scholarly journals Polyphosphate Kinase Terminal Modifications Alter Enzymatic Activity and Affect Stress Recovery in E. coli

2021 ◽  
Vol 35 (S1) ◽  
Author(s):  
Marvin Bowlin ◽  
Michael Gray
Keyword(s):  
Enzymatic Activity ◽  
Stress Recovery ◽  
Polyphosphate Kinase ◽  
E Coli

scholarly journals Characterization of the enzymatic activity of the serine protease domain of Factor VII activating protease (FSAP)

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Nis V. Nielsen ◽  
Elfie Roedel ◽  
Dipankar Manna ◽  
Michael Etscheid ◽  
Jens Preben Morth ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Serine Protease ◽  
Active Site ◽  
Factor Vii ◽  
Protease Domain ◽  
Functional Changes ◽  
E Coli ◽  
Catalytic Activation ◽  
Serine Protease Domain ◽  
Tissue Factor Pathway

AbstractFactor VII (FVII) activating protease (FSAP) is a circulating serine protease. Human genetic studies, based on the Marburg I (MI) (Gly221Glu, chymotrypsin numbering system) polymorphism, implicate FSAP in the pathogenesis of many diseases. Here, we describe the molecular and functional changes caused by the Gly221Glu substitution in the 220 loop using recombinant proteins expressed in E. coli. The serine protease domain (SPD) of wild type (WT) FSAP displayed auto-catalytic activation whereas the MI isoform displayed very low autocatalytic activation and low proteolytic activity against the chromogenic substrate S-2288, Factor VII, tissue factor pathway inhibitor as well as pro-urokinase. Introduction of a thermolysin cleavage site in the activation position (Arg15Gln) led to cleavage of both WT- and MI-SPD and the resulting WT-SPD, but not the MI-SPD, was active. Mutating the Gly221 position to Asp, Gln and Leu led to a loss of activity whereas the Ala substitution was partially active. These results suggest a disturbance of the active site, or non-accessibility of the substrate to the active site in MI-SPD. With respect to regulation with metal ions, calcium, more than sodium, increased the enzymatic activity of WT-SPD. Thus, we describe a novel method for the production of recombinant FSAP-SPD to understand the role of the MI-single nucleotide polymorphism (SNP) in the regulation of its activity.

scholarly journals Enzyme Characteristics of β-d-Galactosidase- and β-d-Glucuronidase-Positive Bacteria and Their Interference in Rapid Methods for Detection of Waterborne Coliforms andEscherichia coli

1998 ◽  
Vol 64 (3) ◽  
pp. 1018-1023 ◽  
Author(s):  
I. Tryland ◽  
L. Fiksdal
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Enzymatic Activity ◽  
Environmental Water ◽  
Coliform Bacteria ◽  
Galactosidase Activity ◽  
Fluorogenic Substrates ◽  
Rapid Methods ◽  
E Coli ◽  
Aerococcus Viridans

ABSTRACT Bacteria which were β-d-galactosidase and β-d-glucuronidase positive or expressed only one of these enzymes were isolated from environmental water samples. The enzymatic activity of these bacteria was measured in 25-min assays by using the fluorogenic substrates 4-methylumbelliferyl-β-d-galactoside and 4-methylumbelliferyl-β-d-glucuronide. The enzyme activity, enzyme induction, and enzyme temperature characteristics of target and nontarget bacteria in assays aimed at detecting coliform bacteria and Escherichia coli were investigated. The potential interference of false-positive bacteria was evaluated. Several of the β-d-galactosidase-positive nontarget bacteria but none of the β-d-glucuronidase-positive nontarget bacteria contained unstable enzyme at 44.5°C. The activity of target bacteria was highly inducible. Nontarget bacteria were induced much less or were not induced by the inducers used. The results revealed large variations in the enzyme levels of different β-d-galactosidase- and β-d-glucuronidase-positive bacteria. The induced and noninduced β-d-glucuronidase activities ofBacillus spp. and Aerococcus viridans were approximately the same as the activities of induced E. coli. Except for some isolates identified asAeromonas spp., all of the induced and noninduced β-d-galactosidase-positive, noncoliform isolates exhibited at least 2 log units less mean β-d-galactosidase activity than induced E. coli. The noncoliform bacteria must be present in correspondingly higher concentrations than those of target bacteria to interfere in the rapid assay for detection of coliform bacteria.

scholarly journals E. coli-Derived L-Asparaginase Retains Enzymatic and Cytotoxic ActivityIn Vitrofor Canine and Feline Lymphoma after Cold Storage

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Jackie M. Wypij ◽  
Holly C. Pondenis
Keyword(s):  
Enzymatic Activity ◽  
Cold Storage ◽  
Therapeutic Option ◽  
Clinical Importance ◽  
Scientific Basis ◽  
Antineoplastic Activity ◽  
Lymphoma Cells ◽  
E Coli ◽  
Freeze Thaw ◽  
Activity Duration

Background. L-asparaginase is effective in treating canine and feline lymphoma, however chemotherapy poses a significant financial cost to veterinary clients, limiting therapy for many pets. Single dose vials result in significant drug wastage, and drug shortages limit consistent availability for pets.Hypothesis.E. coli-derived asparaginase retains enzymatic and antineoplastic activity in canine and feline lymphoma cells after cold storage.Methods.E. coli-derived asparaginase was cold-stored: refrigeration (7–14 days) and freezing (14 days–six months, one to three freeze/thaw cycles). Enzymatic activity of asparaginase was measured via a modified asparagine assay. Effects of cold-stored asparaginase on cell proliferation and cytotoxicity were measured in feline (MYA-1, F1B) and canine (17–71, OSW) lymphoma cells.Results. Cold-storedE. coli-derived asparaginase retains antineoplastic activity in all four cell lines tested. Cold-storedE. coli-derived L-asparaginase depletes asparagine and retains enzymatic activity. Duration of refrigeration, duration of freezing, and number of freeze-thaw cycles have minimal effect on asparaginase enzyme activity.Conclusions and Clinical Importance. This study establishes a scientific basis for long-term cold storage of reconstitutedE. coli-derived asparaginase that may result in better utilization of limited drug resources and improve financial feasibility ofE. coli-derived asparaginase as a therapeutic option for pets with lymphoma.

Improvement of stability and enzymatic activity by site-directed mutagenesis of E. coli asparaginase II

2014 ◽  
Vol 1844 (7) ◽  
pp. 1219-1230 ◽  
Author(s):  
Shikha Verma ◽  
Ranjit Kumar Mehta ◽  
Prasanta Maiti ◽  
Klaus-Heinrich Röhm ◽  
Avinash Sonawane
Keyword(s):  
Enzymatic Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
E Coli

Effects of various salts on the steady-state enzymatic activity of E. coli alkaline phosphatase

1993 ◽  
Vol 50 (3) ◽  
pp. 173-180 ◽  
Author(s):  
Richard W. Poe ◽  
Vani S. Sangadala ◽  
John M. Brewer
Keyword(s):  
Alkaline Phosphatase ◽  
Steady State ◽  
Enzymatic Activity ◽  
E Coli

scholarly journals Overexpression of Soluble Recombinant Human Lysyl Oxidase by Using Solubility Tags: Effects on Activity and Solubility

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Madison A. Smith ◽  
Jesica Gonzalez ◽  
Anjum Hussain ◽  
Rachel N. Oldfield ◽  
Kathryn A. Johnston ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Enzymatic Activity ◽  
Lysyl Oxidase ◽  
The Other ◽  
Glutathione S Transferase ◽  
E Coli ◽  
N Terminus ◽  
Low Solubility ◽  
Total Enzyme ◽  
Matrix Enzyme

Lysyl oxidase is an important extracellular matrix enzyme that has not been fully characterized due to its low solubility. In order to circumvent the low solubility of this enzyme, three solubility tags (Nus-A, Thioredoxin (Trx), and Glutathione-S-Transferase (GST)) were engineered on the N-terminus of mature lysyl oxidase. Total enzyme yields were determined to be 1.5 mg for the Nus-A tagged enzyme (0.75 mg/L of media), 7.84 mg for the Trx tagged enzyme (3.92 mg/L of media), and 9.33 mg for the GST tagged enzyme (4.67 mg/L of media). Enzymatic activity was calculated to be 0.11 U/mg for the Nus-A tagged enzyme and 0.032 U/mg for the Trx tagged enzyme, and no enzymatic activity was detected for the GST tagged enzyme. All three solubility-tagged forms of the enzyme incorporated copper; however, the GST tagged enzyme appears to bind adventitious copper with greater affinity than the other two forms. The catalytic cofactor, lysyl tyrosyl quinone (LTQ), was determined to be 92% for the Nus-A and Trx tagged lysyl oxidase using the previously reported extinction coefficient of 15.4 mM−1 cm−1. No LTQ was detected for the GST tagged lysyl oxidase. Given these data, it appears that Nus-A is the most suitable tag for obtaining soluble and active recombinant lysyl oxidase from E. coli culture.

scholarly journals Dynamic Polar Sequestration of Excess MurG May Regulate Enzymatic Function

2010 ◽  
Vol 192 (18) ◽  
pp. 4597-4605 ◽  
Author(s):  
Allison M. Michaelis ◽  
Zemer Gitai
Keyword(s):  
Enzymatic Activity ◽  
Protein Function ◽  
Cell Biology ◽  
Protein Localization ◽  
Lipid Binding ◽  
Storage Mechanism ◽  
E Coli ◽  
Peptidoglycan Biosynthesis ◽  
Enzymatic Function ◽  
Active Pool

ABSTRACT Advances in bacterial cell biology have demonstrated the importance of protein localization for protein function. In general, proteins are thought to localize to the sites where they are active. Here we demonstrate that in Escherichia coli, MurG, the enzyme that mediates the last step in peptidoglycan subunit biosynthesis, becomes polarly localized when expressed at high cellular concentrations. MurG only becomes polarly localized at levels that saturate MurG's cellular requirement for growth, and E. coli cells do not insert peptidoglycan at the cell poles, indicating that the polar MurG is not active. Fluorescence recovery after photobleaching (FRAP) and single-cell biochemistry experiments demonstrate that polar MurG is dynamic. Polar MurG foci are distinct from inclusion body aggregates, and polar MurG can be remobilized when MurG levels drop. These results suggest that polar MurG represents a temporary storage mechanism for excess protein that can later be remobilized into the active pool. We investigated and ruled out several candidate pathways for polar MurG localization, including peptidoglycan biosynthesis, the MreB cytoskeleton, and polar cardiolipin, as well as MurG enzymatic activity and lipid binding, suggesting that polar MurG is localized by a novel mechanism. Together, our results imply that inactive MurG is dynamically sequestered at the cell poles and that prokaryotes can thus utilize subcellular localization as a mechanism for negatively regulating enzymatic activity.

Localization of adenylation site on glutamine synthetase of E. coli by immunoelectron microscopy and image averaging

1984 ◽  
Vol 42 ◽  
pp. 642-643
Author(s):  
T. Wagenknecht ◽  
G. Bronstein ◽  
J. Frank ◽  
R. Frink ◽  
D. Eisenberg ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Enzymatic Activity ◽  
Immunoelectron Microscopy ◽  
Electron Micrographs ◽  
Single Particle ◽  
Tyrosine Residue ◽  
E Coli ◽  
Averaging Methods ◽  
Correlation Methods ◽  
Image Averaging

The glutamine synthetase (GS) from E. coli is a donut-shaped molecule comprising 12 identical subunits arranged in two opposing hexameric layers (dihedral D6 symmetry). In one mode of regulating enzymatic activity, each of the subunits is subject to adenylation at a specific tyrosine residue. A previous study used the technique of immunoelectron microscopy to map the position of adenylation on the surface of the molecule. We describe here a refinement of the analysis be means of computerized single particle averaging methods.Electron micrographs were digitized with a .78 nm sampling distance and images of GS in the six-fold orientation were selected and brought into mutual alignment by correlation methods. The images were divided into two classes depending on whether or not an antibody was visible at the periphery of the molecule. Selected examples of control and antibody-labeled molecules are shown in Fig. la,b respectively.

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