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Shedding light on the mitochondrial permeability transition
No full textThe mitochondrial permeability transition is an increase of permeability of the inner mitochondrial membrane to ions and solutes with an exclusion size of about 1500 Da. It is generally accepted that the permeability transition is due to opening of a high-conductance channel, the permeability transition pore. Although the molecular nature of the permeability transition pore remains undefined, a great deal is known about its regulation and role in pathophysiology. This review specifically covers the characterization of the permeability transition pore by chemical modification of specific residues through photoirradiation of
mitochondria after treatment with porphyrins. The review also illustrates the basic principles of the photodynamic effect and the mechanisms of phototoxicity and discusses the unique properties of singlet oxygen generated by specific porphyrins in discrete mitochondrial domains. These experiments provided remarkable information on the role, interactions and topology of His and Cys residues in permeability transition pore modulation and defined an important role for the outer membrane 18 kDa translocator
protein (formerly known as the peripheral benzodiazepine receptor) in regulation of the permeability transition
Interaction of human serum albumin with hematoporphyrin and its Zn(2)+-and Fe(3)+-derivatives.
No full textEfficient photoinactivation of methicillin-resistant Staphylococcus aureus by a novel porphyrin incorporated into a poly-cationic liposome
No full textAntimicrobial photodynamic therapy is emerging as a promising therapeutic modality for bacterial infections. Our studies aim at identifying strategies for optimizing the antibacterial activity of porphyrin-type photosensitisers. The photoinactivation properties of a novel, positively charged meso-substituted porphyrin, namely 5-[4-(1-dodecanoylpyridinium)]-10,15,20-triphenyl-porphyrin were tested against a typically antibiotic-resistant pathogen, such as methicillin-resistant Staphylococcus aureus. This porphyrin is characterized by an unusually large quantum yield (0.95) for the generation of the hyper-reactive oxygen species, singlet oxygen. In spite of this, it exhibits a relatively low photosensitising activity against bacteria when dissolved in a homogeneous aqueous solution or incorporated into neutral lipid vesicles. On the contrary, a dramatic potentiation of the photocydal effect takes place when polycationic agents such as liposomes of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride are used as carriers. The cationic carrier primarily acts as a disorganizing agent for the native three-dimensional architecture of the bacterial wall, thereby enhancing its permeability to the photosensitiser. Consequently, the drug can deeply penetrate into the plasma membrane, and rapidly impair selected enzymic activities leading to cell death. Thus, the combination of positively charged drugs and cationic delivery systems appears to represent an innovative modality for achieving an efficient antimicrobial activity and opens new avenues for the development of this phototherapeutic application. (C) 2007 Elsevier Ltd. All rights reserved
Removal of copper from Octopus vulgaris hemocyanin. Preparation of half-apo and apo-derivatives.
No full textThe two copper ions bound in the active site of Octopus vulgaris haemocyanin can be removed by cyanide. The two metal ions react with the ligand sequentially. In this paper the preparation of Octopus half-apo-haemocyanin, containing at the active site a single copper ion, is described. Moreover, the conditions to obtain Octopus apohaemocyanin, containing less than 3% of copper- still bound, are given
COBALT SUBSTITUTION STUDIES ON BOVINE ERYTHROCYTE SUPEROXIDE-DISMUTASE - EVIDENCE FOR A NOVEL COBALT-SUPEROXIDE DISMUTASE DERIVATIVE
No full textThe kinetics of the reaction of Octopus vulgaris hemocyanin with cyanide. Its significance for the structure of the 11 S subunit of molluscan hemocyanin.
No full textNative Octopus vulgaris hemocyanin (Hc) reacts with cyanide stepwise. The first step involves the formation of a complex HcCN with O2 displacement. This complex reacts further with cyanide causing the removal of one copper ion from the active site. The same reaction sequence occurs for the extraction of the second metal ion. The formation of the HcCN complex and the removal of the first and the second copper ion can be differentiated according to the KCN concentration. The rate of metal removal is slightly affected by KCN concentration. The kinetics are dominated by site-site interactions. The kinetic curves show only slight differences when the protein is in the 11 S or 49 S aggregation states, suggesting that the site-site interactions are restricted mainly within the 11 S structure. A kinetic model describing the removal of the first copper ion is proposed assuming that the 11 S component (MW 250,000) is an annular-shaped structure made by five equivalent functional subunits (MW 50,000). An explanation for the incomplete copper removal from molluscan Hc is given. The results are compared with those previously reported for Carcinus maenas Hc
Singlet Oxygen Produced by Photodynamic Action Causes Inactivation of the Mitochondrial Permeability Transition Pore.
No full textWe have studied the effects of singlet oxygen produced by photodynamic action on the cyclosporin A-sensitive permeability transition (PT) in isolated rat liver mitochondria. Mitochondria were incubated with 3 microM hematoporphyrin and irradiated at 365 nm with a fluence rate of 25 watts/m2. For short durations of irradiation (60 s) the adenine nucleotide translocase was inactivated, but mitochondria retained their ability to form a proton electrochemical gradient and accumulated Ca2+ and Pi at the same rate as non-irradiated controls. Strikingly, however, the oxidative effects of photodynamic action prevented opening of the PT pore which is normally induced by Ca2+ plus Pi or by treatment with diethyl pyrocarbonate (a histidine reagent) or diamide (a thiol oxidant). We show that the most likely targets for photodynamic action are critical histidines that undergo degradation. Irradiated, hematoporphyrin-loaded mitochondria treated with diethyl pyrocarbonate or diamide still undergo the PT when treated with phenylarsine oxide, which reacts with a critical dithiol involved in pore modulation (Petronilli, V., Costantini, P., Scorrano, L., Colonna, R., Passamonti, S., and Bernardi, P. (1994) J. Biol. Chem. 269, 16638-16642). These data suggest (i) that the dithiol cysteines are not oxidized by photodynamic action, but rather became inaccessible to oxidants; and (ii) that irradiation of hematoporphyrin-loaded mitochondria does not lead to pore denaturation, but rather to site-selective inactivation of discrete pore functional domains
The reaction between cyanide and the hemocyanin of Carcinus maenas. A kinetic study.
No full textThe kinetics of the reaction between Carcinus maenas hemocyanin and cyanide has been studied at various KCN concentrations and a different temperatures (21° and 4°C) by following the decrease of the copper-peroxide absorption band, centered at 337 nm, of the copper still bound to the protein and the intrinsic fluorescence changes as functions of time. In all conditions used, the absorption band completely disappears and KCN concentration affects only the rate of the process. The reaction is kinetically homogeneous indicating no site-site interaction. The apparent rate constant increases with the square of cyanide concentration and the inverse of O2 concentration. The copper still bound decreases at a rate slower than the 337 nm absorption and the process is not kinetically homogeneous. The fluorescence of the protein increases after an induction period showing an inflection point at about 50% of the total effect. A kinetic model has been proposed on the assumption that the two metal ions are removed sequentially from the active site. The experimental data are in agreement with the theoretical equations derived from the model. The equilibrium constants for the formation of the complex between the first and the second copper ion with cyanide and the rate constants of their decomposition have been calculated. The rate-limiting process for the removal of the second copper ion is the formation of the complex with cyanide
Circular dichroism and fluorescence studies to probe the conformational properties of Rhus vernicifera laccase
No full textThe conformational properties of native, apo- and type-2 copper depleted laccase from Rhus vernicifera have been investigated by circular dichroism and fluorescence spectroscopies. Circular dichroism experiments reveal a high prevalence of random-coil structure in all laccase derivatives, the content of α-helix and β-sheet not exceeding 8% and 21%, respectively. Nevertheless, the microenvironment of the tryptophan residues is deeply shielded from the external medium and exhibits a marked stability against pH-denaturation. Fluorescence data suggest that a class of tryptophan residues close to type-2 site contributes 50% to the overall fluorescence emitted by apo- and type-2 copper depleted laccase. These residues are masked in the native enzyme, due to quenching effects by copper ions and adjacent amino acid side chains. The interaction of 1-anilino-8-naphthalene sulfonate (ANS) with laccase has been studied by following the fluorescence changes of the dye upon binding. Native laccase d..
STEADY-STATE AND TIME-RESOLVED SPECTROSCOPIC STUDIES ON THE HEMATOPORPHYRIN LIPOPROTEIN COMPLEX
No full textThe interaction of hematoporphyrin (Hp) with the isolated rabbit lipoprotein fractions very low density lipoproteins, low-density lipoproteins, and high-density lipoproteins has been studied by steady-state and time-resolved spectroscopy. The porphyrin appears to be bound to both the apoprotein and the lipid phase. The two populations of lipoprotein-bound Hp molecules can be distinguished on the basis of the fluorescence excitation spectrum, decay constants of the lowest excited singlet and triplet states, and accessibility to oxygen. Upon Hp binding, the intrinsic fluorescence emission of apolipoproteins is quenched at least in part via singlet-singlet energy transfer from tryptophyl residues to the porphyrin moiety. The binding of Hp with the protein matrix can be adequately described on the basis of Scatchard analysis, whereas the interaction of Hp with the lipid core can be described as the partitioning of the dye between a hydrophobic and an aqueous phase. The Hp binding capacity of lipoproteins is maximal for very low density lipoproteins
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