scholarly journals RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS

1972 ◽  
Vol 53 (1) ◽  
pp. 1-23 ◽  
Author(s):  
R. Scharff ◽  
R. W. Hendler ◽  
N. Nanninga ◽  
A. H. Burgess
Keyword(s):  
Escherichia Coli ◽  
Nadh Oxidase ◽  
High Capacity ◽  
Chemical Components ◽  
Membrane Envelope ◽  
Major Chemical ◽  
Nadh Oxidase Activity ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
Very High ◽  
Soluble Components

Membrane-envelope fragments have been isolated from Escherichia coli by comparatively mild techniques. The use of DNAase, RNAase, detergents, sonication, lysozyme, and ethylenediaminetetraacetate were avoided in the belief that rather delicate, but metabolically important, associations may exist between the plasma membrane and various cytoplasmic components. The membrane-envelope fragments have been characterized in terms of their content of major chemical components as well as their electron microscope appearance. Fractions containing membrane-envelope fragments were found to possess appreciable DNA- and protein-synthesizing activities. The fragments were rich in membrane content as determined by reduced nicotinamide adenine dinucleotide (NADH) oxidase activity and deficient in soluble components as measured by NADH dehydrogenase activity. The particulate fraction obtained between 20,000 g and 105,000 g and usually considered a ribosomal fraction was rich in membrane content and had a relatively high capacity for DNA synthesis. Envelope fragments sedimenting at 20,000 g attained very high levels of incorporation of amino acids into protein.

scholarly journals RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS

1972 ◽  
Vol 55 (2) ◽  
pp. 266-281 ◽  
Author(s):  
Richard W. Hendler ◽  
Amelia H. Burgess
Keyword(s):  
Escherichia Coli ◽  
Nicotinamide Adenine Dinucleotide ◽  
Oxidase Activity ◽  
Nadh Oxidase ◽  
Molecular Size ◽  
Reduced Form ◽  
Lactate Oxidase ◽  
Membrane Envelope ◽  
Nadh Oxidase Activity ◽  
Sepharose 4B

Membranes obtained from Escherichia coli have been solubilized with deoxycholate. The solubilized dehydrogenases and cytochromes are not sedimented at 105,000 g. These components readily penetrate the "included space" of Sepharose 4B (Pharmacia Fine Chemicals Inc., Uppsala, Sweden) and polyacrylamide gels and have been fractionated on the basis of molecular size. Solubilization destroys nicotinamide adenine dinucleotide, reduced form (NADH) oxidase and D-lactate oxidase activities, but leaves an appreciable part of the original succinoxidase activity intact. Evidence for a succinate dehydrogenase-cytochrome b1 complex is given. Menadione added to the solubilized preparation does not elicit NADH oxidase activity nor stimulate the existing succinoxidase activity, but does provoke an active D-lactate oxidase activity. This D-lactate oxidase activity, however, does not use cytochromes and is not sensitive to cyanide.

scholarly journals RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS

1970 ◽  
Vol 44 (2) ◽  
pp. 376-384 ◽  
Author(s):  
Richard W. Hendler ◽  
Amelia H. Burgess ◽  
Raymond Scharff
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Oxygen Uptake ◽  
Unsaturated Fatty Acids ◽  
Nadh Oxidase ◽  
Saturated Fatty Acids ◽  
Oxygen Electrode ◽  
Reduced Form ◽  
E Coli ◽  
Membrane Envelope

Fatty acids inhibited the ability of Escherichia coli membrane-envelope fragments to catalyze the oxidation of succinate and nicotinamide adenine dinucleotide, reduced form (NADH) and also inhibited the response of the Clark oxygen electrode to nonenzymatic oxygen uptake. In all cases, unsaturated fatty acids were much more inhibitory than saturated fatty acids. Albumin afforded complete protection from inhibition in the nonenzymatic oxygen-uptake experiments but only partial protection for the respiratory activities of the membrane fragments. The succinoxidase activity was totally inhibited by bovine serum albumin at concentrations that inhibited succinate dehydrogenase only slightly and NADH oxidase not at all. The E. coli acellular preparation showed no dehydrogenase or oxidase activity for any of the fatty acids under a variety of conditions. These conditions included variations of pH, concentration of fatty acids, and the presence or absence of albumin, CoA, ATP, NAD, cysteine, succinate, and carnitine. It thus appears that E. coli grown in the absence of fatty acid can not use fatty acids as an energy source.

scholarly journals Energy-linked reduction of nicotinamide–adenine dinucleotide in membranes derived from normal and various respiratory-deficient mutant strains of Escherichia coli K12

1974 ◽  
Vol 144 (1) ◽  
pp. 77-85 ◽  
Author(s):  
Robert K. Poole ◽  
Bruce A. Haddock
Keyword(s):  
Escherichia Coli ◽  
Ionic Strength ◽  
Succinate Dehydrogenase ◽  
High Speed ◽  
Nadh Oxidase ◽  
Supernatant Fraction ◽  
Deficient Mutant ◽  
Glycerophosphate Dehydrogenase ◽  
Nadh Oxidase Activity ◽  
Low Ionic Strength

1. Assay conditions are described for the ATP-dependent, uncoupler-sensitive, energy-linked reduction of NAD+ by succinate, dl-α-glycerophosphate or d-lactate in membranes from aerobically grown Escherichia coli. 2. The reaction may be demonstrated in electron-transport particles (ET particles) from cells grown in glycerol, but not in depleted particles washed in low-ionic-strength buffer, or in ET particles from cells grown in glucose. 3. The latter two classes of particles have low specific activities of ATPase (adenosine triphosphatase), succinate dehydrogenase, dl-α-glycerophosphate dehydrogenase and d-lactate dehydrogenase relative to undepleted ET particles from cells grown in glycerol. 4. Reconstitution of energy-linked NAD+ reduction in particles from cells grown in glucose was done by: (a) addition of the high-speed supernatant fraction from sonicates of the same cells; (b) addition of a protein fraction, precipitated by (NH4)2SO4 from this supernatant, or (c) addition of an (NH4)2SO4-precipitated fraction from the low-ionic-strength wash of particles from cells grown in glycerol. 5. The use of (NH4)2SO4-precipitated fractions from ATPase- or succinate dehydrogenase-deficient mutants grown in glycerol in the above reconstitution indicated that failure to demonstrate the reaction in particles from cells grown in glucose was a result of inadequate activities of appropriate dehydrogenases, rather than of ATPase. 6. Energy-linked NAD+ reduction could be demonstrated in particles from a ubiquinone-deficient mutant only after restoration of NADH oxidase activity by adding ubiquinone-1. 7. The measured rate of the energy-linked reaction in particles from a haem-deficient mutant, however, was not stimulated after the ATP- and haematin-dependent acquisition of functional cytochromes. 8. Results are interpreted as evidence of the ubiquinone-dependent, but cytochrome-independent, nature of the site I region of the respiratory chain in E. coli.

scholarly journals RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS

1971 ◽  
Vol 51 (3) ◽  
pp. 664-673 ◽  
Author(s):  
Richard W. Hendler
Keyword(s):  
Escherichia Coli ◽  
Nadh Oxidase ◽  
Protein Complexes ◽  
Nonheme Iron ◽  
Paramagnetic Resonance ◽  
Iron Protein ◽  
Membrane Envelope ◽  
Epr Signal ◽  
Almost All ◽  
Electron Paramagnetic

The sensitivity of nicotinamide adenine dinucleotide (NADH) oxidase and succinoxidase to metal chelators, the generation of an electron paramagnetic resonance (EPR) signal upon addition of these substrates, and the rate of formation of the EPR signal relative to the rate of the cytochrome reduction suggest the participation of nonheme iron proteins in the respiratory process of Escherichia coli. The most inhibitory metal chelator, thenoyltrifluoro acetone, inhibited the reduction of nonheme iron and cytochromes but did not prevent the reoxidation of the reduced forms. The EPR signal, dehydrogenase, and oxidase activities evoked by NADH are considerably greater than the corresponding activities evoked by succinate. Because both substrates can reduce almost all of the cytochromes, a model in which fewer succinate dehydrogenase-nonheme iron protein complexes are linked to a common cytochrome chain than NADH dehydrogenase-nonheme iron protein complexes is considered likely.

Isolation and HPLC Determination of the Chemical Components of Gentianella acuta (Michx.) Hulten

2018 ◽  
Vol 15 (1) ◽  
pp. 21-33
Author(s):  
Ying Wei ◽  
Yongqiao Liu ◽  
Yifan Hele ◽  
Weiwei Sun ◽  
Yang Wang ◽  
...  
Keyword(s):  
Medicinal Plant ◽  
High Speed ◽  
Chemical Constituents ◽  
Folk Medicine ◽  
Gradient Elution ◽  
Chemical Components ◽  
Isolation And Characterization ◽  
Major Chemical ◽  
Main Components ◽  
First Time

Background: Gentianella acuta (Michx.) Hulten is an important type of medicinal plant found in several Chinese provinces. It has been widely used in folk medicine to treat various illnesses. However, there is not enough detailed information about the chemical constituents of this plant or methods for their content determination. Objective: The focus of this work is the isolation and characterization of the major chemical constituents of Gentianella acuta, and developing an analytical method for their determination. Methods: The components of Gentianella acuta were isolated using (1) ethanol extraction and adsorption on macroporous resin. (2) and ethyl acetate extraction and high speed countercurrent chromatography. A HPLC-DAD method was developed using a C18 column and water-acetonitrile as the mobile phase. Based on compound polarities, both isocratic and gradient elution methods were developed. Results: A total of 29 compounds were isolated from this plant, of which 17 compounds were isolated from this genus for the first time. The main components in this plant were found to be xanthones. The HPLC-DAD method was developed and validated for their determination, and found to show good sensitivity and reliability. Conclusion: The results of this work add to the limited body of work available on this important medicinal plant. The findings will be useful for further investigation and development of Gentianella acuta for its valuable medicinal properties.

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