BBA - General Subjects (v.1623, #1)

Oxidative stresses elevate the expression of cytochrome c peroxidase in Saccharomyces cerevisiae by Minsuk Kwon; Seonha Chong; Sanghwa Han; Kyunghoon Kim (1-5).
Cytochrome c peroxidase (CcP) uses hydrogen peroxide as an electron acceptor to oxidize cytochrome c (Cc) in the mitochondrial intermembrane space. A null allele of yeast CCP1 gene encoding CcP was created by one-step gene disruption method in a diploid yeast strain. Haploid yeast cells with the disrupted CCP1 gene were viable and able to grow in a medium containing lactic acid or glycerol as an energy source, indicating that CcP is not essential for both cell viability and respiration. However, CCP1-disrupted cells were more sensitive to H2O2 than wild-type cells. We also constructed a CCP1–lacZ fused gene and integrated this gene into yeast chromosomal DNA to monitor the expression of CCP1 gene. We found that expression of CCP1 gene increases under respiratory culture conditions and by treatments with H2O2. These results hint that the biological function of CcP is to reduce H2O2 generated during aerobic respiratory process. Moreover, expression of CCP1 gene increased by treatments with peroxynitrite, indicating that CcP may act as a peroxynitrite scavenger.
Keywords: Cytochrome c peroxidase; Hydrogen peroxide; Saccharomyces cerevisiae; Peroxynitrite; Reactive oxygen species;

The transit time and a characteristic time constant for the transition time are formulated for a pathway unit of Michaelis–Menten mechanism in metabolic chains with mass-action-type dependence. The pathway unit in a chain is assigned to each metabolite and two consecutive Michaelis–Menten-type reactions associated with it. All the relevant functions such as control coefficients, elasticities and transit time for the pathway unit are expressed in terms of the flux J and other system parameters. The transition time is characterized by a time constant for a first-order system expressed as the derivative of the total concentration of the internal metabolite with respect to the flux J.
Keywords: Transit time; Transition time; Pathway unit; Michaelis–Menten mechanism; Metabolic control analysis;

Several cyclic disulfide α-melanocyte stimulating hormone (α-MSH) analogues containing the aromatic fluorescent amino acid β-(2-naphthyl)-d-alanine (d-Nal) have high affinity and selectivity for the melanocortin (MC)-4 receptor. Considering the possible relevant role played by the lipid phase in the peptide-receptor interaction, the structures of two cyclic α-MSH analogues, containing both Trp and d-Nal fluorophores, were investigated by steady-state and time-resolved fluorescence spectroscopy, in aqueous solution and in the presence of dimyristoyl phosphatidylglycerol (DMPG) vesicles, and compared with that of the natural peptide. The amino acid d-Nal gives a unique de-excitation fluorescence profile, with an excited state lifetime much longer than those of Trp, allowing good distinction between the two fluorophores. The cyclic analogues' aqueous structures seem to be adequate for membrane penetration, as Trp fluorescence indicates that, in both aqueous and lipid media, the Trp environment in the cyclic peptides is similar to that of α-MSH when incorporated in lipid bilayers. Trp, in the cyclic analogues, seems to penetrate deeper in the bilayer than in the native peptide. The amino acid d-Nal was also found to penetrate deep into the lipid bilayer, having its excited-state lifetime drastically changed from aqueous solution to lipid medium. The present work shows that d-Nal may serve as a fluorescent probe for studies of MC peptides and suggests that the high affinity and selectivity of the cyclic peptides to the MC4 membrane receptor could be related to their deeper penetration into the bilayer core.
Keywords: α-MSH; d-Nal; Fluorescence; Structural property; Cyclic analogue;

Physico-chemical and immunological properties and partial amino acid sequencing of a new metalloprotease: endoprotease Thr-N by Sébastien Niamké; Olivier Guionie; Laetitia Guével-David; Claire Moallic; Soumaila Dabonne; Jean-Pierre Sine; Bernard Colas (21-28).
Previous studies have described the isolation of a new metalloprotease with a strict specificity for the amide bonds of peptide substrates having a threonine residue at the P1′ position [Biochem. Biophys. Res. Commun. 256 (1999) 307]. The present work reports the physico-chemical properties of the enzyme which enable the optimal conditions for the digestion of proteins by the protease to be determined. At pH 8.2 and up to 37 °C, the enzyme possesses a good proteolytic activity and is stable for at least 12 h. The protease is sensitive to detergents and dithiol-reducing agents so that these chemicals must be eliminated after treatment of the protein substrate when this needs to be denatured and reduced before its hydrolysis by the enzyme. An increase in the enzymatic activity is observed in the presence of urea up to a 2.0 M concentration, beyond which the activity decreases. The enzyme can also be used in the presence of organic solvents such as acetonitrile, isopropanol or dioxane (10%, v/v) without loss of activity. Studies performed with antibodies raised against the purified endoprotease Thr-N indicated the absence of cross-immunoinactivation and cross-immunoprecipitation with all tested proteases. Also, no homology of sequence was found with the proteases indexed in the databases. Thus, our results show that endoprotease Thr-N not only represents an original protease by its unique specificity but also by its immunological and molecular properties.
Keywords: Metalloprotease; Endoprotease Thr-N; Enzymatic specificity; Digestive enzyme; Snail (Achatina);

An electrochemical investigation of ligand-binding abilities of film-entrapped myoglobin by Wenjun Zhang; Chunhai Fan; Yuting Sun; Genxi Li (29-32).
Film-entrapped myoglobin exhibits well-defined electrochemistry which, upon ligand binding, displays a titratable redox potential shift. This effect has been observed to be highly dependent on the charged state of involved films. We have demonstrated that this approach may act as a model system for studies of molecular recognition between proteins and ligands.
Keywords: Protein film voltammetry; Myoglobin; Imidazole;

Effects induced by hydroxyl radicals on salmon calcitonin: a RP-HPLC, CD and TEM study by Maria Cristina Gaudiano; Marco Diociaiuti; Paola Bertocchi; Luisa Valvo (33-40).
The effects of hydroxyl radical attack on a peptidic drug were studied in vitro. Different chemico-physical techniques were used to investigate structural damage induced by oxidative stress conditions in salmon calcitonin (sCT), a peptide hormone used in treating osteoporosis. Reversed-phase liquid chromatography (RP-HPLC), circular dichroism (CD) and transmission electron microscopy (TEM) were applied to measure formation of oxidation/degradation products and to reveal the conformational and ultrastructural modifications in the presence of OH· free radicals. Hydroxyl radicals were obtained from ferrous sulfate and ascorbic acid mixtures. The RP-HPLC results revealed the formation of new chromatographic peaks indicating a number of degradation/oxidation products formed in the presence of OH· free radicals. CD spectra showed slight protein conformational modifications as well as aggregation. TEM confirmed sCT aggregation and suggested the formation of fibrillar aggregates.
Keywords: Oxidative stress; Hydroxyl radicals; Salmon calcitonin; RP-HPLC; CD; TEM;

Homocysteine induces vascular endothelial growth factor expression in differentiated THP-1 macrophages by Makiko Maeda; Isamu Yamamoto; Yasushi Fujio; Junichi Azuma (41-46).
Hyperhomocysteinemia has been reported to be an independent risk factor for atherosclerosis and atherothrombosis. However, the molecular mechanism by which hyperhomocysteinemia can lead to atherosclerosis and atherothrombosis has not been completely described. Vascular endothelial growth factor (VEGF) has been proposed to play an important role in the progression of atherosclerosis. In the present study, we hypothesized that hyperhomocysteinemia might be associated with VEGF expression in atherosclerotic lesions. We investigated VEGF mRNA expression and VEGF secretion by homocysteine (Hcy) in differentiated THP-1 macrophages. As a result, it has been revealed that VEGF mRNA was upregulated by Hcy in a dose- and time-dependent manner in THP-1 macrophages with the increase in VEGF secretion. Importantly, other sulfur compounds, such as methionine and cysteine, showed no effect on VEGF expression, indicating that homocysteine specifically induced VEGF. Our findings suggest that hyperhomocysteinemia could promote the development of atherosclerotic lesions through VEGF induction in macrophages.
Keywords: Homocysteine; Vascular endothelial growth factor; THP-1; mRNA; Macrophage;

NMR studies of a ferredoxin from Haloferax mediterranei and its physiological role in nitrate assimilatory pathway by Rosa M. Martı́nez-Espinosa; Frutos C. Marhuenda-Egea; Antonio Donaire; Marı́a José Bonete (47-51).
Haloferax mediterranei is a halophilic archaeon that can grow in aerobic conditions with nitrate as sole nitrogen source. The electron donor in the aerobic nitrate reduction to ammonium was a ferredoxin. This ferredoxin has been purified and characterised. Air-oxidized H. mediterranei ferredoxin has a UV–visible absorption spectra typical of 2Fe-type ferredoxins with an A 420/A 280 of 0.21. The nuclear magnetic resonance (NMR) spectra of the ferredoxin showed similarity to those of ferredoxins from plant and bacteria, containing a [2Fe–2S] cluster. The physiological function of ferredoxin might be to serve as an electron donor for nitrate reduction to ammonium by assimilatory nitrate (EC 1.6.6.2) and nitrite reductases (EC 1.7.7.1). The apparent molecular weight (Mr) of the ferredoxin was estimated to be 21 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
Keywords: Halophile; Archaea; Ferredoxin; Iron–sulfur cluster; Assimilatory nitrate pathway; Nuclear magnetic resonance;