scholarly journals A comparative study of the regulation of Ca2+ of the activities of the 2-oxoglutarate dehydrogenase complex and NAD+-isocitrate dehydrogenase from a variety of sources

1981 ◽  
Vol 196 (2) ◽  
pp. 619-624 ◽  
Author(s):  
J G McCormack ◽  
R M Denton
Keyword(s):  
Escherichia Coli ◽  
Comparative Study ◽  
Isocitrate Dehydrogenase ◽  
Human Heart ◽  
Flight Muscle ◽  
Rat Tissues ◽  
Similar Extent ◽  
E Coli ◽  
Oxoglutarate Dehydrogenase ◽  
Dehydrogenase Complex

Ca2+ was shown to activate oxoglutarate dehydrogenase and NAD+-isocitrate dehydrogenase from heart and other rat tissues by markedly decreasing the Km values of the enzymes for their respective substrates [see Denton & McCormack (1980) FEBS Lett. 119, 1-8]. Similar effects of Ca2+ were observed in the present study with both enzymes from other vertebrate sources (pigeon, trout, frog and human heart), but not with the enzymes from blowfly or locust flight muscle, or potato or Escherichia coli. In contrast, the Km values of the oxoglutarate dehydrogenases were affected by ADP, ATP and H+ to a similar extent in every case, except for the enzyme from E. coli, which was not sensitive to regulation by these agents.

scholarly journals Limited proteolysis and proton n.m.r. spectroscopy of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli

1981 ◽  
Vol 199 (3) ◽  
pp. 733-740 ◽  
Author(s):  
R N Perham ◽  
G C K Roberts
Keyword(s):  
Escherichia Coli ◽  
Lipoic Acid ◽  
Polypeptide Chain ◽  
Pyruvate Dehydrogenase Complex ◽  
Proton Nuclear Magnetic Resonance ◽  
Resonance Spectroscopy ◽  
Multienzyme Complex ◽  
Oxoglutarate Dehydrogenase ◽  
Polypeptide Chains ◽  
Dehydrogenase Complex

The 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli was treated with trypsin at pH 7.0 at 0 degrees C. Loss of the overall catalytic activity was accompanied by rapid cleavage of the lipoate succinyltransferase polypeptide chains, this apparent Mr falling from 50 000 to 36 000 as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. A slower shortening of the 2-oxoglutarate decarboxylase chains was also observed, whereas the lipoamide dehydrogenase chains were unaffected. The inactive trypsin-treated enzyme had lost the lipoic acid-containing regions of the lipoate succinyltransferase polypeptide chains, yet remained a highly assembled structure, as judged by gel filtration and electron microscopy. The lipoic acid-containing regions are therefore likely to be physically exposed in the complex, protruding from the structural core formed by the lipoate succinyltransferase component between the subunits of the other component enzymes. Proton nuclear magnetic resonance spectroscopy of the 2-oxoglutarate dehydrogenase complex revealed the existence of substantial regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after removal of the lipoic acid-containing regions by treatment of the complex with trypsin. By analogy with the comparably mobile regions of the pyruvate dehydrogenase complex of E. coli, it is likely that the highly mobile regions of polypeptide chain in the 2-oxoglutarate complex are in the lipoate succinyltransferase component and encompass the lipoyl-lysine residues. It is clear, however, that the mobility of this polypeptide chain is not restricted to the immediate vicinity of these residues.

Comparative Study of AFM and FESEM for Imaging the Single Cell of Escherichia Coli Bacteria

2015 ◽  
Vol 34 ◽  
pp. 61-66
Author(s):  
Kamarulazizi Ibrahim ◽  
Mohammad Hafiz Khalid ◽  
Mohamed Hassan Eisa ◽  
Mohd Nazalan Najimudin ◽  
Mohammad A. Al Rajhi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Comparative Study ◽  
Single Cell ◽  
Food Poisoning ◽  
Rapid Identification ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Identification Of Bacteria ◽  
Escherichia Coli Bacteria

In this work, a comparative study using atomic force microscopy (AFM) and field emission–scanning electron microscopy (FESEM) has been carried out to assess the morphology of single cellEscherichia colibacteria (E-coli).E-colibacteria are a major concern for public health. Attention was focused on the certain strains ofE-colibacteria, because some strains can be toxic and cause food poisoning. TheE-colibacteria have attracted much research interest because this bacterium is easily to get, cheap and rapid reproductively. Imaging ofE-colirecently, was improved by using high resolution microscopy. Current techniques for detection such as, AFM and FESEM has attracted great interest and emerging as a potentially powerful whole-organism fingerprinting tool for the rapid identification of bacteria. The obtained results of AFM and FESEM techniques have been compared to show the image quality of single cellE-coli.

scholarly journals Expression and lipoylation in Escherichia coli of the inner lipoyl domain of the E2 component of the human pyruvate dehydrogenase complex

1993 ◽  
Vol 289 (1) ◽  
pp. 81-85 ◽  
Author(s):  
J Quinn ◽  
A G Diamond ◽  
A K Masters ◽  
D E Brookfield ◽  
N G Wallis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Structural Studies ◽  
Gene Encoding ◽  
E Coli ◽  
Inner Lipoyl Domain ◽  
Dehydrogenase Complex ◽  
Lipoyl Domain

The dihydrolipoamide acetyltransferase subunit (E2p) of mammalian pyruvate dehydrogenase complex has two highly conserved lipoyl domains each modified with a lipoyl cofactor bound in amide linkage to a specific lysine residue. A sub-gene encoding the inner lipoyl domain of human E2p has been over-expressed in Escherichia coli. Two forms of the domain have been purified, corresponding to lipoylated and non-lipoylated species. The apo-domain can be lipoylated in vitro with partially purified E. coli lipoate protein ligase, and the lipoylated domain can be reductively acetylated by human E1p (pyruvate dehydrogenase). Availability of the two forms will now allow detailed biochemical and structural studies of the human lipoyl domains.

scholarly journals Replication of Colonic Crohn's Disease Mucosal Escherichia coli Isolates within Macrophages and Their Susceptibility to Antibiotics

2007 ◽  
Vol 52 (2) ◽  
pp. 427-434 ◽  
Author(s):  
Sreedhar Subramanian ◽  
Carol L. Roberts ◽  
C. Anthony Hart ◽  
Helen M. Martin ◽  
Steve W. Edwards ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Colonic Mucosa ◽  
Human Monocyte ◽  
Fold Increase ◽  
Serum Concentrations ◽  
Similar Extent ◽  
E Coli ◽  
K 12

ABSTRACT There is increasing evidence that Escherichia coli organisms are important in Crohn's disease (CD) pathogenesis. In CD tissue they are found within macrophages, and the adherent-invasive CD ileal E. coli isolate LF82 can replicate inside macrophage phagolysosomes. This study investigates replication and antibiotic susceptibility of CD colonic E. coli isolates inside macrophages. Replication of CD colonic E. coli within J774-A1 murine macrophages and human monocyte-derived macrophages (HMDM) was assessed by culture and lysis after gentamicin killing of noninternalized bacteria and verified by electron microscopy (EM). All seven CD colonic isolates tested replicated within J774-A1 macrophages by 3 h (6.36-fold ± 0.7-fold increase; n = 7 isolates) to a similar extent to CD ileal E. coli LF82 (6.8-fold ± 0.8-fold) but significantly more than control patient isolates (5.2-fold ± 0.25-fold; n = 6; P = 0.006) and E. coli K-12 (1.0-fold ± 0.1-fold; P < 0.0001). Replication of CD E. coli HM605 within HMDM (3.9-fold ± 0.7-fold) exceeded that for K-12 (1.4-fold ± 0.2-fold; P = 0.03). EM showed replicating E. coli within macrophage vacuoles. Killing of HM605 within J774-A1 macrophages following a 3-h incubation with antibiotics at published peak serum concentrations (C max) was as follows: for ciprofloxacin, 99.5% ± 0.2%; rifampin, 85.1% ± 6.6%; tetracycline, 62.8% ± 6.1%; clarithromycin, 62.1% ± 5.6% (all P < 0.0001); sulfamethoxazole, 61.3% ± 7.0% (P = 0.0007); trimethoprim, 56.3% ± 3.4% (P < 0.0001); and azithromycin, 41.0% ± 10.5% (P = 0.03). Ampicillin was not effective against intracellular E. coli. Triple antibiotic combinations were assessed at 10% C max, with ciprofloxacin, tetracycline, and trimethoprim causing 97% ± 0.0% killing versus 86% ± 2.0% for ciprofloxacin alone. Colonic mucosa-associated E. coli, particularly CD isolates, replicate within macrophages. Clinical trials are indicated to assess the efficacy of a combination antibiotic therapy targeting intramacrophage E. coli.

ColiComplete® Substrate-Supporting Disc Method for Confirmed Detection of Total Coliforms and Escherichia coli in All Foods: Comparative Study

1994 ◽  
Vol 77 (1) ◽  
pp. 58-63 ◽  
Author(s):  
Philip T Feldsine ◽  
Maria T Falbo-Nelson ◽  
David L Hustead
Keyword(s):  
Escherichia Coli ◽  
Comparative Study ◽  
Total Coliform ◽  
Coliform Bacteria ◽  
Most Probable Number ◽  
Probable Number ◽  
Total Coliforms ◽  
E Coli ◽  
Total Coliform Bacteria ◽  
Better Than

Abstract The ColiComplete® substrate-supporting disc (SSD) method for simultaneous confirmed total coliform count and Escherichia coli determination in all foods was compared with the AOAC most probable number (MPN) methods 966.23 and 966.24. In this comparative study, 20 water and food types were analyzed; 7 of these foods were naturally contaminated with coliform bacteria, 6 food types were naturally contaminated with E. coli, and the remaining foods were inoculated with coliform bacteria and/or E. coli. Data were analyzed separately for total coliform bacteria and for E. coli. Mean log MPN counts were determined by the SSD method and the appropriate AOAC MPN procedure. Results were then analyzed for mean log MPN differences and variance, according to methods described by AOAC INTERNATIONAL Results for both total conforms and E. coli indicate that the SSD method is equivalent to or better than AOAC MPN methods 966.23 and 966.24.

scholarly journals The role of loop and β-turn residues as structural and functional determinants for the lipoyl domain from the Escherichia coli 2-oxoglutarate dehydrogenase complex

2007 ◽  
Vol 409 (2) ◽  
pp. 357-366 ◽  
Author(s):  
D. Dafydd Jones ◽  
Richard N. Perham
Keyword(s):  
Escherichia Coli ◽  
Structural Integrity ◽  
Pyruvate Decarboxylase ◽  
Covalent Attachment ◽  
E Coli ◽  
Loop Sequence ◽  
Equivalent Residue ◽  
Oxoglutarate Dehydrogenase ◽  
And Function ◽  
Lipoyl Domain

The lipoyl domain of the dihydrolipoyl succinyltransferase (E2o) component of the 2OGDH (2-oxoglutarate dehydrogenase) multienzyme complex houses the lipoic acid cofactor through covalent attachment to a specific lysine side chain residing at the tip of a β-turn. Residues within the lipoyl-lysine β-turn and a nearby prominent loop have been implicated as determinants of lipoyl domain structure and function. Protein engineering of the Escherichia coli E2o lipoyl domain (E2olip) revealed that removal of residues from the loop caused a major structural change in the protein, which rendered the domain incapable of reductive succinylation by 2-oxoglutarate decarboxylase (E1o) and reduced the lipoylation efficiency. Insertion of a new loop corresponding to that of the E. coli pyruvate dehydrogenase lipoyl domain (E2plip) restored lipoylation efficiency and the capacity to undergo reductive succinylation returned, albeit at a lower rate. Exchange of the E2olip loop sequence significantly improved the ability of the domain to be reductively acetylated by pyruvate decarboxylase (E1p), retaining approx. 10-fold more acetyl groups after 25 min than wild-type E2olip. Exchange of the β-turn residue on the N-terminal side of the E2o lipoyl-lysine DKA/V motif to the equivalent residue in E2plip (T42G), both singly and in conjunction with the loop exchange, reduced the ability of the domain to be reductively succinylated, but led to an increased capacity to be reductively acetylated by the non-cognate E1p. The T42G mutation also slightly enhanced the lipoylation rate of the domain. The surface loop is important to the structural integrity of the protein and together with Thr42 plays an important role in specifying the interaction of the lipoyl domain with its partner E1o in the E. coli 2OGDH complex.

scholarly journals The binding of Ca2+ ions to pig heart NAD+-isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex

1989 ◽  
Vol 263 (2) ◽  
pp. 453-462 ◽  
Author(s):  
G A Rutter ◽  
R M Denton
Keyword(s):  
Dissociation Constant ◽  
Isocitrate Dehydrogenase ◽  
Binding Sites ◽  
Tight Binding ◽  
Variable Number ◽  
Apparent Dissociation Constant ◽  
Oxoglutarate Dehydrogenase ◽  
Dissociation Constant Kd ◽  
Flow Dialysis ◽  
Dehydrogenase Complex

1. The binding of Ca2+ ions to purified pig heart NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase, freed of contaminating Ca2+ by parvalbumin/polyacrylamide chromatography, has been studied by flow dialysis and by the use of fura-2. 2. For the 2-oxoglutarate dehydrogenase complex, 3.5 mol of Ca2+-binding sites/mol of complex were apparent, with an apparent dissociation constant (Kd value) for Ca2+ of 2.0 microM. These values were little affected by Mg2+ ions, ADP or 2-oxoglutarate. 3. By contrast, binding of Ca2+ to NAD+-isocitrate dehydrogenase (Kd = 14 microM) required ADP, isocitrate and Mg2+ ions. The number of Ca2+-binding sites associated with NAD+-isocitrate dehydrogenase was then 0.9 mol/mol of tetrameric enzyme. 4. The 2-oxoglutarate dehydrogenase complex bound ADP (as ADP3-) to a group of tight-binding sites (Kd = 3.1 microM) with a stoichiometry, 3.3 mol/mol of complex, similar to that for the binding of Ca2+; a variable number of much weaker sites (Kd = 100 microM) for ADP3- was also apparent.

A Comparative Study of the Treatment of Urinary Infection

1969 ◽  
Vol 14 (3) ◽  
pp. 71-75 ◽  
Author(s):  
A. C. Kennedy ◽  
M. E. M. Allison ◽  
J. D. Briggs ◽  
J. McGeachie
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Comparative Study ◽  
Recurrence Rate ◽  
Statistical Evaluation ◽  
Urinary Infection ◽  
Elevated Blood ◽  
Initial Infection ◽  
E Coli ◽  
Lower Recurrence Rate

Fifty-five female patients aged 12 to 76 years were found to have a significant urinary tract infection on culture of 2 or more consecutive mid-stream specimens of urine (i.e. over 100,000 organisms/ml.). Ninety per cent were due to Escherichia coli and 50 per cent were asymptomatic. A comparative study of the value of 4 regimes (alkali alone, sulphonamide plus alkali, streptomycin plus alkali, nitrofurantoin) in eradicating the initial infection and on the recurrence rate was carried out. Alkali alone had no effect on bacteriuria, nitrofurantoin had a 100 per cent 5-day sterility rate, while sulphonamide and streptomycin had 85 and 88 per cent 5-day sterility rates respectively, differences which are not statistically significant. All of the failures had E. coli infections, none had pyelographic evidence of pyelonephritis or had an elevated blood urea, and only 2 had an antibody titre against the infecting organism over 1/320. There was an overall recurrence rate, mainly due to re-infection, of 53 per cent within the first 6 months. Sulphonamide therapy seemed associated with a lower recurrence rate than nitrofurantoin or streptomycin but the numbers involved are too small to allow statistical evaluation.

scholarly journals Roles of Charged Residues of Rotor and Stator in Flagellar Rotation: Comparative Study using H+-Driven and Na+-Driven Motors in Escherichia coli

2006 ◽  
Vol 188 (4) ◽  
pp. 1466-1472 ◽  
Author(s):  
Toshiharu Yakushi ◽  
Junghoon Yang ◽  
Hajime Fukuoka ◽  
Michio Homma ◽  
David F. Blair
Keyword(s):  
Escherichia Coli ◽  
Comparative Study ◽  
Electrostatic Interactions ◽  
General Feature ◽  
Flagellar Motor ◽  
Motor Patterns ◽  
E Coli ◽  
Flagellar Motors ◽  
Charged Residues ◽  
Flagellar Rotation

ABSTRACT In Escherichia coli, rotation of the flagellar motor has been shown to depend upon electrostatic interactions between charged residues of the stator protein MotA and the rotor protein FliG. These charged residues are conserved in the Na+-driven polar flagellum of Vibrio alginolyticus, but mutational studies in V. alginolyticus suggested that they are relatively unimportant for motor rotation. The electrostatic interactions detected in E. coli therefore might not be a general feature of flagellar motors, or, alternatively, the V. alginolyticus motor might rely on similar interactions but incorporate additional features that make it more robust against mutation. Here, we have carried out a comparative study of chimeric motors that were resident in E. coli but engineered to use V. alginolyticus stator components, rotor components, or both. Charged residues in the V. alginolyticus rotor and stator proteins were found to be essential for motor rotation when the proteins functioned in the setting of the E. coli motor. Patterns of synergism and suppression in rotor/stator double mutants indicate that the V. alginolyticus proteins interact in essentially the same way as their counterparts in E. coli. The robustness of the rotor-stator interface in V. alginolyticus is in part due to the presence of additional charged residues in PomA but appears mainly due to other factors, because an E. coli motor using both rotor and stator components from V. alginolyticus remained sensitive to mutation. Motor function in V. alginolyticus may be enhanced by the proteins MotX and MotY.

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