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This paper reports notable observations regarding the ion charge states of thermally stable cytochrome c, generated using an alternating current (AC) electrospray ionization (ESI) device. An AC ESI sprayer entrains low‐mobility ions to accumulate at the meniscus cone tip prior to the ejection of detached aerosols to produce analyte ions. Therefore, as the solvent acidity varies, protein ions entrained in the AC cone tip are found to change conformation less significantly compared with those in the direct current (DC) cone. We acquired the AC ESI mass spectra of cytochrome c at pH range from 2 to 4. Unlike the DC ESI mass spectra showing clear conformation changes due to denaturing, the AC spectra indicated that only partial denaturing occurs even at extremely acidic pH 2. More native cytochrome c in lower charge states therefore remained. Moreover, with a solvent mixture of aqueous buffer and acetonitrile (70:30), partially denatured cytochrome c was still preserved at pH 2 by using AC ESI. Completely denatured proteins are observed at pH 2 by using DC ESI. [ABSTRACT FROM AUTHOR]

Presents several articles to be published in the "European Journal of Biochemistry." "An Endonuclease From Yeast Mitochondrial Fractions," by R. Morosoli and C. V. Lusena; "Antigenic Relationships Between Pore Proteins of Escherichia coli K12," by N. Overbeeke, G. van Scharrenburg and B. Lugtenberg; "Respiratory Properties of Cytochrome-c-Deficient Mutants of Azotobacter vinelandii," by P.S. Hoffman, T. V. Morgan and D. V. DerVartanian; "Hydroxyl-Ion-Induced Subunit Dissociation of Yeast Cytoplasmic Pyruvate Decarboxylase. A Circular Dichroism Study," by R. F. W. Hopmann; Others.

Air pollution mostly originating from car exhaust and industrial emissions is seriously endangering human health. The study of toxic effects of particulate matter (PM) is a subject of very active research that covers a broad field from biology to chemistry and technology. The main issue of these works is the understanding of the oxidative properties of PM. In this paper, we present a quantitative analytical method based on the resonance Raman spectroscopy of cytocrome c that can be used to monitor the oxidative reactions that may take place between biological samples and PM. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Size-exclusion chromatography in line with small-angle X-ray scattering (SEC-SAXS) has emerged as an important method for investigation of heterogeneous and self-associating systems, but presents specific challenges for data processing including buffer subtraction and analysis of overlapping peaks. This paper presents novel methods based on singular value decomposition (SVD) and Guinier-optimized linear combination (LC) to facilitate analysis of SEC-SAXS data sets and high-quality reconstruction of protein scattering directly from peak regions. It is shown that Guinier-optimized buffer subtraction can reduce common subtraction artifacts and that Guinier-optimized linear combination of significant SVD basis components improves signal-to-noise and allows reconstruction of protein scattering, even in the absence of matching buffer regions. In test cases with conventional SAXS data sets for cytochrome c and SEC-SAXS data sets for the small GTPase Arf6 and the Arf GTPase exchange factors Grp1 and cytohesin-1, SVD-LC consistently provided higher quality reconstruction of protein scattering than either direct or Guinier-optimized buffer subtraction. These methods have been implemented in the context of a Python-extensible Mac OS X application known as Data Evaluation and Likelihood Analysis ( DELA), which provides convenient tools for data-set selection, beam intensity normalization, SVD, and other relevant processing and analytical procedures, as well as automated Python scripts for common SAXS analyses and Guinier-optimized reconstruction of protein scattering. [ABSTRACT FROM AUTHOR]

Hybrid architectures which combine a MIP with an immobilized 'affinity ligand' or a biocatalyst sum up the advantages of both components. In this paper, hybrid architectures combining a layer of a molecularly imprinted electropolymer with a mini-enzyme or a self-assembled monolayer will be presented. (i) Microperoxidase-11 (MP-11) catalyzed oxidation of the drug aminopyrine on a product-imprinted sublayer: The peroxide dependent conversion of the analyte aminopyrine takes place in the MP-11 containing layer on top of a product-imprinted electropolymer on the indicator electrode. The hierarchical architecture resulted in the elimination of interfering signals for ascorbic acid and uric acid. An advantage of the new hierarchical structure is the separation of MIP formation by electropolymerization and immobilization of the catalyst. In this way it was for the first time possible to integrate an enzyme with a MIP layer in a sensor configuration. This combination has the potential to be transferred to other enzymes, e.g. P450, opening the way to clinically important analytes. (ii) Epitope-imprinted poly-scopoletin layer for binding of the C-terminal peptide and cytochrome c (Cyt c): The MIP binds both the target peptide and the parent protein almost eight times stronger than the non-imprinted polymer with affinities in the lower micromolar range. Exchange of only one amino acid in the peptide decreases the binding by a factor of five. (iii) MUA-poly-scopoletin MIP for cytochrome c: Cyt c bound to the MIP covered gold electrode exhibits direct electron transfer with a redox potential and rate constant typical for the native protein. The MIP cover layer suppresses the displacement of the target protein by BSA or myoglobin. The combination of protein imprinted polymers with an efficient electron transfer is a new concept for characterizing electroactive proteins such as Cyt c. The competition with other proteins shows that the MIP binds its target Cyt c preferentially and that molecular shape and the charge of protein determine the binding of interfering proteins. [ABSTRACT FROM AUTHOR]

This paper reports the first tests of the suitability of the standardized mitochondrial cytochrome c oxidase subunit I (COI) barcoding system for the identification of Canadian deerflies and horseflies. Two additional mitochondrial molecular markers were used to determine whether unambiguous species recognition in tabanids can be achieved. Our 332 Canadian tabanid samples yielded 650 sequences from five genera and 42 species. Standard COI barcodes demonstrated a strong A + T bias (mean 68.1%), especially at third codon positions (mean 93.0%). Our preliminary test of this system showed that the standard COI barcode worked well for Canadian Tabanidae: the target DNA can be easily recovered from small amounts of insect tissue and aligned for all tabanid taxa. Each tabanid species possessed distinctive sets of COI haplotypes which discriminated well among species. Average conspecific Kimura two-parameter (K2P) divergence (0.49%) was 12 times lower than the average divergence within species. Both the neighbour-joining and the Bayesian methods produced trees with identical monophyletic species groups. Two species, Chrysops dawsoni Philip and Chrysops montanus Osten Sacken (Diptera: Tabanidae), showed relatively deep intraspecific sequence divergences (∼10 times the average) for all three mitochondrial gene regions analysed. We suggest provisional differentiation of Ch. montanus into two haplotypes, namely, Ch. montanus haplomorph 1 and Ch. montanus haplomorph 2, both defined by their molecular sequences and by newly discovered differences in structural features near their ocelli. [ABSTRACT FROM AUTHOR]

Oxygen is a vital substrate for plant energy metabolism. Since plants do not have a sophisticated mechanism to deliver oxygen to those sites where it is actually needed, a plant cell has to continuously cope with changes of the oxygen tension within the tissue. The actual internal oxygen concentration will depend on the resistance for oxygen diffusion through the tissue, as well as on the actual respiratory activity. This paper discusses the current state of knowledge on the regulation of respiration by the oxygen availability. Contradicting opinions from the literature on plant respiration are reviewed and commented upon. Also, knowledge about the regulation of respiration in animal mitochondria is included. Apart from changes in glycolytic flux, the role of both the cytochrome-c oxidase (COX) and the alternative oxidase (AOX) in the adaptive response of respiration to changes in the oxygen availability are discussed. One hypothesis is formulated which describes an alternative or additional role for AOX. It is suggested that AOX could play a role in maintaining oxygen homeostasis within the mitochondrion. Because of the relative low affinity for oxygen of AOX as compared to COX, the alternative oxidase will not interfere with COX activity, but AOX activity will reduce the free oxygen concentration, thereby decreasing the production of reactive oxygen species (ROS) inside the mitochondrion. [ABSTRACT FROM AUTHOR]

In addition to the conventional cytochrome c oxidase, mitochondria of all plants studied to date contain a second cyanide-resistant terminal oxidase or alternative oxidase (AOX). The AOX is located in the inner mitochondrial membrane and branches from the cytochrome pathway at the level of the quinone pool. It is non-protonmotive and couples the oxidation of ubiquinone to the reduction of oxygen to water. For many years, the AOX was considered to be confined to plants, fungi and a small number of protists. Recently, it has become apparent that the AOX occurs in wide range of organisms including prokaryotes and a moderate number of animal species. In this paper, we provide an overview of general features and current knowledge available about the AOX with emphasis on structure, the active site and quinone-binding site. Characterisation of the AOX has advanced considerably over recent years with information emerging about the role of the protein, regulatory regions and functional sites. The large number of sequences available is now enabling us to obtain a clearer picture of evolutionary origins and diversity. [ABSTRACT FROM AUTHOR]

Background information. Caspase-dependent and -independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53wt (wild-type p53) HeLa cells compared with p53mt (mutated p53) C-33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase-9/caspase-3 mitochondrial-dependent apoptotic pathway and via a delayed caspase-independent pathway. In contrast with p53wt cells, p53mt C-33A cells exhibit firstly caspase-8 activation leading to caspase-3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP-1 [poly(ADP-ribose) polymerase-1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z-VAD-fmk points toward a major involvement of a caspase-dependent pathway in staurosporine-induced apoptosis in p53wt HeLa cells, which is not the case in p53mt C-33A cells. Meanwhile, the use of 3-aminobenzamide, a PARP-1 inhibitor known to prevent AIF (apoptosis-inducing factor) release, significantly decreases staurosporine-induced death in these p53mt carcinoma cells, suggesting a preferential implication of caspase-independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin-α, isolated as a suppressor of p53-mediated transactivation, or by PRIMA-1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio-nuclear AIF translocation in staurosporine-induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase-8 or caspase-9 signalling cascades and via caspase-independent cell death, as well as through p53 activity. [ABSTRACT FROM AUTHOR]

Pancreatitis is a severe and frequently lethal disorder, a major cause of which is alcohol abuse. Parenchymal cell death is a major complication of pancreatitis. In experimental models of (non-alcoholic) acute pancreatitis, acinar cells have been shown to die through both necrosis and apoptosis, the two principal pathways of cell death. The severity of experimental acute pancreatitis correlates directly with the extent of necrosis and inversely with apoptosis. Thus, understanding the regulation of apoptosis and necrosis, and whether it is possible to manipulate the pattern of death responses, is becoming exceedingly important in investigations of the pathogenesis and treatment of pancreatitis. The effects of alcohol on cell death responses of pancreatitis, and the mechanisms that may mediate these effects, are just starting to be explored. This paper reviews the signaling pathways mediating the balance between apoptosis and necrosis in acute pancreatitis, and alcohol’s effects on cell death responses in pancreatitis. [ABSTRACT FROM AUTHOR]

Methamphetamine (METH) is an illicit drug that causes neurodegenerative effects in humans. In rodents, METH induces apoptosis of striatal glutamic acid decarboxylase (GAD) -containing neurons. This paper provides evidence that METH-induced cell death occurs consequent to interactions of ER stress and mitochondrial death pathways. Specifically, injections of METH are followed by an almost immediate activation of proteases calpain and caspase-12, events consistent with drug-induced ER stress. Involvement of ER stress was further supported by observations of increases in the expression of GRP78/BiP and CHOP. Participation of the mitochondrial pathway was demonstrated by the transition of AIF, smac/DIABLO, and cytochrome c from mitochondrial into cytoplasmic fractions. These changes occur before the apoptosome-associated pro-caspase-9 cleavage. Effector caspases-3 and -6, but not -7, were cleaved with the initial time of caspase-3 activation occurring before caspase 9 cleavage; this suggests possible earlier cleavage of caspase-3 by caspase-12. These events preceded proteolysis of the caspase substrates DFF-45, lamin A, and PARP in nuclear fractions. These findings indicate that METH causes neuronal apoptosis in part via cross-talks between ER- and mitochondria-generated processes, which cause activation of both caspase-dependent and -independent pathways. [ABSTRACT FROM AUTHOR]

Microperoxidase 8 (MP8) is a heme octapeptide obtained by hydrolytic digestion of horse heart cytochrome c. At pH below 9, the heme iron is axially coordinated to the imidazole side chain of His18 and to a water molecule. Replacement of this weak ligand by H2O2 allows the formation of high-valent iron-oxo species which are responsible for both peroxidase-like and cytochrome P450-like activities of MP8. This paper shows that MP8 is able to catalyze the nitration of phenol by nitrite. The reaction requires H2O2 and is inhibited by ligands having a high affinity for the iron, catalase and radical scavengers. This suggests that the nitrating species could be NO2• radicals formed by the oxidation of nitrite by high-valent iron-oxo species. This new activity of MP8 opens a new access to nitro-aromatic compounds under mild conditions and validates the use of this minienzyme to mimick heme peroxidases, especially in the reactions of NO-derived species with biomolecules under oxidative stress conditions. [ABSTRACT FROM AUTHOR]

Reports in the literature indicate that the flavin prosthetic group of Chromatium cytochrome c552 is not acid-extractable but is dissociated from the protein by various treatments, including prolonged incubation with urea solution, which are not expected to break covalent bonds. In the present paper it is established by isolation of peptic and of tryptic-chymotryptic flavin peptides that the flavin component is covalently linked to the protein. The peptide chain is attached to the 8α group of the FAD, as in other enzymes containing covalently bound flavin, as evidenced by a hypochromic shift of the near ultraviolet absorption maximum, the characteristics of the hyperfine EPR spectrum, and the release of 8-carboxy-FMN from the cytochrome on oxidation with cold performic acid with concomitant hydrolysis of the pyrophosphate linkage. The fluorescence of the flavin peptides, at the FAD, FMN and riboflavin level, is extensively quenched, with less than 1% of the quantum yield of fluorescence, compared with riboflavin, in the peptic peptide. On oxidation of the tryptic-chymotryptic peptide with performic acid the fluorescence increases to 50% of that given by an equimolar concentration of riboflavin. This increase is accompanied by a further hypochromic shift of the near ultraviolet absorption maximum. This behavior, the tendency of the flavin peptide to undergo autooxidation, and the positive chloroplatinic acid test resemble the properties of cysteinylflavin thioether and its peptides and suggest that the flavin is bonded to a cysteine residue, as in monoamine oxidase. The presence of cysteine in both flavin peptides from cytochrome c552 has been verified by the liberation of cysteine on acid hydrolysis. Despite these similarities to peptides containing a cysteinyl flavin thioether. the peptic and tryptic-chymotryptic peptides from cytochrome c552 show several properties which preclude a thioether linkage. Evidence is summarized to indicate that the flavin is linked via a thiohemiacetal bond to a cysteinyl residue in the polypeptide chain. Thus, the flavin released from the peptides by acid hydrolysis is in every respect identical to 8-formylriboflavin. Further, two flavin components were detected on high voltage electrophoresis after aminopeptidase M digestion of the peptic peptide, which had mobilities expected for the aminoacylflavin and a thiazolidine derivative. A thiohemiacetal structure also ac-counts for the much greater lability of the flavin peptide linkage than that found for monoamine oxidase. Thus from the evidence presented, it is concluded that the structure of the covalently bound flavin in Chromatium cytochrome c552 is 8α-S-cysteinyl-8α-hydroxy-FAD. [ABSTRACT FROM AUTHOR]

We have developed a new technique for the study of redox-linked conformational changes in proteins, by the combination of two established techniques. Fourier-transform infrared spectroscopy has been used together with direct electrochemistry of the protein at a modified metal electrode surface. The technique has been evaluated with cytochrome c. because of its well-characterized electrochemistry and because the availability of X-ray crystallographic and NMR studies of both redox states of the protein provides a reference against which our data can be compared. In electrochemical control experiments, it was confirmed that the spectroelectrochemical cell design allows fast, accurate and reproducible control of the redox poise of the protein. The resulting reduced-minus-oxidized infrared difference spectra show the changes in the frequencies and intensities of molecular vibrations which arise from the redox-linked conformational change. In contrast to the absolute infrared spectra of proteins, such difference spectra can be sufficiently straightforward to allow interpretation at the level of individual bonds. A complete interpretation of the spectra is beyond the scope of the present paper: however, on the basis of the data presented, we are able to suggest assignments for all except one of the major bands between 1500 cm-1 and 1800 cm -1. [ABSTRACT FROM AUTHOR]

The protomeric chain of Hansenula anomala flavocytochrome b2 was previously shown to be built as the covalent association of two functional domains: an L-lactate dehydrogenase domain and a cytochrome c reductase domain, joined together by a proteolytically sensitive zone. This paper concerns the specific cleavage of this latter zone with a H. anomala proteinase(s) preparation and the purification of the resulting L-lactate dehydrogenase moiety of the molecule with at least 25% recovery, (i.e. one order of magnitude more than for the previously published method). A preliminary characterization of this dehydrogenase domain indicates that it is a tetramer (Mr = 4 × 39000) containing FMN as expected and not heme. It has high L-lactate:ferricyanide oxidoreductase activity (about 70% that of the whole flavocytochrome b2) and the same Km for L(+)-lactate as flavocytochrome b2, but it has no L-lactate:cytochrome c oxidoreductase activity. Its flavin semiquinone is stabilized in the presence of pyruvate as in fiavocytochrome b2. The subcellular origin of the H. anomala proteinase in the preparation has not yet been elucidated. [ABSTRACT FROM AUTHOR]

The reversible association of the Zn2+-substituted Hansenula anomala cytochrome c dimer (Thomas et al., preceding paper in this issue) to flavocytochrome b2 in oxidized or lactate-reduced state has been investigated by fluorimetry. The same method has been used for the determination of Zn-cytochrome c complexing to defined proteolytic fragments of flavocytochromc b2, either heme-b2-containing monomers or a flavin-finked tetramer. All these fragments but the isolated cytochrome b2 core showed binding stoichiometries, Kd values and ionic strength dependences quite similar to those found for native flavocytochrome b2. These data allowed localization of the single high-affinity binding site of cytochrome c on a particular globule in the dehydrogenase domain of the flavocytochrome b2 protomers. Quenching of the Zn-porphyrin c fluorescence in the various complexes occurred with only minor changes of the fluorescence lifetime and did not show any direct relationship to the presence or the redox state of the heine b2 group. [ABSTRACT FROM AUTHOR]

A model is proposed to explain the biphasic reduction of ferricytochrome c at alkaline pH assuming an equilibrium distribution between a reducible and non-reducible conformer. An analysis of this model is given in an appendix to this paper. Protein denaturants, increasing pH and temperature are found to disturb the conformer equilibrium in favour of the irreducible form. The thermodynamic parameters governing this equilibrium have been determined. Kinetic parameters defined in the model were measured under a variety of conditions. The kinetic and equilibrium data strongly suggests that only the reducible conformer possesses the 695 nm band. Possible origins of the 695 nm band are discussed. [ABSTRACT FROM AUTHOR]

Amino acid analyses of L-lactate dehydrogenase from baker's show that the minimum molecular weight (53 000 daltons) of the protein is much lower than found in the literature (80 000). This result, combined with those reported in the following papers, leads to a revision of the dimeric model generally accepted for cytochrome b2 [ABSTRACT FROM AUTHOR]

A functional respiratory chain is dependent on protein components encoded by both mtDNA and nuclear DNA. Isolated cytochrome c oxidase (COX) deficiency is often caused by mutations in nuclear genes regulating the assembly of the 13 protein subunits of this complex. The accompanying paper by Zeman and co-workers reports that mutations in SCO2 are common in infantile COX deficiency and are associated with a very poor prognosis. Conclusion: Molecular diagnosis is often feasible in patients with COX deficiency and particular attention should he paid to mutations in COX assembly genes. [ABSTRACT FROM AUTHOR]

In this paper we tested the hypothesis that interrupting reperfusion (ischemic postconditioning) reduces the neuron death after transient global cerebral ischemia/reperfusion. Ten min transient cerebral ischemia was induced by four-vessel occlusion in male Sprague-Dawley rats. The animals underwent postconditioning consisting of 3 cycles of 15s/15s, 30s/30s or 60s/15s reperfusion/occlusion, respectively, which were applied after the start of reperfusion. Cytosolic expression of cytochrome c and caspase-3 was evaluated by immunohistochemistry. Ten min ischemia and 7 days reperfusion destroyed approximately 79% of CA1 neurons and 58% parietal cortex neurons, respectively. Three cycles of 30s/30s reperfusion/ischemia postconditioning decreased the neuron loss to 7% in CA1, and 10% in cortex. Another two postconditioning protocols failed to rescue the CA1 neuron death, but decreased the loss of cortex neurons. The expression of cytochrome c at 5h and 12h and caspase-3 at 12h increased significantly after reperfusion, which was prevented by three cycles of 30s/30s reperfusion/ischemia postconditioning. Our data provide the evidence that appropriate interrupting reperfusion is neuroprotective against global cerebral ischemia/reperfusion injury, and this protective effect is through preventing the activation of caspase and then neuronal apoptosis. [ABSTRACT FROM AUTHOR]

An increase in mitochondrial membrane permeability is central to cell death including apoptosis and necrosis. During apoptosis, permeabilization of outer mitochondrial membrane leads to the release of several apoptogenic factors, such as cytochrome c and Smac/Diablo, into the cytoplasm that activate downstream death programs, including apoptotic proteases called caspases, although the detailed mechanism of outer mitochondrial membrane permeabilization remains elusive. Although the mitochondrial membrane permeability transition (MPT), resulting in Δψ loss, mitochondrial swelling and rupture of the outer membrane has initially been proposed as a general mechanism for apoptotic permeabilization of outer mitochondrial membrane, the recent studies with cyclophilin D-deficient mice indicate that MPT regulates some forms of necrotic death, but not apoptotic death, and that MPT is involved in ischemia–reperfusion injury in heart and brain. Anti-apoptotic proteins, Bcl-2 and Bcl-xL, efficiently block not only apoptotic mitochondrial permeabilization but also MPT. The present paper focuses on the mechanisms by which Bcl-2 family members control the permeability of mitochondrial membrane during apoptosis and necrosis. [ABSTRACT FROM AUTHOR]