RESUMEN
The review considers the problem of hydrogen peroxide decomposition and hydroxyl radical formation in the presence of iron in vivo and in vitro. Analysis of the literature data allows us to conclude that, under physiological conditions, transport of iron, carried out with the help of carrier proteins, minimizes the possibility of appearance of free iron ions in cytoplasm of the cell. Under pathological conditions, when the process of transferring an iron ion from a donor protein to an acceptor protein can be disrupted due to modifications of the carrier proteins, iron ions can enter cytosol. However, at pH values close to neutral, which is typical for cytosol, iron ions are converted into water-insoluble hydroxides. This makes it impossible to decompose hydrogen peroxide according to the mechanism of the classical Fenton reaction. A similar situation is observed in vitro, since buffers with pH close to neutral are used to simulate free radical oxidation. At the same time, iron hydroxides are able to catalyze decomposition of hydrogen peroxide with formation of a hydroxyl radical. Decomposition of hydrogen peroxide with iron hydroxides is called Fenton-like reaction. Studying the features of Fenton-like reaction in biological systems is the subject of future research.
Asunto(s)
Peróxido de Hidrógeno , Radical Hidroxilo , Radical Hidroxilo/química , Hierro/química , Hidróxidos , Oxidación-Reducción , Proteínas PortadorasRESUMEN
αH-Crystallin, a high molecular weight form of α-crystallin, is one of the major proteins in the lens nucleus. This high molecular weight aggregate (HMWA) plays an important role in the pathogenesis of cataracts. We have shown that the chaperone-like activity of HMWA is 40% of that of α-crystallin from the lens cortex. Refolding with urea significantly increased-up to 260%-the chaperone-like activity of α-crystallin and slightly reduced its hydrodynamic diameter (Dh). HMWA refolding resulted in an increase in chaperone-like activity up to 120% and a significant reduction of Dh of protein particles compared with that of α-crystallin. It was shown that the chaperone-like activity of HMWA, α-crystallin, and refolded α-crystallin but not refolded HMWA was strongly correlated with the denaturation enthalpy measured with differential scanning calorimetry (DSC). The DSC data demonstrated a significant increase in the native protein portion of refolded α-crystallin in comparison with authentic α-crystallin; however, the denaturation enthalpy of refolded HMWA was significantly decreased in comparison with authentic HMWA. The authors suggested that the increase in the chaperone-like activity of both α-crystallin and HMWA could be the result of the correction of misfolded proteins during renaturation and the rearrangement of protein supramolecular structures.
Asunto(s)
Catarata , Cristalinas , alfa-Cristalinas , Humanos , Hidrodinámica , Rastreo Diferencial de CalorimetríaRESUMEN
ßL-crystallin aggregation due to oxidative damage in the presence of H2O2 and ferric chloride was studied in-vitro under conditions close to physiological. It was shown that the protein aggregation characterized by the nucleation time and the aggregation rate significantly depended on the composition of the isoosmotic buffers used, and decreased in the series HEPES buffer > Tris buffer > PBS. Ferric chloride at neutral pH was converted into water-insoluble iron hydroxide III (≡FeIIIOH). According to the data of scanning electron microscopy the ≡FeIIIOH particles formed in HEPES buffer, Tris buffer, and PBS practically did not differ in structure. However, the sizes of ≡FeIIIOH floating particles measured by dynamic light scattering differed significantly and were 44 ± 28 nm, 93 ± 66 nm, 433 ± 316 nm (Zaver ± SD) for HEPES buffer, Tris buffer, and PBS, respectively. It was found by the spin trap method that the ability of ≡FeIIIOH to decompose H2O2 with the formation of a â¢OH decreases in the series HEPES buffer, Tris buffer, and PBS. The authors suggest that the ability to generate â¢OH during the decomposition of H2O2 is determined by the total surface area of ≡FeIIIOH particles, which significantly depends on the composition of the buffer in which these particles are formed.
Asunto(s)
Cristalinas , Compuestos de Hierro , HEPES/química , Trometamina , Peróxido de Hidrógeno , Estrés Oxidativo , Tampones (Química) , Oxidación-ReducciónRESUMEN
The absence of cellular organelles in fiber cells and very high cytoplasmic protein concentration (up to 900 mg/ml) minimize light scattering in the lens and ensure its transparency. Low oxygen concentration, powerful defense systems (antioxidants, antioxidant enzymes, chaperone-like protein alpha-crystallin, etc.) maintain lens transparency. On the other hand, the ability of crystallins to accumulate age-associated post-translational modifications, which reduce the resistance of lens proteins to oxidative stress, is an important factor contributing to the cataract formation. Here, we suggest a mechanism of cataractogenesis common for the action of different cataractogenic factors, such as age, radiation, ultraviolet light, diabetes, etc. Exposure to these factors leads to the damage and death of lens epithelium, which allows oxygen to penetrate into the lens through the gaps in the epithelial layer and cause oxidative damage to crystallins, resulting in protein denaturation, aggregation, and formation of multilamellar bodies (the main cause of lens opacification). The review discusses various approaches to the inhibition of lens opacification (cataract development), in particular, a combined use of antioxidants and compounds enhancing the chaperone-like properties of alpha-crystallin. We also discuss the paradox of high efficiency of anti-cataract drugs in laboratory settings with the lack of their clinical effect, which might be due to the late use of the drugs at the stage, when the opacification has already formed. A probable solution to this situation will be development of new diagnostic methods that will allow to predict the emergence of cataract long before the manifestation of its clinical signs and to start early preventive treatment.
Asunto(s)
Catarata , Cristalinas , Cristalino , alfa-Cristalinas , Antioxidantes/metabolismo , Catarata/etiología , Cristalinas/análisis , Cristalinas/metabolismo , Humanos , Cristalino/metabolismo , Chaperonas Moleculares/metabolismo , Oxígeno/metabolismo , alfa-Cristalinas/análisis , alfa-Cristalinas/química , alfa-Cristalinas/metabolismoRESUMEN
The chronically exposure of eye lenses to ultra violet and visible light of the solar radiation is an important risk factor for development of the senile cataract diseases. Various photosensitizer molecules including riboflavin (RF) play a significant role in photo-oxidative damages of lens proteins underlying development of opacity in the lenticular tissues. In the current study, RF-mediated photo-oxidation of human αA-crystallin (αA-Cry) was assessed using SDS-PAGE analysis, dynamic light scattering and other spectroscopic assessments. The RF-photosensitized reactions led to non-disulfide covalent cross-linking, oligomerization and significant structural changes in αA-Cry. The photo-damaging of αA-Cry under solar radiation was also accompanied by the reduction in both Trp and Tyr fluorescence intensities which followed by the formation of new photosensitizer chromophores. The solvent exposed hydrophobic patches, secondary structures and chaperone-like activity of αA-Cry were significantly altered after exposure to the solar radiation in the presence of RF. Although glutathione and ascorbate were capable to partially protect the photo-induced structural damages of human αA-Cry, they also disrupted its chaperone function when co-exposed with this protein to the solar radiation. Also, the most promising data were obtained with cysteine which its availability in the lenticular tissues is a rate limiting factor for the biosynthesis of glutathione. Overall our results suggest that glutathione and ascorbate, as the major anti-oxidant compounds within lenticular tissues, demonstrate controversial effect on structure and chaperone-like activity of human αA-Cry. Elucidation of this effect may demand further experiments.
Asunto(s)
Antioxidantes/química , Cristalino/química , Multimerización de Proteína/efectos de la radiación , Luz Solar , Cadena A de alfa-Cristalina/química , Antioxidantes/metabolismo , Cisteína/química , Cisteína/metabolismo , Glutatión/química , Glutatión/metabolismo , Humanos , Cristalino/metabolismo , Dominios Proteicos , Cadena A de alfa-Cristalina/metabolismoRESUMEN
α-Crystallin is the major eye lens protein that has been shown to support lens transparency by preventing the aggregation of lens proteins. The 3D structure of α-crystallin is largely unknown. Electron microscopy, single-particle 3D reconstruction, size exclusion chromatography, dynamic light scattering, and analytical ultracentrifugation were used to study the structure of the native α-crystallin. Native α-crystallin has a wide distribution in size. The shape of mass distribution is temperature-dependent, but the oligomers with a sedimentation coefficient of ~22â¯S (750-830â¯kDa) strongly prevailed at all temperatures used. A 3D model of native α-crystallin with resolution of ~2â¯nm was created. The model is asymmetrical, has an elongated bean-like shape 13â¯×â¯19â¯nm with a dense core and filamentous "kernel". It does not contain a central cavity. The majority of α-crystallin particles regardless of experimental conditions are 13â¯×â¯19â¯nm, which corresponds to 22S sedimentation coefficient, hydrodynamic diameter 20â¯nm and mass of 750-830â¯kD. These particles are in dynamic equilibrium with particles of smaller and larger sizes.
Asunto(s)
Cristalinas/química , Modelos Moleculares , Conformación Proteica , alfa-Cristalinas/química , Animales , Bovinos , Cromatografía en Gel , Dispersión Dinámica de Luz , Temperatura , Ultracentrifugación , alfa-Cristalinas/ultraestructuraRESUMEN
Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C) in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, Uhr) is characterized by a high rate of aggregation. Aggregation of Uhr leads to the formation of primary aggregates with the hydrodynamic radius (Rh,1) of 10.3 nm. The second form (low reactive unfolded form, Ulr) participates in the aggregation process by its attachment to the primary aggregates produced by the Uhr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (Ast). At complete exhaustion of Ulr, secondary aggregates with the hydrodynamic radius (Rh,2) of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates.
Asunto(s)
Desnaturalización Proteica , Albúmina Sérica Bovina/química , Área Bajo la Curva , Rastreo Diferencial de Calorimetría , Cromatografía en Gel , Calor , Cinética , Microscopía Electrónica de Transmisión , Análisis Espectral/métodos , UltracentrifugaciónRESUMEN
Ultraviolet radiation is a risk factor for cataractogenesis. It is believed that enhanced rates of lens opacification and cataract formation are the results of gradual loss of chaperone-like efficiency of α-crystallin upon exposure to UV light. To characterize chaperone-like activity of α-crystallin damaged by UV irradiation, a test system based on dithiothreitol-induced aggregation of holo-α-lactalbumin from bovine milk was used. The adsorption capacity of α-crystallin (AC0) with respect to the target protein (α-lactalbumin) was used as a measure of anti-aggregation activity of α-crystallin. The data on SDS-PAGE testify that UV irradiation of α-crystallin results in covalent cross-linking of subunits in α-crystallin oligomers. The dependence of AC0 value on the irradiation dose was compared with the UV-induced diminution of the portion of native α-crystallin estimated from the data on differential scanning calorimetry. On the basis of such comparison a conclusion has been made that the loss in chaperone-like activity is mainly due to UV-induced denaturation of α-crystallin subunits. Cross-linking of remaining native subunits leads to an additional decrease in anti-aggregation activity.
Asunto(s)
Agregación Patológica de Proteínas/tratamiento farmacológico , Rayos Ultravioleta , alfa-Cristalinas/química , alfa-Cristalinas/farmacología , Animales , Bovinos , Cromatografía en Gel , Ditiotreitol/farmacología , Electroforesis en Gel de Poliacrilamida , Cinética , Lactalbúmina/química , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
The effect of protein and chemical chaperones and crowders on thermal stability and aggregation of apoform of rabbit muscle glycogen phosphorylase b (apoPhb) has been studied at 37°C. Proline suppressed heat-induced loss in ability of apoPhb to reconstitution at 37°C, whereas α-crystallin did not reveal a protective action. To compare the antiaggregation activity of intact and crosslinked α-crystallins, an adsorption capacity (AC) of a protein chaperone with respect to a target protein was estimated. This parameter is a measure of the antiaggregation activity. Crosslinking of α-crystallin results in 11-fold decrease in the initial AC. The nonlinear character of the relative initial rate of apoPhb aggregation versus the [intact α-crystallin]/[apoPhb] ratio plot is indicative of the decrease in the AC of α-crystallin with increasing the [α-crystallin]/[apoPhb] ratio and can be interpreted as an evidence for dynamic chaperone structure and polydispersity of α-crystallin-target protein complexes. As for chemical chaperones, a semisaturation concentration of the latter was used as a characteristic of the antiaggregation activity. A decrease in the semisaturation concentration for proline was observed in the presence of the crowders (polyethylene glycol and Ficoll-70).
Asunto(s)
Apoproteínas/metabolismo , Calor , Sustancias Macromoleculares/farmacología , Chaperonas Moleculares/farmacología , Fosforilasa b/metabolismo , Agregado de Proteínas/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacos , Animales , Área Bajo la Curva , Bovinos , Reactivos de Enlaces Cruzados/farmacología , Cinética , Polietilenglicoles/farmacología , Prolina/farmacología , Conejos , alfa-Cristalinas/farmacologíaRESUMEN
The methodology for quantification of the anti-aggregation activity of protein and chemical chaperones has been elaborated. The applicability of this methodology was demonstrated using a test-system based on dithiothreitol-induced aggregation of bovine serum albumin at 45°C as an example. Methods for calculating the initial rate of bovine serum albumin aggregation (v agg) have been discussed. The comparison of the dependences of v agg on concentrations of intact and cross-linked α-crystallin allowed us to make a conclusion that a non-linear character of the dependence of v agg on concentration of intact α-crystallin was due to the dynamic mobility of the quaternary structure of α-crystallin and polydispersity of the α-crystallin-target protein complexes. To characterize the anti-aggregation activity of the chemical chaperones (arginine, arginine ethyl ester, arginine amide and proline), the semi-saturation concentration [L]0.5 was used. Among the chemical chaperones studied, arginine ethyl ester and arginine amide reveal the highest anti-aggregation activity ([L]0.5â=â53 and 58 mM, respectively).
Asunto(s)
Ditiotreitol/farmacología , Chaperonas Moleculares/metabolismo , Albúmina Sérica Bovina/química , Animales , Arginina/farmacología , Bovinos , Cromatografía en Gel , Reactivos de Enlaces Cruzados/farmacología , Electroforesis en Gel de Poliacrilamida , Fraccionamiento de Campo-Flujo , Cinética , Luz , Chaperonas Moleculares/química , Tamaño de la Partícula , Prolina/farmacología , Unión Proteica/efectos de los fármacos , Estructura Cuaternaria de Proteína , Desplegamiento Proteico/efectos de los fármacos , Refractometría , Dispersión de Radiación , Albúmina Sérica Bovina/metabolismo , Ultracentrifugación , Viscosidad/efectos de los fármacos , alfa-Cristalinas/química , alfa-Cristalinas/metabolismoRESUMEN
It was shown that the rate of reconstruction of muscle glycogen phosphorylase b (Phb) from apoenzyme and pyridoxal 5'-phosphate decreased under crowding conditions. The effect of crowding was counteracted by chaperones (α-crystallin and proline). Sedimentation analysis shows that crowding stimulates the formation of high-molecular-weight associates at 25 °C, whereas chaperones stabilize small oligomers. The study of the kinetics of apoPhb aggregation at 37 °C showed that the anti-aggregation activity of chaperones decreased under crowding conditions. When studying the sedimentation behavior of the mixture of apoPhb and α-crystallin, the complexes between unfolded apoPhb and dissociated forms of α-crystallin were observed. It is assumed that these complexes are responsible for realization of the chaperone-like activity of α-crystallin under crowding conditions.
Asunto(s)
Chaperonas Moleculares/metabolismo , Fosforilasa b/química , Fosforilasa b/metabolismo , Animales , Cinética , Fosforilasa b/aislamiento & purificación , Prolina/farmacología , Fosfato de Piridoxal , Conejos , Temperatura , alfa-Cristalinas/farmacologíaRESUMEN
An aggregation test system based on the aggregation of UV-irradiated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit skeletal muscle has been proposed. On the basis of the measurements of the enzyme activity and differential scanning calorimetry data a conclusion has been made that UV radiation results in formation of damaged protein molecules with lower thermostability. It was shown that the order of aggregation rate for UV-irradiated GAPDH with respect to the protein was close to 2. This means that such a test system allows detecting the effect of various agents exclusively on the stage of aggregation of unfolded protein molecules. The influence of α-crystallin and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) on aggregation of UV-irradiated GAPDH was studied. Despite the fact that HP-ß-CD accelerates thermal aggregation of non-irradiated GAPDH, in the case of aggregation of UV-irradiated GAPDH HP-ß-CD reveals a purely protective effect.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Músculo Esquelético/enzimología , Rayos Ultravioleta , 2-Hidroxipropil-beta-Ciclodextrina , Animales , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Cinética , Chaperonas Moleculares/química , Desnaturalización Proteica , Estabilidad Proteica , Conejos , Temperatura , alfa-Cristalinas/química , beta-Ciclodextrinas/químicaRESUMEN
The effect of histidine-containing dipeptides-carnosine and N-acetylcarnosine-on preventing and treating of cataracts of various etiologic origins has been demonstrated in many studies in vivo, while the precise molecular mechanism of their action is actually obscure. Cataract has been recently attributed to conformational diseases due to the association of lens structure protein aggregation with cataract pathogenesis. In our study, effect of histidine-containing dipeptides-carnosine, N-acetylcarnosine, and anserine-on the UV induced ßL-crystallin aggregation was studied in vitro. It was first demonstrated that N-acetylcarnosine and anserine (10-40 mM) considerably suppressed UV induced aggregation of ßL-crystallin, while carnosine exerted no effect. Positive correlation between anti-aggregating activity of the compounds used and their hydrophobicity was obtained. It was revealed that N-acetylcarnosine and anserine inhibited the initial stages of the protein photochemical damage. A decrease in the size of protein aggregates was detected in the presence of N-acetylcarnosine and anserine. UV irradiation of ßL-crystallin resulted in a significant increase in the number of protein carbonyl groups, and the dipeptides studied did not affect this process. We suppose that N-acetylcarnosine and anserine inhibit ßL-crystallin aggregation via formation of a protein-dipeptide complex that prevents macromolecular conformational changes and ensuing protein aggregation.
Asunto(s)
Dipéptidos/metabolismo , Cristalino/metabolismo , Chaperonas Moleculares/metabolismo , beta-Cristalinas/metabolismo , Animales , Anserina/metabolismo , Carnosina/análogos & derivados , Carnosina/metabolismo , Catarata/metabolismo , Bovinos , Dipéptidos/química , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Chaperonas Moleculares/química , Carbonilación Proteica/efectos de la radiación , Conformación Proteica , Rayos Ultravioleta , beta-Cristalinas/química , beta-Cristalinas/efectos de la radiaciónRESUMEN
The effect of crowding on the chaperone-like activity of α-crystallin has been studied using aggregation of UV-irradiated glycogen phosphorylase b (Phb) from rabbit skeletal muscle as an aggregation test system. The merit of this test system is the possibility of testing agents that directly affect the stage of aggregation of the protein molecules. It was shown that the solution of Phb denatured by UV contained aggregates with a hydrodynamic radius of 10.4 nm. These aggregates are relatively stable at 20 °C; however, they reveal a tendency to stick further in the presence of crowding agents. The study of the effect of α-crystallin on the aggregation of UV-irradiated Phb in the presence of the crowding agents by dynamic light scattering at 37 °C showed that under crowding conditions the antiaggregation ability of α-crystallin was weakened. On the basis of the analytical ultracentrifugation, size-exclusion chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the scheme of interaction of UV-irradiated Phb and α-crystallin has been proposed. It is assumed that chaperone-target protein complexes of two types are formed, namely, the complexes of dissociated forms of α-crystallin with a protein substrate and high-mass α-crystallin-denatured protein complexes. The complexes of the first type reveal a weak propensity to aggregate even under crowding conditions. The complexes of the second type are characterized by the lower rate of aggregation in comparison with that of original UV-irradiated Phb. However, crowding stimulates the rate of aggregation of these complexes, resulting in the above-mentioned decrease in the chaperone-like activity of α-crystallin.
Asunto(s)
Fosforilasa b/metabolismo , alfa-Cristalinas/metabolismo , Animales , Bovinos , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Masculino , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Fosforilasa b/efectos de la radiación , Desnaturalización Proteica , Conejos , Dispersión de Radiación , Ultracentrifugación , Rayos Ultravioleta , alfa-Cristalinas/químicaRESUMEN
Thermal denaturation and aggregation of UV-irradiated ß(L)-crystallin from eye lenses of steers have been studied. The data on size-exclusion chromatography and SDS-PAGE indicated that UV irradiation of ß(L)-crystallin at 10 °Ð¡ resulted in fragmentation of the protein molecule and formation of cross-linked aggregates. Fluorescence data showed that tryptophan fluorescence in the irradiated protein decreased exponentially with the UV dose. Decrease in tryptophan fluorescence is a result of photochemical destruction, but not of conformational changes of protein, because there is no red shift in the fluorescence maximum. The differential scanning calorimetry (DSC) profiles of the samples of UV-irradiated and wild type ß(L)-crystallin were registered. The area under curves, which is proportional to the amount of the native protein, decreased exponentially with increasing the irradiation dose. The shape of the DSC profiles for the samples of UV-irradiated ß(L)-crystallin was identical to that for wild type ß(L)-crystallin. The DSC data allowed estimating the portion of UV-denatured ß(L)-crystallin, which is not registered by DSC, and the portion of the combined fraction consisting of native and UV-damaged molecules retaining the native structure. A conclusion has been made that UV-induced denaturation of ß(L)-crystallin follows the one-hit model. The study of the kinetics of thermal aggregation of UV-irradiated ß(L)-crystallin at 37 °Ð¡ using dynamic light scattering showed that the initial stage of aggregation was that of formation of the start aggregates with the hydrodynamic radius of 20 nm. Further sticking of the start aggregates proceeded in the regime of reaction-limited cluster-cluster aggregation. Splitting of the aggregate population into two components occurred above a definite point in time.
Asunto(s)
Rayos Ultravioleta , beta-Cristalinas/química , beta-Cristalinas/efectos de la radiación , Animales , Rastreo Diferencial de Calorimetría , Bovinos , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Cristalino/química , Luz , Desnaturalización Proteica/efectos de la radiación , Dispersión de Radiación , Espectrometría de FluorescenciaRESUMEN
The kinetics of dithiothreitol (DTT)-induced aggregation of alpha-lactalbumin from bovine milk has been studied using dynamic light-scattering technique. Analysis of the distribution of the particles formed in the solution of alpha-lactalbumin after the addition of DTT by size showed that the initial stage of the aggregation process was the stage of formation of the start aggregates with the hydrodynamic radius (R(h)) of 80-100nm. Further growth of the protein aggregates proceeds as a result of sticking of the start aggregates. Suppression of alpha-lactalbumin aggregation by alpha-crystallin is mainly due to the increase in the duration of the lag period on the kinetic curves of aggregation. It is assumed that the initially formed complexes of unfolded alpha-lactalbumin with alpha-crystallin were transformed to the primary clusters prone to aggregation as a result of the redistribution of the denatured protein molecules on the surface of the alpha-crystallin particles.
Asunto(s)
Ditiotreitol/farmacología , Lactalbúmina/metabolismo , alfa-Cristalinas/farmacología , Animales , Bovinos , Relación Dosis-Respuesta a Droga , Cinética , Lactalbúmina/química , Luz , Unión Proteica/efectos de los fármacos , Desnaturalización Proteica , Dispersión de Radiación , Espectrometría de Fluorescencia , Propiedades de Superficie , alfa-Cristalinas/metabolismoRESUMEN
Effects of alpha-crystallin and GroEL on the kinetics of thermal aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been studied using dynamic light scattering and analytical ultracentrifugation. The analysis of the initial parts of the dependences of the hydrodynamic radius of protein aggregates on time shows that in the presence of alpha-crystallin or GroEL the kinetic regime of GAPDH aggregation is changed from the regime of diffusion-limited cluster-cluster aggregation to the regime of reaction-limited cluster-cluster aggregation, wherein the sticking probability for the colliding particles becomes lower the unity. In contrast to alpha-crystallin, GroEL does not interfere with formation of the start aggregates which include denatured GAPDH molecules. On the basis of the analytical ultracentrifugation data the conclusion has been made that the products of dissociation of GAPDH and alpha-crystallin or GroEL play an important role in the interactions of GAPDH and chaperones at elevated temperatures.
Asunto(s)
Proteínas de Escherichia coli/química , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Proteínas de Choque Térmico/química , Músculo Esquelético/enzimología , alfa-Cristalinas/química , Animales , Bovinos , Cinética , Masculino , Músculo Esquelético/química , Conformación Proteica , Conejos , TemperaturaRESUMEN
Effect of alpha-crystallin on thermal inactivation, denaturation and aggregation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been in the focus of this study. Acceleration of heat-induced inactivation of mAAT was demonstrated in the presence of alpha-crystallin. According to the data of differential scanning calorimetry, alpha-crystallin induces destabilization of the mAAT molecule. The size of protein aggregates formed at heating of mAAT at a constant rate (1 degree C/min) has been defined by dynamic light scattering. The obtained data show that aggregation of mAAT in the presence of alpha-crystallin proceeds in the regime of reaction-limited cluster-cluster aggregation.
Asunto(s)
Aspartato Aminotransferasas/química , Mitocondrias/enzimología , Temperatura , alfa-Cristalinas/farmacología , Aspartato Aminotransferasas/metabolismo , Difusión , Activación Enzimática/efectos de los fármacos , Estabilidad de Enzimas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacosRESUMEN
Thermal aggregation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been studied at various temperatures and various protein concentrations by dynamic light scattering. The character of the dependence of protein aggregate size on time indicates that aggregation of mAAT proceeds in the regime of diffusion-limited cluster-cluster aggregation. Suppression of mAAT aggregation by alpha-crystallin is due to transition of the aggregation process into the regime of reaction-limited cluster-cluster aggregation. Realization of this regime of aggregation means that the sticking probability for the colliding particles is less than unity.
Asunto(s)
Aspartato Aminotransferasa Mitocondrial/química , Aspartato Aminotransferasa Mitocondrial/metabolismo , Temperatura , alfa-Cristalinas/farmacología , Animales , Bovinos , Cinética , Luz , Unión Proteica/efectos de los fármacos , Dispersión de RadiaciónRESUMEN
Kinetics of thermal aggregation of yeast alcohol dehydrogenase I (yADH) have been studied using dynamic light scattering at a fixed temperature (56 degrees C) and under the conditions where the temperature was elevated at a constant rate (1 K/min). The initial parts of the dependences of the hydrodynamic radius on time (or temperature) follow the exponential law. At rather high values of time splitting of the population of aggregates into two components occurs. It is assumed that such peculiarities of the kinetics of thermal aggregation of yADH are due to the presence of a sequence -YSGVCHTDLHAWHGDWPLPVK- in the polypeptide chain possessing chaperone-like activity. Thermodynamic parameters for thermal denaturation of yADH have been calculated from the differential scanning calorimetry data.