Estudo epidemiológico pré-cirúrgico de cadelas portadoras de neoplasias mamárias

O artigo não apresenta resumo.

Tryptic digestion of soybean lipoxygenase-1 generates a 60 kDa fragment with improved activity and membrane binding ability.

Lipoxygenases are key enzymes in the metabolism of unsaturated fatty acids. Soybean lipoxygenase-1 (LOX-1), a paradigm for lipoxygenases isolated from different sources, is composed of two domains: a approximately 30 kDa N-terminal domain and a approximately 60 kDa C-terminal domain. We used limited proteolysis and gel-filtration chromatography to generate and isolate a approximately 60 kDa fragment of LOX-1 ("mini-LOX"), produced by trypsin cleavage between lysine 277 and serine 278. Mini-LOX was subjected to N-terminal sequencing and to electrophoretic, chromatographic, and spectroscopic analysis. Mini-LOX was found to be more acidic and more hydrophobic than LOX-1, and with a higher content of alpha-helix. Kinetic analysis showed that mini-LOX dioxygenates linoleic acid with a catalytic efficiency approximately 3-fold higher than that of LOX-1 (33.3 x 10(6) and 10.9 x 10(6) M(-1) x s(-1), respectively), the activation energy of the reaction being 4.5 +/- 0.5 and 8.3 +/- 0.9 kJ x mol(-1) for mini-LOX and LOX-1, respectively. Substrate preference, tested with linoleic, alpha-linolenic, and arachidonic acids, and with linoleate methyl ester, was the same for LOX-1 and mini-LOX, and also identical was the regio- and stereospecificity of the products generated thereof, analyzed by reversed-phase and chiral high-performance liquid chromatography, and by gas chromatography/mass spectrometry. Mini-LOX was able to bind artificial vesicles with higher affinity than LOX-1, but the binding was less affected by calcium ions than was that of LOX-1. Taken together, these results suggest that the N-terminal domain of soybean lipoxygenase-1 might be a built-in inhibitor of catalytic activity and membrane binding ability of the enzyme, with a possible role in physio(patho)logical conditions.

Conformational changes at the active site of pantetheine hydrolase during denaturation by guanidine hydrochloride.

Conformational changes at the active site of pantetheine hydrolase (EC3.5.1.-) during guanidine hydrochloride (GndHCl) denaturation were investigated by UV and circular dichroism spectroscopy and by electron spin resonance spectroscopy, following the spectral behaviour of the nitroxide radicals (N-(1-oxyl-2,2,5,5,-tetramethyl-3-pyrrolidinyl) iodacetamide) covalently linked to the two active site cysteine residues. At low denaturant concentrations (0.2 M) no conformational changes may be observed, whereas the catalytic activity, is strongly affected. The results indicate that the active site of pantetheine hydrolase is labile and unfolds under conditions in which no global tertiary structure modifications can be observed.

Paracoccus denitrificans cytochrome c oxidase: a kinetic study on the two- and four-subunit complexes.

Cytochrome c oxidase from Paracoccus denitrificans has been purified in two different forms differing in polypeptide composition. An enzyme containing polypeptides I-IV is obtained when the purification procedure is performed in beta-d-dodecylmaltoside. If, however, Triton X-100 is used to purify the enzyme under otherwise identical conditions, an enzyme is obtained containing only subunits I-II. The two enzymes are undistinguishable by optical spectroscopy but show significant differences in the transient and steady-state time regimes, as studied by stopped-flow spectroscopy. The observed differences, however, are not due to removal of subunits III and IV, but rather to a specific effect of Triton X-100 which appears to affect cytochrome c binding. From these results it is not expected that subunits III and IV play any significant role in cytochrome c binding and, possibly, in the subsequent electron transfer processes. The results also suggest that both electrostatic and hydrophobic interactions may be important in the initial electron transfer process from cytochrome c.

Electron entry in a CuA mutant of cytochrome c oxidase from Paracoccus denitrificans. Conclusive evidence on the initial electron entry metal center.

A cytochrome c oxidase subunit II C216S mutant from Paracoccus denitrificans in which the CuA site was changed by site-directed mutagenesis to a mononuclear copper site [Zickermann, V., Wittershagen, A., Kolbesen, B.O. and Ludwig, B. Biochemistry 36 (1997) 3232-3236] was investigated by stopped-flow spectroscopy. Contrary to the behavior of the wild type enzyme, in this mutant cytochrome a cannot be reduced by excess cytochrome c in the millisecond time scale in which cytochrome c oxidation is observed. The results conclusively identify and establish CuA as the initial electron entry site in cytochrome c oxidase. Partial rapid reduction (ca. 20%) of the modified CuA site suggests that the mononuclear copper ion has a redox potential ca. 100 mV lower than the wild type, and that internal electron transfer to cytochrome a is > or = 10(3)-fold slower than with the wild type enzyme.

Investigating the mechanism of electron transfer to the binuclear center in Cu-heme oxidases.

Novel experimental evidence is presented further supporting the hypothesis that, starting with resting oxidized cytochrome c oxidase, the internal electron transfer to the oxygen binding site is kinetically controlled. The reduction of the enzyme was followed spectroscopically and in the presence of NO or CO, used as trapping ligands for reduced cytochrome a3; ruthenium hexamine was used as a spectroscopically silent electron donor. Consistent with the high combination rate constant for reduced cytochrome a3, NO proved to be a very efficient trapping ligand, while CO did not. The results are discussed in view of two alternative (thermodynamic and kinetic) hypotheses of control of electron transfer to the binuclear (cyt.a3-CuB) center. Fulfilling the prediction of the kinetic control hypothesis: i) the reduction of cytochrome a3 and ligation are synchronous and proceed at the intrinsic rate of cytochrome a3 reduction, ii) the measured rate of formation of the nitrosyl derivative is independent of the concentration of both the reductant and NO.

Cytochrome-c-binding site on cytochrome oxidase in Paracoccus denitrificans.

To monitor the docking site for cytochrome c on cytochrome oxidase from Paracoccus denitrificans, a series of site-directed mutants in acidic residues exposed on the three largest subunits was constructed, and the purified enzymes were assayed for their steady-state kinetic parameters, their ionic strength dependence, and their fast electron entry kinetics by stopped-flow measurements. Increasing the ionic strength, the maximum of the bell-shaped dependence of the steady-state rate observed for wild type shifts the maximum to lower ionic strength in most of the mutants. The Km determined in steady-state experiments under different conditions is largely increased for most of the subunit II and one of the subunit I mutants, giving evidence that binding is impaired, whereas subunit III residues do not seem to contribute significantly. In addition, the bimolecular rate constant for cytochrome c oxidation under pre-steady state conditions was measured using stopped flow spectroscopy. Taken together, the results demonstrate that the initial interaction of cytochrome c and oxidase is mediated through glutamates and aspartates mainly located in subunit II. The crystal structure of oxidase reveals that the participating residues are clustered, creating an extended, negatively charged patch. We propose this clustering to be a decisive factor in the recognition of positively charged patches on the surface of cytochrome c.

Tryptophan 121 of subunit II is the electron entry site to cytochrome-c oxidase in Paracoccus denitrificans. Involvement of a hydrophobic patch in the docking reaction.

To investigate the contribution of hydrophobic residues to the molecular recognition of cytochrome c with cytochrome oxidase, we mutated several hydrophobic amino acids exposed on subunit II of the Paracoccus denitrificans oxidase. KM and kcat values and the bimolecular rate constant were determined under steady- or presteady-state conditions, respectively. We present evidence that Trp-121 which is surrounded by a hydrophobic patch is the electron entry site to oxidase. Mutations in this cluster do not affect the binding of cytochrome c as the KM remains largely unchanged. Rather, the kcat is reduced, proposing that these hydrophobic residues are required for a fine tuning of the redox partners in the initial collisional complex to obtain a configuration optimal for electron transfer.

Electron-transfer properties of Pseudomonas aeruginosa [Lys44, Glu64]azurin.

In the hydrophobic patch of azurin from Pseudomonas aeruginosa, an electric dipole was created by changing Met44 into Lys and Met64 into Glu. The effect of this dipole on the electron-transfer properties of azurin was investigated. From a spectroscopic characterization (NMR, EPR and ultraviolet-visible) it was found that both the copper site and the overall structure of the [Lys44, Glu64]azurin were not disturbed by the two mutations. A small perturbation of the active site at high pH, similar to that observed for [Lys44]azurin, occurs in the double mutant. At neutral pH the electron-self-exchange rate constant of the double mutant shows a decrease of three orders of magnitude compared with the wild-type value. The possible reasons for this decrease are discussed. Electron transfer with the proposed physiological redox partners cytochrome c551 and nitrite reductase have been investigated and the data analyzed in the Marcus framework. From this analysis it is confirmed that the hydrophobic patch of azurin is the interaction site with both partners, and that cytochrome c551 uses its hydrophobic patch and nitrite reductase a negatively charged surface area for the electron transfer.

The terminal oxidase in the marine bacterium Pseudomonas nautica 617.

The molecular properties of a novel membrane quinol oxidase from the marine bacterium Pseudomonas nautica 617 are presented. The protein contains 2b hemes/mole which may be distinguished by EPR spectroscopy but not by optical spectroscopy and electrochemistry. Respiration, though being cyanide insensitive, is not inhibited by carbon monoxide and oxygen reduction is carried out only half-way with production of hydrogen peroxide. The terminal oxidase represents, therefore, a unique example in the large family of terminal oxidases known up to date.

Probing the high-affinity site of beef heart cytochrome c oxidase by cross-linking.

A covalent complex between cytochrome c oxidase and Saccharomyces cerevisiae iso-1-cytochrome c (called caa3) has been prepared at low ionic strength. Subunit III Cys-115 of beef heart cytochrome c oxidase cross-links by disulphide bond formation to thionitrobenzoate-modified yeast cytochrome c, a derivative shown to bind into the high-affinity site for substrate [Fuller, Darley-Usmar and Capaldi (1981) Biochemistry 20, 7046-7053]. Stopped-flow experiments show that (1) covalently bound yeast cytochrome c cannot donate electrons to cytochrome oxidase, whereas oxidation of exogenously added cytochrome c and electron transfer to cytochrome a are only slightly affected; (2) the steady-state reduction levels of cytochrome c and cytochrome a in the covalent complex caa3 are higher than those found in the native aa3 enzyme. However, (3) K(m) and Vmax values obtained from the non-linear Eadie-Hofstee plots are very similar in both caa3 and aa3. The results imply that cytochrome c bound to the high-affinity site is not in a configuration optimal for electron transfer.

Structure and function of a molecular machine: cytochrome c oxidase.

Cytochrome c is responsible for over 90% of the dioxygen consumption in the living cell and contributes to the build-up of a proton electrochemical gradient derived by the vectorial transfer of electrons between cytochrome c and molecular oxygen. The metal ions found in cytochrome oxidases play a crucial role in these processes and have been extensively studied. In this review we present and discuss some of the relevant spectroscopic and kinetic properties of the prosthetic groups of cytochrome c oxidase.

Sulfolobus acidocaldarius terminal oxidase. A kinetic investigation and its structural interpretation.

The thermoacidophilic archaebacterium Sulfolobus acidocaldarius possesses a very unusual terminal oxidase. We report original kinetic experiments on membranes of this microorganism carried out by stopped flow, using time-resolved optical spectroscopy combined with singular value decomposition analysis. The reduced-oxidized kinetic difference spectrum of the Sulfolobus membranes is characterized by three significant peaks in the visible region at 605, 586, and 560 nm. The 605-nm peak and part of the 586-nm peak (cytochrome aa3-type quinol oxidase) are reduced synchronously by both ascorbate plus N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) and dithionite, and they are very rapidly oxidized by molecular oxygen. A second pool of cytochromes seems to contribute to the 586-nm peak which is not reduced by ascorbate plus TMPD and reacts very slowly with dithionite. The b-type cytochromes (560 nm peak) are reduced by both reductants and are essentially "non-autoxidizable" at room temperature. Only one CO binding site with spectral features, kinetic properties, and ligand affinity not very dissimilar from those of mammalian cytochrome oxidase can be detected in the ascorbate-reduced membranes. On the contrary, a second CO binding site having unusual properties for aa3 terminal oxidases can be detected in the dithionite-reduced membranes.

Probing the oxygen binding site of cytochrome c oxidase by cyanide.

Cyanide binding to cytochrome c oxidase has been investigated by sequential mixing and rapid scan stopped flow spectroscopy. Double mixing experiments confirm earlier reports that cyanide binds rapidly to partially reduced enzyme species formed in turnover. The absorbance/time/wavelength matrices, captured during the onset of cyanide inhibition of cytochrome c oxidase by rapid scan stopped flow, were analyzed by singular value decomposition and the spectral contributions of the chromophores separated. Examination of the time courses and amplitudes of the spectral signals provide evidence that entry of a single (1-1.3) electron into the enzyme is sufficient to trigger rapid cyanide binding. This electron resides predominantly on the cytochrome a/CuA pair in the inhibited enzyme. In addition, although cytochrome a3 remains oxidized, it does not appear to be the site for the initial inhibitory binding of cyanide. Our data suggest that CuB2+ is the initial binding site.

Electron transfer and ligand binding in terminal oxidases. Impact of recent structural information.

A consensus structure for the active site of terminal oxidases has been recently proposed by Hosler et al. [(1993) J. Bioenerg. Biomem. 25, 121-135]. We exploit the novel structural information to propose a hypothesis for the large difference in the rate of internal electron transfer found when experiments are started either with the reduced or with the oxidized enzyme. This rationale also allows us to discuss the oxidation state of the prevailing oxygen reacting species with reference to the concentration of the two substrates (oxygen and cytochrome c) and to the structural state of the oxidase.

Lonidamine-mediated respiratory changes in rat heart myocytes: a re-examination of the functional response of mitochondrial cytochrome c oxidase.

Respiratory activity of intact cardiac myocytes isolated from rats treated with lonidamine (LND) has been examined under conditions where cytochrome oxidase turns over at its maximal rate. Compared to myocytes isolated from control rat hearts, those treated with LND displayed a 60% increase in the cytochrome oxidase-dependent rate of respiration; electron microscopy revealed, in agreement with the literature, that the membrane structure of the mitochondrion had become disorganized. The increase in the rate of oxygen consumption was correlated with the (partial) impairment of the membrane ability to maintain the proton electrochemical potential gradient which normally inhibits oxidase activity. Results are discussed with reference to previous reports showing no effect of LND on cytochrome c oxidase activity. The evidence reported better clarifies the contribution of cytochrome oxidase to the demonstrated energetic failure displayed by cells treated with LND.

Release of cytochromes from hypoxic and reoxygenated guinea pig heart.

Isolated, perfused hearts from guinea pigs were subjected to hypoxia for 30 minutes followed by reoxygenation for 30 minutes. Cellular damage was assessed by measuring the release of the cytoplasmic enzyme lactate dehydrogenase and the mitochondrial markers cytochrome c and cytochrome oxidase. The release of the enzymes was correlated with electron microscopy. Hypoxia induced an increase in the release of lactate dehydrogenase and cytochrome c. During reoxygenation, the release of lactate dehydrogenase was exacerbated while that of cytochrome c decreased, suggesting a partial recovery of the mitochondria. Cytochrome oxidase was not detectable in the extracellular space during hypoxia or reoxygenation. It is suggested that cytochrome c is a specific marker for damage to mitochondria caused by hypoxia and its loss may affect respiratory chain function.

Proton pumping by cytochrome oxidase as studied by time-resolved stopped-flow spectrophotometry.

The H+/e- stoichiometry for the proton pump of cytochrome c oxidase reportedly varies between 0 and 1, depending on experimental conditions. In this paper, we report the results obtained by a combination of transient optical spectroscopy with a time resolution of 10 ms and a singular value decomposition analysis to follow the kinetics, separate the observed spectral components, and quantitate the stoichiometry of the pump. By using cytochrome oxidase reconstituted into small unilamellar vesicles, we show that the time courses of ferrocytochrome c oxidation and phenol red acidification or alkalinization fit a simple kinetic scheme. The fitting procedure leads to unbiased and objective determination of the H+/e- ratio under various experimental conditions. The proton-pumping stoichiometry was found to be 1.01 +/- 0.10, independent of the number of turnovers, proton back-leak rate, or type of experiment (oxidant or reductant pulse).

Time-resolved optical spectroscopy on intact myocytes.

Myocytes prepared from perfused rat heart were studied spectroscopically using a photodiode array spectrophotometer adapted to a rapid mixing stopped-flow apparatus. The isolated cells were found to be viable for 3 to 4 hours, i.e. over the total time of the experiments. Sodium ascorbate and tetramethyl-para-phenylenediamine were used as exogenous reductants. Cytoplasmic and mitochondrial membranes were found to be freely permeable to tetramethyl-para-phenylenediamine. The use of singular value decomposition proved to be powerful in resolving the spectral contributions of the chromophoric components within the overall absorption spectrum. Spectral resolution was improved by adding carbon monoxide at a concentration that kept myoglobin fully saturated without affecting the activity of cytochrome c oxidase. The redox state of cytochrome c and cytochrome a was observed during the steady-state consumption of oxygen and during the reduction following the exhaustion of oxygen. The redox state of the two chromophores was found to be approximately equal and close to 25-30% oxidized during steady-state respiration; during the final reduction they changed simultaneously. These experiments suggest that in living cells, as in the purified enzyme, the rate limiting step of the turnover of cytochrome oxidase is the internal transfer of electrons from cytochrome a to cytochrome a3.

The oxygen reactive species of cytochrome-c-oxidase: an alternative view.

In a recent review article Babcok and Wikström (Nature, 1992, 356, 301-309) proposed that the species of cytochrome-c-oxidase which binds molecular oxygen during turnover is the so-called mixed valence enzyme, in which the binuclear center cytochrome a3-CuB is reduced, while the cytochrome a/CuA sites are oxidized. This proposal is based on earlier work (Morgan and Wikström, Biochemistry 1991, 30, 948-958) in which it was found that the steady-state reduction levels of cytochrome c and cytochrome a in respiring rat liver mitochondria (sustained by ascorbate and TMPD) are quite different, the latter being much more oxidized than the former; evaluation of the steady-state reduction levels demanded a large correction due to the optical contribution of oxidized TMPD+ which overlaps with the cytochromes. We report below that application of transient spectroscopy and SVD analysis to respiring rat heart myocytes, under conditions in which the contribution of TMPD+ is very small or absent, allows to show that the steady-state reduction levels of cytochrome c and cytochrome a are comparable at all times accessible to measurement in the rapid-scanning stopped-flow spectrophotometer. Our conclusion, in agreement with previous results, is that mixed valence cytochrome-c-oxidase as defined above is not the prevailing oxygen binding species of cytochrome-c-oxidase, unless electron donation to cytochrome c becomes rate limiting.