced specific chitinase-like enzymes not observed otherwise. Enzymatic activities and profiles of the isolates, however, were strain and substrate specific rather than habitat specific. However, a sagebrush rhizosphere soil had a larger actinomycete community with higher chitinolytic activities than the nearby bulk soil. The use of PAGE to compare chitinase-like proteins induced in media with and without FCW was useful for identifying chitinase-like enzymes potentially involved in antifungal activity.Key words: chitinase, actinomycetes, hydrolytic enzymes, rhizosphere, antifungal.

Native polyacrylamide gel electrophoresis of membrane proteins: Glutaminase detection afterin situ specific activity staining

1993 ◽  
Vol 14 (1) ◽  
pp. 88-93 ◽  
Author(s):  
Juan C. Aledo ◽  
Simón Gómez-Biedma ◽  
Juan A. Segura ◽  
Manuel Molina ◽  
Ignacio Núñez de Castro ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Activity Staining ◽  
Native Polyacrylamide Gel Electrophoresis

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

A tribute to Gwen Leighty

2006 ◽  
Vol 19 (2) ◽  
pp. 84-86
Author(s):  
Jennifer Paustenbaugh
Keyword(s):  
Web Sites ◽  
Academic Libraries ◽  
Design Methodology ◽  
Fund Raising ◽  
Personal Knowledge ◽  
Valuable Contribution ◽  
Content Type ◽  
History Of ◽  
Development Network ◽  
Fund Raiser

PurposeThe purpose of the paper is to provide a tribute to the life and work of library fund‐raiser Gwen Leighty.Design/methodology/approachThe paper uses personal knowledge and references to Academic Libraries Advancement and Development Network (ALADN) and LIBDEV web sites.FindingsThe paper finds that fundraising is connecting with people and the journey that each development officer must make while raising funds for their library.Originality/valueThe paper presents a brief history of ALADN and the valuable contribution one person made to the cause of library fund‐raising.

scholarly journals The Native Monomer of Bacillus Pumilus Ribonuclease Does Not Exist Extracellularly

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Olga Ilinskaya ◽  
Vera Ulyanova ◽  
Irina Lisevich ◽  
Elena Dudkina ◽  
Nataliya Zakharchenko ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Concentration ◽  
Bacillus Pumilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Culture Fluid ◽  
Size Exclusion ◽  
High Protein Concentration ◽  
Exclusion Chromatography ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Hypochromic Effect

Supported by crystallography studies, secreted ribonuclease of Bacillus pumilus (binase) has long been considered to be monomeric in form. Recent evidence obtained using native polyacrylamide gel electrophoresis and size-exclusion chromatography suggests that binase is in fact dimeric. To eliminate ambiguity and contradictions in the data we have measured conformational changes, hypochromic effect, and hydrodynamic radius of binase. The immutability of binase secondary structure upon transition from low to high protein concentration was registered, suggesting the binase dimerization immediately after translocation through the cell membrane and leading to detection of binase dimers only in the culture fluid regardless of ribonuclease concentration. Our results made it necessary to take a fresh look at the binase stability and cytotoxicity towards virus-infected or tumor cells.

Glycogen phosphorylase in fish muscle: demonstration of three interconvertible forms

1990 ◽  
Vol 258 (2) ◽  
pp. C344-C351 ◽  
Author(s):  
H. Schmidt ◽  
G. Wegener
Keyword(s):  
Glycogen Phosphorylase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Muscular Activity ◽  
Fish Muscle ◽  
Kinetic Properties ◽  
Phosphorylase Activity ◽  
Tissue Extracts

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.

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