A cytochrome c mutant with high electron transfer and antioxidant activities but devoid of apoptogenic effect

2002 ◽  
Vol 362 (3) ◽  
pp. 749-754 ◽  
Author(s):  
Ziedulla Kh. ABDULLAEV ◽  
Marina E. BODROVA ◽  
Boris V. CHERNYAK ◽  
Dmitry A. DOLGIKH ◽  
Ruth M. KLUCK ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Cytochrome C ◽  
Antioxidant Activities ◽  
H2o2 Production ◽  
High Electron ◽  
Wild Type ◽  
Low Concentrations ◽  
Rat Heart Mitochondria ◽  
The Difference ◽  
Α Helix

A cytochrome c mutant lacking apoptogenic function but competent in electron transfer and antioxidant activities has been constructed. To this end, mutant species of horse and yeast cytochromes c with substitutions in the N-terminal α-helix or position 72 were obtained. It was found that yeast cytochrome c was much less effective than the horse protein in activating respiration of rat liver mitoplasts deficient in endogenous cytochrome c as well as in inhibition of H2O2 production by the initial segment of the respiratory chain of intact rat heart mitochondria. The major role in the difference between the horse and yeast proteins was shown to be played by the amino acid residue in position 4 (glutamate in horse, and lysine in yeast; horse protein numbering). A mutant of the yeast cytochrome c containing K4E and some other ‘horse’ modifications in the N-terminal α-helix, proved to be (i) much more active in electron transfer and antioxidant activity than the wild-type yeast cytochrome c and (ii), like the yeast cytochrome c, inactive in caspase stimulation, even if added in 400-fold excess compared with the horse protein. Thus this mutant seems to be a good candidate for knock-in studies of the role of cytochrome c-mediated apoptosis, in contrast with the horse K72R, K72G, K72L and K72A mutant cytochromes that at low concentrations were less active in apoptosis than the wild-type, but were quite active when the concentrations were increased by a factor of 2–12.

scholarly journals A cytochrome c mutant with high electron transfer and antioxidant activities but devoid of apoptogenic effect

2002 ◽  
Vol 362 (3) ◽  
pp. 749 ◽  
Author(s):  
Ziedulla Kh. ABDULLAEV ◽  
Marina E. BODROVA ◽  
Boris V. CHERNYAK ◽  
Dmitry A. DOLGIKH ◽  
Ruth M. KLUCK ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Cytochrome C ◽  
Antioxidant Activities ◽  
High Electron

scholarly journals The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2

1993 ◽  
Vol 291 (1) ◽  
pp. 89-94 ◽  
Author(s):  
P White ◽  
F D C Manson ◽  
C E Brunt ◽  
S K Chapman ◽  
G A Reid
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Cytochrome C ◽  
Kinetic Properties ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Cytochrome C Reductase ◽  
Flavocytochrome B2 ◽  
Cytochrome C Reduction

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this ‘hinge-swap’ enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; kcat. for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the kcat. for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-2H]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

Inhibition of Mitochondrial Electron Transfer in Rats by Ethanethiol and Methanethiol

1979 ◽  
Vol 56 (2) ◽  
pp. 147-156 ◽  
Author(s):  
T. Vahlkamp ◽  
A. J. Meijer ◽  
J. Wilms ◽  
R. A. F. M. Chamuleau
Keyword(s):  
Electron Transfer ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Rat Hepatocytes ◽  
Rat Liver Mitochondria ◽  
Dimethyl Sulphide ◽  
Submitochondrial Particles ◽  
Isolated Rat Hepatocytes ◽  
The Difference ◽  
Isolated Rat

1. We have investigated the effects of ethanethiol, methanethiol and dimethyl sulphide on some metabolic processes of isolated rat hepatocytes, isolated mitochondria from liver and brain and ox-heart submitochondrial particles. 2. Ethanethiol, but not dimethyl sulphide, inhibited both gluconeogenesis and ureogenesis from various substrates in rat hepatocytes, depressed cellular ATP content and caused an increased reduction of the mitochondria. 3. Ethanethiol inhibited respiration in isolated rat-liver mitochondria with several substrates, both in the presence of ADP and phosphate or in the presence of an uncoupling agent. Ethanethiol also inhibited respiration in isolated rat-brain mitochondria. Dimethyl sulphide was much less effective in inhibiting mitochondrial respiration. 4. In ox-heart submitochondrial particles ethanethiol inhibited electron transfer between cytochrome c and oxygen. 5. Purified cytochrome c oxidase was inhibited by ethanethiol in a non-competitive manner. 6. Methanethiol inhibited cytochrome c oxidase and was an effective inhibitor of mitochondrial electron transfer, both in liver and brain. 7. The difference in inhibitory properties between ethanethiol, methanethiol and dimethyl sulphide observed in our experiments coincides with the difference in potency to elicit coma in rats. We suggest that inhibition of mitochondrial electron transfer by mercaptans may be relevant to the mechanism by which energy production in brain is depressed during hepatic coma.

Electrocatalytic proton reduction by zinc phthalocyanine in nafion and poly(4-vinylpyridine-co-styrene) matrices

2000 ◽  
Vol 04 (01) ◽  
pp. 31-36 ◽  
Author(s):  
FENG ZHAO ◽  
JIAN ZHANG ◽  
DIETER WÖHRLE ◽  
MASAO KANEKO
Keyword(s):  
Electron Transfer ◽  
Bimolecular Reaction ◽  
Zinc Phthalocyanine ◽  
Turnover Number ◽  
Proton Reduction ◽  
Rate Determining Step ◽  
Low Concentrations ◽  
The Matrix ◽  
Electron Propagation ◽  
The Difference

Electrochemical proton reduction was catalysed by zinc phthalocyanine ( ZnPc ) incorporated in a Nafion or poly(4-vinylpyridine-co-styrene) (P(VP-St)) film coated on a graphite electrode. The turnover number (TN) of the complex to catalyse H 2 evolution was 104 h-1. The TN of Nafion[ ZnPc ] was about two times higher than that of P(VP-St)[ ZnPc ]. The difference was ascribed to the electron propagation in the film influenced by the interaction of the complex with the matrix and by the counterion migration between the matrix and the electrolyte solution. The electron transfer in the reduction of the ZnPc complex in the matrix was concluded to be the rate-determining step for the proton reduction. The TN was independent of the ZnPc concentration in the matrix at low concentrations although H 2 formation was regarded to be a bimolecular reaction process, which was explained by the electron transfer through the matrix via a monomolecular diffusion which was the rate-determining step.

Factor IX-Padua enhances the fibrinolytic resistance of plasma clots

2014 ◽  
Vol 111 (02) ◽  
pp. 226-232 ◽  
Author(s):  
Concetta Ammollo ◽  
Fabrizio Semeraro ◽  
Mario Colucci ◽  
Paolo Simioni
Keyword(s):  
Thrombin Generation ◽  
Factor Ix ◽  
Clot Lysis ◽  
Healthy Controls ◽  
Wild Type ◽  
Thrombin Activatable Fibrinolysis Inhibitor ◽  
Thrombotic Risk ◽  
Low Concentrations ◽  
Fibrinolysis Inhibitor ◽  
The Difference

SummaryHypercoagulable conditions may determine a hypofibrinolytic state by increasing the activation of thrombin-activatable fibrinolysis inhibitor (TAFI). Factor (F)IX-Padua is a mutated FIX with an eight-fold increased clotting activity and associates with a higher venous thrombotic risk. We evaluated the influence of FIX-Padua on TAFI-mediated regulation of fibrinolysis. A subject hemizygous for FIX-Padua, two family members (heterozygous and normal) and six healthy controls were studied. Clot lysis, TAFI activation and thrombin generation were evaluated in contact-inhibited plasma challenged with low concentrations of tissue factor. Fibrinolysis times were significantly longer in FIX-Padua carriers than controls. The difference disappeared when activated TAFI (TAFIa) was inhibited, when TAFI activation was avoided or when clotting was made independent of FIX. TAFIa generation was markedly enhanced in FIX-Padua carriers as compared to controls, and this could be explained by a greater thrombin generation in the former. Hyperactive FIX, but not wild-type FIX, enhanced fibrinolytic resistance also when the FXI-dependent positive feedback was blocked by a neutralising anti-FXI antibody. This thrombin-mediated, TAFI-dependent down-regulation of fibrinolysis provides new clues for explaining the heightened thrombotic risk in subjects carrying the FIXPadua mutation.

scholarly journals Effects of pH on kinetics of the structural rearrangement that gates the electron-transfer reaction between zinc cytochrome c and plastocyanin: Analysis of protonation states in a diprotein complex

2003 ◽  
Vol 68 (4-5) ◽  
pp. 327-337
Author(s):  
Milan Crnogorac ◽  
Nenad Kostic
Keyword(s):  
Electron Transfer ◽  
Cytochrome C ◽  
Structural Rearrangement ◽  
Electron Transfer Reaction ◽  
Salt Bridges ◽  
Wild Type ◽  
Redox Active ◽  
Effects Of Ph ◽  
In The Wild ◽  
Ph Range

Electron transfer from zinc cytochrome c to copper(II)plastocyanin in the electrostatically- stabilized complex [Crnogorac MM, Shen C, Young S Hansson O, Kostic NM (1996) Biochemistry 35, 16465?74]. We study this rearrangement in four complexes Zncyt/pc(II), which zinc cytochrome c makes with the wild-type form and the single mutants Asp42Asn, Glu59Gln, and Glu60Gln of plastocyanin. The rate constant for the rearrangement, kF differs for the four forms of plastocyanin but is independent of pH from 5.4 to 9.0 in all four cases. That kF is affected by the single mutations but not by pH changes suggests that the residues Asp 42, Glu59, and Glu60 in the wild-type plastocyanin remain deprotonated (i.e., as anions) within the Zncyt/pc(II) complex throughout the pH range examined. This fact agrees with the notion that loss of salt bridges in the initial (redox-inactive) configuration of the complex is compensated by formation of new salt bridges in the rearranged (redox-active) configuration.

scholarly journals Distribution and Severity of Placental Lesions Caused by the Chlamydia abortus 1B Vaccine Strain in Vaccinated Ewes

Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 543
Author(s):  
Sergio Gastón Caspe ◽  
Javier Palarea-Albaladejo ◽  
Clare Underwood ◽  
Morag Livingstone ◽  
Sean Ranjan Wattegedera ◽  
...  
Keyword(s):  
Disease Outbreaks ◽  
Principal Component ◽  
Placental Pathology ◽  
Wild Type ◽  
Cell Infiltrate ◽  
Chlamydia Abortus ◽  
Vaccine Type ◽  
The Difference ◽  
Enzootic Abortion Of Ewes

Chlamydia abortus infects livestock species worldwide and is the cause of enzootic abortion of ewes (EAE). In Europe, control of the disease is achieved using a live vaccine based on C. abortus 1B strain. Although the vaccine has been useful for controlling disease outbreaks, abortion events due to the vaccine have been reported. Recently, placental pathology resulting from a vaccine type strain (vt) infection has been reported and shown to be similar to that resulting from a natural wild-type (wt) infection. The aim of this study was to extend these observations by comparing the distribution and severity of the lesions, the composition of the predominating cell infiltrate, the amount of bacteria present and the role of the blood supply in infection. A novel system for grading the histological and pathological features present was developed and the resulting multi-parameter data were statistically transformed for exploration and visualisation through a tailored principal component analysis (PCA) to evaluate the difference between them. The analysis provided no evidence of meaningful differences between vt and wt strains in terms of the measured pathological parameters. The study also contributes a novel methodology for analysing the progression of infection in the placenta for other abortifacient pathogens.

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