The use of bee venom melittin to assess the topography of membrane vesicles derived from Paracoccus denitrificans

1980 ◽  
Vol 58 (10) ◽  
pp. 996-1003 ◽  
Author(s):  
Jeanette R. Pik ◽  
Hugh G. Lawford
Keyword(s):  
Nadh Dehydrogenase ◽  
Paracoccus Denitrificans ◽  
Intact Cell ◽  
Respiratory Control ◽  
Membrane Vesicles ◽  
Bee Venom ◽  
Respiratory Enzymes ◽  
Protein Ratio ◽  
Membrane Topography ◽  
Considerable Controversy

There exists considerable controversy regarding membrane topography in vesicles derived by osmotic lysis of spheroplasts of Gram-negative bacteria. It has been reported by others that bee venom can be used to quantitate the portion of a heterogeneous vesicle population with an inside-out orientation by determining the degree of loss of crypticity of NADH dehydrogenase activity. We have demonstrated that a major component of bee venom, melittin, causes an increase in the activity of several different respiratory enzymes in isolated membrane vesicles of Paracoccus denitrificans. The degree of stimulation produced by melittin is dependent upon (i) the nature of the respiratory substrates, (ii) the pH, (iii) the presence of Mg2+, (iv) the melittin: membrane protein ratio, and (v) the growth history of the cells from which the membrane vesicles were derived. Melittin-induced enhancement of TMPD: ascorbate and cytochrome c oxidase activities cannot be accounted for by increased accessibility of non-permeant substrate to the interior of the vesicle. The stimulatory effect of melittin may rely in part on its ability to alter the proton permeability of the membrane thereby abolishing respiratory control. Collectively these observations call into question the usefulness of bee venom melittin in quantitative analyses of membrane topography. These results are consistent with the postulated existence of a homogeneous vesicle population in which the topography of the NADH dehydrogenase is different from that of the intact cell.

Heterogeneity of phospholipid synthesis in rat liver endoplasmic reticulum during proliferation of smooth membranes

1976 ◽  
Vol 22 (1) ◽  
pp. 173-197
Author(s):  
J.A. Higgins
Keyword(s):  
Endoplasmic Reticulum ◽  
Uniform Distribution ◽  
Specific Activity ◽  
Intact Cell ◽  
Smooth Endoplasmic Reticulum ◽  
Membrane Phospholipid ◽  
Phospholipid Synthesis ◽  
Protein Ratio ◽  
Cytochemical Localization ◽  
Heterogeneous Distribution

During proliferation of smooth endoplasmic reticulum (SER) induced by phenobarbital the specific activity of acyltransferases of the smooth microsomes increases, there is a transient rise in the phospholipid/protein ratio of these membranes, and an increased incorporation of [14C]glycerol into smooth-membrane phospholipid. Microsomes separated into subfractions on 2 gradients exhibited a heterogeneous distribution of these characteristics, indicating a non-uniform distribution of the site of phospholipid synthesis in the ER under these conditions. Cytochemical localization of acyltransferases on whole liver and smooth and rough microsomes confirmed this heterogeneity, and indicated that the distribution of this activity was not restricted to any morphologically distinct site in the ER of the intact cell. After 4 days of phenobarbital treatment the increased membrane is restricted to lighter subfractions and is similar in distribution to that of increased acyltransferase activity. These results indicate that the synthesis of membrane phospholipid and the growth of the SER in response to phenobarbital is not uniform but occurs at randomly dispersed sites in the SER while proteins may be added preferentially at these sites resulting in a final uniform distribution.

Coupling between oxidative metabolism and active transport in the midgut of tobacco hornworm

1980 ◽  
Vol 238 (1) ◽  
pp. C1-C9 ◽  
Author(s):  
L. J. Mandel ◽  
D. F. Moffett ◽  
T. G. Riddle ◽  
M. M. Grafton
Keyword(s):  
Active Transport ◽  
Oxidative Metabolism ◽  
Consumption Rate ◽  
Respiratory Control ◽  
Tobacco Hornworm ◽  
Respiratory Enzymes ◽  
Endogenous Substrates ◽  
Redox Level ◽  
Mitochondrial Uncoupler ◽  
K Transport

Active K transport (Isc) in the midgut of tobacco hornworm Manduca sexta has been shown to be highly dependent on oxidative metabolism. However, the oxygen consumption rate (rO2) was not altered by conditions that drastically affect Isc. Respiration was normally maximal, inasmuch as uncouplers did not increase rO2. This rate could be maintained without any added substrate probably by oxidation of endogenous substrates. Additional succinate increased rO2 by 17%. Simultaneous monitoring of Isc and the redox level of the respiratory chain components demonstrated that 1) succinate (5 mM) reduced all the respiratory enzymes while increasing Isc by 17%; 2) sesamol (5 mM), a mitochondrial uncoupler, reoxidized all respiratory enzymes and inhibited Isc by about 50%; 3) cyanide (1 mM) fully reduced the cytochromes and completely inhibited Isc. These redox responses indicate that the mitochondria in this tissue are normally coupled, even if respiration is maximal and is not modulated by active transport. Mitochondria isolated from the midgut show coupling and respiratory control by ADP, appearing to behave like mitochondria from other tissues. Therefore, a cytoplasmic constraint must exist in this tissue that continually elicits an unmodulated maximal respiratory rate.

scholarly journals Control of respiration rate in non-growing cells of Paracoccus denitrificans

1987 ◽  
Vol 246 (3) ◽  
pp. 779-782 ◽  
Author(s):  
I Kucera ◽  
L Lampardová ◽  
V Dadák
Keyword(s):  
Respiration Rate ◽  
Membrane Fraction ◽  
Nadh Dehydrogenase ◽  
Paracoccus Denitrificans ◽  
Direct Determination ◽  
Limiting Factor ◽  
Control Of Respiration ◽  
Ubiquinone Oxidoreductase ◽  
Nadh Ubiquinone Oxidoreductase

By means of fluorimetric measurement and by direct determination of intracellular NAD+ and NADH contents, it was proved that the respiration rate of Paracoccus denitrificans cells utilizing glucose is limited by processes preceding NADH oxidation in the respiratory chain, so that the membrane NADH dehydrogenase is not saturated by its substrate. In the separated membrane fraction on saturation with exogenous NADH the main limiting factor is represented by NADH: ubiquinone oxidoreductase.

scholarly journals Modifications of the Aerobic Respiratory Chain of Paracoccus Denitrificans in Response to Superoxide Oxidative Stress

2019 ◽  
Vol 7 (12) ◽  
pp. 640 ◽  
Author(s):  
Vojtěch Sedláček ◽  
Igor Kučera
Keyword(s):  
Oxidative Stress ◽  
Respiratory Chain ◽  
Nadh Dehydrogenase ◽  
Paracoccus Denitrificans ◽  
Mrna Level ◽  
Oxygen Species ◽  
Stress Perception ◽  
Iron Sulfur Clusters ◽  
Iron Sulfur ◽  
Mammalian Mitochondria

Paracoccus denitrificans is a strictly respiring bacterium with a core respiratory chain similar to that of mammalian mitochondria. As such, it continuously produces and has to cope with superoxide and other reactive oxygen species. In this work, the effects of artificially imposed superoxide stress on electron transport were examined. Exposure of aerobically growing cells to paraquat resulted in decreased activities of NADH dehydrogenase, succinate dehydrogenase, and N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) oxidase. Concomitantly, the total NAD(H) pool size in cells was approximately halved, but the NADH/NAD+ ratio increased twofold, thus partly compensating for inactivation losses of the dehydrogenase. The inactivation of respiratory dehydrogenases, but not of TMPD oxidase, also took place upon treatment of the membrane fraction with xanthine/xanthine oxidase. The decrease in dehydrogenase activities could be fully rescued by anaerobic incubation of membranes in a mixture containing 2-mercaptoethanol, sulfide and ferrous iron, which suggests iron–sulfur clusters as targets for superoxide. By using cyanide titration, a stress-sensitive contribution to the total TMPD oxidase activity was identified and attributed to the cbb3-type terminal oxidase. This response (measured by both enzymatic activity and mRNA level) was abolished in a mutant defective for the FnrP transcription factor. Therefore, our results provide evidence of oxidative stress perception by FnrP.

NADH Dehydrogenase and NADh Oxidation in Membrane Vesicles from Bacillus subtilis

1981 ◽  
Vol 120 (3) ◽  
pp. 599-606 ◽  
Author(s):  
Jack BERGSMA ◽  
Rein STRIJKER ◽  
Jet Y. E. ALKEMA ◽  
Hendrik G. SEIJEN ◽  
Wil N. KONINGS
Keyword(s):  
Bacillus Subtilis ◽  
Nadh Dehydrogenase ◽  
Membrane Vesicles ◽  
Nadh Oxidation

Biochemical Characterization of Chlorophyll-Free Mitochondria From Pea Leaves

1985 ◽  
Vol 12 (3) ◽  
pp. 219 ◽  
Author(s):  
DA Day ◽  
M Neuburger ◽  
R Douce
Keyword(s):  
Biochemical Characterization ◽  
Membrane Integrity ◽  
Phospholipid Composition ◽  
Respiratory Control ◽  
Matrix Proteins ◽  
Green Leaf ◽  
Protein Ratio ◽  
Linear Gradient ◽  
The Matrix

Mitochondria from pea leaves were purified by centrifugation on a self-generated Percoll gradient which contained a linear gradient of polyvinylpyrrolidone-25 (0-10%, w/v). The chlorophyll content of the purified mitochondria was less than 1 �g per mg protein. All substrates were rapidly oxidized by these mitochondria, the rate of glycine oxidation being between 200 and 300 nmol O2 min-1 mg-1 protein, depending on the age of the leaves used. These rates did not vary significantly over a period of 20 h, provided NAD+ was supplied exogenously, when the mitochondria were stored on ice. Respiratory control, ADP/O ratios and outer membrane integrity (always more than 95%) were also maintained during storage. The phospholipid composition of the membranes from the leaf mitochondria was virtually identical to that of mitochondria from non-photosynthetic tissues although their lipid to protein ratio was slightly lower. The polypeptide pattern of the membranes from green leaf mitochondria and those from etiolated leaves and hypocotyls were also similar, but marked differences were observed between the matrix proteins from the different tissues. In particular, intensely stained bands at 94, 51,41 and 15.5 kDa which were present in the matrix of green leaf mitochondria were missing or present in much smaller quantities in the non-photosynthetic tissues. This difference was correlated with the ability of the mitochondria to oxidize glycine, suggesting that the four polypeptides may be associated with the glycine decarboxylase complex.

scholarly journals The reconstitution of l-3-glycerophosphate-cytochrome c oxidoreductase from l-3-glycerophosphate dehydrogenase, ubiquinone-10 and ubiquinol—cytochrome c oxidoreductase

1980 ◽  
Vol 192 (1) ◽  
pp. 19-31 ◽  
Author(s):  
I R Cottingham ◽  
C I Ragan
Keyword(s):  
Cytochrome C ◽  
Nadh Dehydrogenase ◽  
Brain Mitochondria ◽  
Complex Iii ◽  
Protein Ratio ◽  
Bovine Heart ◽  
Glycerophosphate Dehydrogenase ◽  
Reconstituted System ◽  
Ubiquinone 10 ◽  
Self Aggregation

Purified L-3-glycerophosphate dehydrogenase from pig brain mitochondria interacts with ubiquinone-10 and ubiquinol-cytochrome c oxidoreductase (Complex III) from bovine heart mitochondria to reconstitute antimycin-sensitive L-3-glycerophosphate- cytochrome c oxidoreductase. This activity is completely dependent on the two enzymes and largely dependent on ubiquinone-10. Reconstitution requires that the two enzymes should be simultaneously present in the same membranous aggregate produced by removal of detergent from the enzymes. Reconstitution by removing detergent by dialysis or dilution is inefficient because of self-aggregation of the dehydrogenase. Highly efficient reconstitution can be achieved if the enzymes are co-precipitated by addition of ethanol. The rate with reconstituted enzyme approaches that expected from the turnover of the dehydrogenase with ubiquinone-1 as acceptor. The behaviour of the reconstituted system shows some of the characteristics expected for a stoicheiometric association of one molecule of dehydrogenase with one molecule of Complex III. On raising the phospholipid/protein ratio, the dehydrogenase and Complex III appear to operate as independent enzymes acting in sequence. These effects are very similar to those observed for the interaction of NADH dehydrogenase and Complex III and are explained in terms of the model proposed by Heron, Ragan & Trumpower [(1978) biochem. J. 174, 791-800].

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