RESUMEN
AIM: To attempt to identify the primary targets of carbamylation in bovine lenses incubated under physiological condition. METHODS: Fresh intact bovine lenses were incubated with [(14)C]-labelled potassium cyanate for seven days. The water-soluble proteins (WSP) of both cortex and nucleus lens were isolated by size-exclusion chromatography on a Sephacryl S-300HR column. The higher radioactive fractions were pooled and freeze-dried, and separated further by loading on an Affinity Blue column to separate some enzymes. In addition, WSP from cortex was separated directly by affinity chromatography. The most reactive fractions with higher radioactivity from [(14)C]-cyanate were further analyzed by SDS-gels and mass spectrometry. RESULTS: The majority of protein incorporating [(14)C]-labelled potassium cyanate was in the water-soluble fractions, and much more in the cortex than in the nucleus. Chromatography results demonstrated that the major incorporated [(14)C]-carbamylated crystallins were fractions corresponding to α-crystallin, ß-crystallin and ξ-crystallin in the cortex, but ß-crystallin and γ-crystallin in the nucleus. The SDS gels showed that bound fractions of cortex crystallins after Affi-Gel Blue separation were abundant with 20 and 35kDa proteins. However, the bound fractions of nucleus crystallins mainly showed 20kDa proteins. Mass spectrometry analysis of these higher radioactivity fractions and a database search revealed that the proteins were originated from bovine α-crystallin A and B chains and ξ-crystallin in the cortex; ßA1 and αB-crystallins with a little γB-crystallin in the nucleus respectively. Further analysis suggested the location of this carbamylation of αB-crystallin in the nucleus to be at Lys 92 and 103. CONCLUSION: α-and ξ-crystallin from cortex can be preferentially targeted by carbamylation during whole lens incubations. Carbamylation of these crystallins at the earlier stage may result in further unfolding and misfolding of lens proteins, leading to aggregation of crystallins and eventually to cataract formation.
RESUMEN
PURPOSE: To investigate the effect of a combination of carnosine and aspirin eye drops on the progression of diabetic cataract formation induced by streptozotocin (STZ). METHODS: Rats were made diabetic with STZ. Animals in the treated groups received carnosine, aspirin, or a combination of carnosine and aspirin as drops to the eyes. Cataract progression was monitored by slit lamp microscope and classified into four stages. At the end of 8 weeks, the animals were killed and biochemical changes were determined. Blood and urine glucose levels, body weights, food, and intake were also determined. RESULTS: About 84.4% of the rats responded to the STZ injection. There were statistically significant differences in the stage of cataract of lenses between the untreated and the treated diabetic animals and between the combination and the aspirin group at the 7th and 8th week. There was a significant decrease in the water-soluble protein in the diabetic groups compared with the control group. The three treatments improved the water-soluble protein levels, and the combination treatment had the greatest effect. The levels of thiol were remarkably decreased in the lenses of diabetic rats, except the combination group. The specific activity of glutathione peroxidase (GPx) was increased and the activities of glutathione reductase (GR) and catalase (CAT) were decreased in all the diabetic groups. CONCLUSIONS: The results indicated that carnosine, aspirin, and a combination eye drops are effective against the onset and development of diabetic cataract in rats. Most important, the effect of combination eye drops is better than aspirin only.
Asunto(s)
Aspirina/uso terapéutico , Carnosina/uso terapéutico , Catarata/complicaciones , Catarata/tratamiento farmacológico , Complicaciones de la Diabetes/tratamiento farmacológico , Soluciones Oftálmicas/uso terapéutico , Animales , Aspirina/farmacología , Glucemia/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Carnosina/farmacología , Catarata/patología , Complicaciones de la Diabetes/inducido químicamente , Complicaciones de la Diabetes/patología , Conducta de Ingestión de Líquido/efectos de los fármacos , Quimioterapia Combinada , Conducta Alimentaria/efectos de los fármacos , Glucosuria/complicaciones , Cristalino/efectos de los fármacos , Cristalino/enzimología , Cristalino/patología , Masculino , Soluciones Oftálmicas/farmacología , Ratas , Ratas Sprague-Dawley , Solubilidad/efectos de los fármacos , Estreptozocina , Compuestos de Sulfhidrilo/metabolismo , Factores de TiempoRESUMEN
PURPOSE: Carbamylation, an important post-translational modification of proteins, inevitably causes conformational changes of lens proteins. It may increase aggregation between crystallin molecules and disrupt the close packing required for transparency thus leading to cataract. The aim of this study was to isolate the primary targets of carbamylation in the lens and identify them by mass spectrometry. MATERIALS AND METHODS: Fresh intact bovine lenses were incubated with 100 mM potassium cyanate for 7 days. The proteins in the water-soluble fractions from the normal control and the cyanate-modified lens proteins were separated by two-dimensional (2-D) gel electrophoresis with identification after silver staining. Protein spots that differed between the normal and carbamylated groups were selected for further analysis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). RESULTS: The 2-D gel results showed that the major lens proteins were in the section of pI 5-8, with relative molecular masses of 20-35 kDa, and changes in the carbamylated fraction like strings of beads indicating modification. The mass spectrometry analysis and a database search identified carbamylated proteins originating from alphaA-crystallin, betaB2- and gammaS-(betaS)-crystallins. CONCLUSIONS: These crystallins may be vulnerable proteins targeted by carbamylation. The accumulated aggregation and loss of chaperone activity may contribute to cataract formation.
Asunto(s)
Cristalino/metabolismo , Espectrometría de Masas , Procesamiento Proteico-Postraduccional , Cadena A de alfa-Cristalina/metabolismo , Cadena B de beta-Cristalina/metabolismo , Animales , Bovinos , Electroforesis en Gel Bidimensional , Técnicas In Vitro , Peso Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Cadena A de alfa-Cristalina/química , Cadena B de beta-Cristalina/química , gamma-Cristalinas/química , gamma-Cristalinas/metabolismoRESUMEN
PURPOSE: Alpha-crystallin, a ubiquitous molecular chaperone, is found in high concentrations in the lens. Its structure and precise mechanism of action, however, are unknown. The purpose of these experiments was to further the understanding of the chaperone function of alpha-crystallin. METHODS: X-ray- and neutron-solution-scattering studies were used to measure the radius of gyration of bovine lens alpha-crystallin when complexed with its target protein beta-crystallin in both normal and heavy-water-based solutions. Spectrophotometry was used as a chaperone assay. RESULTS: The radius of gyration of alpha-crystallin on its own and when mixed with beta-crystallin was 69 +/- 1 A at 35 degrees C and increased with the temperature. In contrast to H2O-buffered solutions, the radius of gyration did not increase significantly in D2O-buffered solutions up to 55 degrees C, and at 70 degrees C was, on average, some 15 to 20 A smaller. CONCLUSIONS: Bovine lens alpha-crystallin in solution can be modeled as a fenestrated spherical shell of diameter 169 A. At physiological temperatures, a weak interaction between alpha- and beta-crystallin occurs, and beta-crystallin is located in the fenestrations. Deuterium substitution indicates that the superaggregation process is controlled by hydrogen bonding. However, the chaperone process and superaggregation appear not to be linked.
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Chaperonas Moleculares/química , Difracción de Neutrones/métodos , Difracción de Rayos X/métodos , alfa-Cristalinas/química , Animales , Bovinos , Unión Proteica , Dispersión del Ángulo Pequeño , beta-Cristalinas/químicaRESUMEN
Glutathione reductase (GR) plays a key role in maintaining thiol groups in the lens, and its activity decreases with aging and cataract formation. Mammalian thioredoxin (Trx) and thioredoxin reductase (TrxR), or the Trx/TrxR system, participates in the repair of oxidatively damaged lens proteins and enzymes. Alpha-crystallin, a molecular chaperone, prevents the aggregation of partially denatured proteins under various stress conditions. Thioltransferase (TTase, or glutaredoxin) can maintain the homeostasis of lens protein thiols thus protecting against oxidative stress. We investigated whether the Trx/TrxR system can revive GR activity in both the cortex and nucleus of human cataract and clear aged lenses and whether alpha-crystallin and TTase can help this effect. The GR activity in the cortex and nucleus of the cataractous lenses was significantly lower than that of the aged clear lenses. The highest activity in the cortex was observed in the clear aged lenses. The combination of Trx and TrxR revived the activity of GR from both the cortex and nucleus of aged clear lenses. However, in cataract lenses (grade II and grade IV), there was a statistically significant recovery of GR activity in the cortex, but not in the nucleus. No recovery was observed when Trx or TrxR were used separately. Alpha-crystallin successfully revived GR activity in the cortex of cataract grade II lenses, but not in the nucleus. The combination of alpha-crystallin and Trx/TrxR gave a further increase of activity. TTase alone revived some of the GR activity but together with the Trx/TrxR system gave no statistically significant enhancement of GR activity. These results indicate that both disulfide bond formation and protein unfolding are responsible for GR inactivation.
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Catarata/enzimología , Glutatión Reductasa/metabolismo , Cristalino/enzimología , Proteína Disulfuro Reductasa (Glutatión)/farmacología , Reductasa de Tiorredoxina-Disulfuro/farmacología , Tiorredoxinas/farmacología , alfa-Cristalinas/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/metabolismo , Sinergismo Farmacológico , Glutarredoxinas , Humanos , Persona de Mediana EdadRESUMEN
PURPOSE: To investigate whether mammalian thioredoxin (Trx) and thioredoxin reductase (TrxR), with or without alpha-crystallin can revive inactivated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in both the cortex and nucleus of human aged clear and cataract lenses. METHODS: The lens cortex (including capsule-epithelium) and the nucleus were separated from human aged clear and cataract lenses (grade II and grade IV) with similar average age. The activity of GAPDH in the water-soluble fraction after incubation with or without Trx or/and TrxR for 60 min at 30 degrees C was measured spectrophotometrically. In addition, the effect of a combination of Trx/TrxR and bovine lens alpha-crystallin was investigated. RESULTS: GAPDH activity was lower in the nucleus of clear lenses than in the cortex, and considerably diminished in the cataractous lenses, particularly in the nucleus of cataract lenses grade IV. Trx and TrxR were able to revive the activity of GAPDH markedly in both the cortex and nucleus of the clear and cataract lenses. The percentage increase of activity in the cortex of the clear lenses was less than that of the nucleus in the presence of Trx and TrxR, whereas it was opposite in the cataract lenses. The revival of activity in both the cortex and nucleus from the cataract lenses grade II was higher than that of the grade IV. Moreover, Trx alone, but not TrxR, efficiently enhanced GAPDH activity. The combination of Trx and TrxR had greater effect than that of either alone. In addition, alpha(L)-crystallin enhanced the activity in the cortex of cataract grade II with Trx and TrxR present. However, it failed to provide a statistically significant increase of activity in the nucleus. CONCLUSIONS: This is the first evidence to show that mammalian Trx and TrxR are able to revive inactivated GAPDH in human aged clear and cataract lenses, and alpha-crystallin helped this effect. The inactivation of GAPDH during aging and cataract development must be caused in part by disulphide formation and in part by unfolding, and can be recovered by reducing agents and a molecular chaperone.
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Envejecimiento/metabolismo , Catarata/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Cristalino/enzimología , Reductasa de Tiorredoxina-Disulfuro/farmacología , Tiorredoxinas/farmacología , alfa-Cristalinas/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Bovinos , Activación Enzimática/efectos de los fármacos , Humanos , Corteza del Cristalino/enzimología , Núcleo del Cristalino/enzimología , Cristalino/química , Persona de Mediana Edad , Extractos de Tejidos/metabolismoRESUMEN
Glycation and other non-enzymic post-translational modifications of proteins have been implicated in the complications of diabetes and other conditions. In recent years there has been extensive progress in the search for ways to prevent the modifications and prevent the consequences of the modifications. These areas are covered in this review together with newer ideas on possibilities of reversing the chemical modifications.
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Glicosilación , Procesamiento Proteico-Postraduccional , Aspirina/química , Carbohidratos/química , Complicaciones de la Diabetes/prevención & control , Diabetes Mellitus/etiología , Productos Finales de Glicación Avanzada/análisis , Humanos , Ibuprofeno/química , Proteínas/química , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/efectos de los fármacosRESUMEN
We have studied the effect of a crowded (macromolecular) solution on reaction rates of the decarboxylating enzymes urease, pyruvate decarboxylase and glutamate decarboxylase. A variety of crowding agents were used including haemoglobin, lysozyme, various dextrans and polyethylene glycol. Enzyme reaction rates of all three enzymes show two different types of effect that separate the globular proteins from the polysaccharides/polymers. Increasing concentration of globular proteins caused a dramatic rise in enzyme activity up to 30% crowding concentration then the activity decreased, whereas the polymers caused a concentration dependent decrease in activity. The viscosities of the globular proteins were low compared to the polymers. The increased activity with proteins may be due to the decreased amount of free water, which leads to higher effective concentration of substrates, or to an increased oligomeric state by self-association. The lower activities of all enzymes with all agents at high concentrations may be explained by a decrease in rates of diffusion. An increase in protein crowding (decrease in cell water content) may contribute to pathological conditions including cataract and Alzheimer's disease.
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Enzimas/química , Polímeros/química , Animales , Bovinos , Pollos , Dextranos/química , Escherichia coli/metabolismo , Fabaceae/enzimología , Glutamato Descarboxilasa/química , Hemoglobinas/química , Muramidasa/química , Polietilenglicoles/química , Proteómica/métodos , Piruvato Descarboxilasa/química , Ureasa/químicaRESUMEN
PURPOSE: Alpha-crystallin, a major structural protein in the lens, prevents heat- and oxidative stress-induced aggregation of proteins and inactivation of enzymes by acting as a molecular chaperone. Modification of alpha-crystallin by some posttranslational modifications results in conformational changes and decreases in chaperone activity, which may contribute to cataractogenesis in vivo. Carnosine (beta-alanyl-L-histidine), an endogenous histidine dipeptide, prevents protein modifications including glycation and oxidation. The purpose of this study was to further explore whether carnosine can protect alpha-crystallin against glycation by a sugar and a sugar phosphate, and in particular to find whether it can protect against its decreased chaperone activity. Additionally, we investigated whether carnosine could directly react with a sugar and a sugar phosphate. METHODS: Bovine lens alphaL-crystallin was separated by size-exclusion chromatography on a Sephacryl S-300 HR column. alphaL-crystallin was incubated with different concentrations of fructose 6-phosphate (F6P) and ribose with or without carnosine for different times. The chaperone activity of alphaL-crystallin was monitored using the prevention of thermal aggregation of betaL-crystallin. The modified alphaL-crystallin was examined by SDS-PAGE and fluorescence measurements. The absorbance spectra of solutions of carnosine and sugars were investigated. RESULTS: Carnosine inhibited the crosslinking of alphaL-crystallin induced by F6P and ribose in a dose- and time-dependent manner. It protected alphaL-crystallin against its decreased chaperone activity induced by 100 mM F6P during four days incubation, but not against ribose-induced change. Control alphaL-crystallin gave 96% protection against aggregation of betaL-crystallin after four days incubation, but only 85% protection was achieved in the presence of F6P, rising to 96% (p=0.0004) in the presence of carnosine. After more extensive modification by sugar and a sugar phosphate, there was no significant protective effect of carnosine on alphaL-crystallin cross-linking or chaperone activity. The tryptophan fluorescence of modified alphaL-crystallin was remarkably decreased in the presence of F6P and ribose. However, the decrease was less when 50 mM carnosine was present during eight days incubation with F6P. Carnosine did not maintain the fluorescence when ribose was used. The nontryptophan fluorescence was increased with a shift to longer wavelengths in a time-dependent manner. Carnosine readily reacted with F6P and ribose thereby inhibiting glycation-mediated protein modification as revealed electrophoretically. The increased absorbance was time-dependent, suggesting adducts may be formed between F6P, ribose, and carnosine. CONCLUSIONS: This is the first report showing that carnosine can protect the chaperone activity of alpha-crystallin. This chaperone may protect against cataractous changes. In addition to demonstrating the effects of carnosine on prevention crosslinking, our studies also bring out important evidence that carnosine reacts with F6P and ribose, which suggests carnosine's potential as a possible nontoxic modulator of diabetic complications.
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Carnosina/farmacología , Fructosafosfatos/farmacología , Cristalino/metabolismo , Chaperonas Moleculares/metabolismo , Ribosa/farmacología , alfa-Cristalinas/metabolismo , Animales , Bovinos , Reactivos de Enlaces Cruzados/farmacología , Fluorescencia , Fructosafosfatos/antagonistas & inhibidores , Chaperonas Moleculares/antagonistas & inhibidores , Ribosa/antagonistas & inhibidores , alfa-Cristalinas/antagonistas & inhibidoresRESUMEN
Glutathione and the related enzymes belong to the defence system protecting the eye against chemical and oxidative stress. This review focuses on GSH and two key enzymes, glutathione reductase and glucose-6-phosphate dehydrogenase in lens, cornea, and retina. Lens contains a high concentration of reduced glutathione, which maintains the thiol groups in the reduced form. These contribute to lens complete transparency as well as to the transparent and refractive properties of the mammalian cornea, which are essential for proper image formation on the retina. In cornea, gluthatione also plays an important role in maintaining normal hydration level, and in protecting cellular membrane integrity. In retina, glutathione is distributed in the different types of retinal cells. Intracellular enzyme, glutathione reductase, involved in reducing the oxidized glutathione has been found at highest activity in human and primate lenses, as compared to other species. Besides the enzymes directly involved in maintaining the normal redox status of the cell, glucose-6-phosphate dehydrogenase which catalyzes the first reaction of the pentose phosphate pathway, plays a key role in protection of the eye against reactive oxygen species. Cornea has a high activity of the pentose phosphate pathway and glucose-6-phosphate dehydrogenase activity. Glycation, the non-enzymic reaction between a free amino group in proteins and a reducing sugar, slowly inactivates gluthathione-related and other enzymes. In addition, glutathione can be also glycated. The presence of glutathione, and of the related enzymes has been also reported in other parts of the eye, such as ciliary body and trabecular meshwork, suggesting that the same enzyme systems are present in all tissues of the eye to generate NADPH and to maintain gluthatione in the reduced form. Changes of glutathione and related enzymes activity in lens, cornea, retina and other eye tissues, occur with ageing, cataract, diabetes, irradiation and administration of some drugs.
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Córnea/enzimología , Glucosafosfato Deshidrogenasa/metabolismo , Glutatión Reductasa/metabolismo , Glutatión/metabolismo , Cristalino/enzimología , Retina/enzimología , Animales , Ojo/enzimología , HumanosRESUMEN
PURPOSE: The aim of this study was to investigate how glutathione reductase (GR) loses its activity during cataract formation and whether it is possible to revive it back to the normal levels. METHOD: In this study, endogenous as well as synthetic reducing systems (GSH, TTase, DTT, captopril) and alpha-crystallin at different concentrations were incubated with the soluble fraction of human cataract lens protein. The activity of glutathione reductase with or without the reducing agents and alpha-crystallin was tested, and the difference in activity gained was calculated. RESULTS: Five agents (GSH, DTT, TTase, captopril, alpha-low crystallin) were able to revive the activity of GR from human cataract lenses to different extents. CONCLUSION: This study shows that human lens GR activity was revived by different reducing agents as well as by a molecular chaperone (alpha-crystallin).
Asunto(s)
Catarata/enzimología , Glutatión Reductasa/metabolismo , Cristalino/enzimología , Chaperonas Moleculares/farmacología , Sustancias Reductoras/farmacología , alfa-Cristalinas/farmacología , Animales , Captopril/farmacología , Bovinos , Ditiotreitol/farmacología , Relación Dosis-Respuesta a Droga , Activación Enzimática , Glutarredoxinas , Glutatión/farmacología , Humanos , Técnicas In Vitro , Chaperonas Moleculares/administración & dosificación , Proteína Disulfuro Reductasa (Glutatión)/farmacología , Sustancias Reductoras/administración & dosificación , alfa-Cristalinas/administración & dosificaciónRESUMEN
Carnosine, an endogenous histidine-containing dipeptide, protects protein from oxidation and glycation, which may contribute to a potential treatment for some conformational diseases including cataract. Glycation, the non-enzymic reaction of sugars with proteins, promotes cross-linking and further aggregation. Prolonged use of glucocorticoids is a risk factor for cataract, as is diabetes. Esterase activity in the lens is decreased in senile cataract and diabetes. Previously, we reported that glycation and a steroid inactivate esterase. Here we tested the inactivation of esterase with fructose, fructose 6-phosphate (F6P) and ribose as model glycation reactions and prednisolone-21-hemisuccinate (P-21-H) as a model steroid and investigated the ability of carnosine to protect esterase against inactivation. The activity of esterase was measured by a spectrophotometric assay using p-nitrophenyl acetate as the substrate. The modified esterase was examined electrophoretically. The esterase was progressively inactivated by F6P, fructose, ribose and P-21-H. P-21-H was more effective than the sugars. Carnosine significantly inhibited the inactivation of esterase induced by all four compounds. Carnosine decreased the extent of the cross-linking. These results provide further evidence for carnosine's role as an anti-glycation compound. It is also proposed that carnosine may be an anti-steroid agent.
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Carboxilesterasa/metabolismo , Carnosina/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Glicosilación/efectos de los fármacos , Prednisolona/análogos & derivados , Prednisolona/farmacología , Esteroides/farmacología , Animales , Carboxilesterasa/antagonistas & inhibidores , Carboxilesterasa/aislamiento & purificación , Carnosina/farmacología , Electroforesis en Gel de Agar , Inhibidores Enzimáticos/farmacología , Fructosa/farmacología , Fructosafosfatos/farmacología , Hígado/enzimología , Ribosa/farmacología , Porcinos , Factores de TiempoRESUMEN
A number of naturally occurring small organic molecules, primarily involved in maintaining osmotic pressure in the cell, display chaperone-like activity, stabilizing the native conformation of proteins and protecting them from various kinds of stress. Most of them are sugars, polyols, amino acids or methylamines. In addition to their intrinsic protein-stabilizing activity, these small organic stress molecules regulate the activity of some molecular chaperones, and may stabilize the folded state of proteins involved in unfolding or in misfolding diseases, such as Alzheimer's and Parkinson's diseases, or alpha1-antitrypsin deficiency and cystic fibrosis, respectively. Similar to molecular chaperones, most of these compounds have no substrate specificity, but some specifically stabilize certain proteins, e.g., 6-aminohexanoic acid (AHA) stabilizes apolipoprotein A. In the present work, the capacity of 6-aminohexanoic acid to stabilize non-specifically other proteins is demonstrated. Both trehalose and AHA significantly protect glucose-6-phosphate dehydrogenase (G6PD) against glycation-induced inactivation, and renatured enzyme already inactivated by glycation and by guanidinium hydrochloride (GuHCl). To the best of our knowledge, there are no data on the effect of these compounds on protein glycation. The correlation between the recovery of enzyme activity and structural changes indicated by fluorescence spectroscopy and Western blotting contribute to better understanding of the protein stabilization mechanism.
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Ácido Aminocaproico/farmacología , Glucosa/metabolismo , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Guanidina/farmacología , Trehalosa/farmacología , Naftalenosulfonatos de Anilina/química , Western Blotting , Desnaturalización Proteica , Pliegue de Proteína , Espectrometría de FluorescenciaRESUMEN
In this study, endogenous as well as synthetic reducing systems were shown to reduce the disulphide bonds formed in glyceraldehyde 3-phosphate dehydrogenase, an important glycolytic enzyme previously reported to have lost its activity in human cataract lenses, resulting in reviving the activity of this enzyme. Disulphide bond formation is a non-specific posttranslational modification of proteins, which leads to a loss of function of the affected protein. When an enzyme is targeted, this harmful effect can be easily detected by monitoring the change of activity. Endogenous reducing systems are responsible for breaking these bonds and returning the protein (enzyme) to its natural state, when these mechanisms fail to do so, the loss of enzyme activity will be permanent.
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Catarata/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/análisis , Cristalino/enzimología , Sustancias Reductoras/farmacología , Anciano , Anciano de 80 o más Años , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Captopril/farmacología , Ditiotreitol/farmacología , Activación Enzimática , Glutarredoxinas , Glutatión/farmacología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Cristalino/efectos de los fármacos , Persona de Mediana Edad , Proteína Disulfuro Reductasa (Glutatión)/farmacología , EspectrofotometríaRESUMEN
The glutathionylation of human lens proteins was examined by Western-blot analysis with an anti-GSH antibody and scanning. Several different glutathionylated proteins were observed, and a 47 kDa band was of particular interest. This band did not appear after SDS/PAGE under reducing conditions, suggesting that it was a glutathionylated fraction. The 47 kDa band was found principally in the outer part of the lens, the cortex, but not in the lens nucleus where older proteins are present. The 47 kDa component was composed of betaB1-, betaB2- and gammaS-crystallin, with the gammaS-crystallin having glutathione bound at Cys-82 and at Cys-22, Cys-24 or Cys-26. We conclude that when glutathione becomes bound to gammaS-crystallin, it causes it to bind in turn to the beta-crystallin polypeptides to form a dimer.
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Disulfuros/química , Disulfuros/metabolismo , Glutatión/química , Glutatión/metabolismo , Cristalino/química , gamma-Cristalinas/química , gamma-Cristalinas/metabolismo , Envejecimiento/fisiología , Secuencia de Aminoácidos , Western Blotting , Catarata/metabolismo , Cisteína/genética , Cisteína/metabolismo , Dimerización , Electroforesis en Gel de Poliacrilamida , Humanos , Datos de Secuencia Molecular , Peso MolecularRESUMEN
Previously we showed that glycation-induced inactivation and loss of antigenicity of enzymes occur simultaneously. Alpha-crystallin, a major structural protein of the mammalian lens, prevents the aggregation of other proteins and protects enzyme function against post-translational modification in vitro. However, it is not known whether alpha-crystallin can also protect against loss of antigenicity of enzymes. Esterase activity in the lens is decreased in senile cataract and diabetes. We investigated the loss of antigenicity of esterase caused by different insults and the ability of alpha-crystallin to protect. Inactivation of carboxylesterase by sugars, fructose 6-phosphate (F6P) and a steroid, prednisolone-21-hemisuccinate (P-21-H), was measured spectrophotometrically in the presence and absence of alpha-crystallin, while loss of antigenicity was monitored simultaneously using an immunoprecipitation method. The esterase was progressively inactivated by fructose, F6P, ribose, and P-21-H. Bovine alpha-crystallin fully protected against inactivation of esterase by all four compounds, and also protected against loss of antigenicity of the esterase by fructose, ribose and P-21-H at a molar ratio of 1:1. The results indicated that alpha-crystallin, under our experimental conditions, clearly exhibited the ability to prevent loss of antigenicity and inactivation of esterase. The protective effect of alpha-crystallin against loss of antigenicity indicates a novel aspect of its chaperoning function.
Asunto(s)
Carbohidratos/inmunología , Carboxilesterasa/metabolismo , Fructosafosfatos/farmacología , Prednisolona/análogos & derivados , Prednisolona/farmacología , alfa-Cristalinas/fisiología , Animales , Carboxilesterasa/antagonistas & inhibidores , Bovinos , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Inhibidores Enzimáticos/inmunología , Inhibidores Enzimáticos/farmacología , Fructosa/farmacología , Glucosa/farmacología , Humanos , Cristalino/química , Cristalino/metabolismo , Pruebas de Precipitina , Ribosa/farmacologíaRESUMEN
Several mechanisms have been proposed for the way in which glucose and its metabolites cause cataract, retinopathy and other complications of diabetes, the most convincing being glycation. Glycation, the reaction of sugars with free amino groups of proteins, is one of a variety of non-enzymic post-translational modifications. The aim of the present study was to identify some of the most reactive proteins in the lens when incubated under physiological conditions. Fresh intact bovine lenses were incubated with [14C]glucose in a conventional tissue-culture medium with added antibiotics. After 3 and 6 days of incubation, the water-soluble proteins were separated by size-exclusion chromatography. Glycated proteins from the water-soluble fractions were separated by using a sugar affinity column (Affi-Gel 601). Then the radioactive fractions were identified on SDS/polyacrylamide gels. In addition, the whole bovine lenses were incubated with 10 mM fructose and glucose for 3 and 6 days. The glycated proteins from the water-soluble fractions in parallel with the radioactive fractions were separated by affinity chromatography, and were identified further by amino-acid sequencing. A progressive uptake of radioactive label showed that the majority of proteins incorporating both glucose and fructose were water-soluble fractions. Chromatography and SDS/polyacrylamide gel results showed that alpha- and gamma-crystallin and some proteins of a mean molecular mass of 36-37 kDa incorporated sugars early during incubation. After 6 days of incubation, more crystallins were glycated compared with 3 days, in particular beta-crystallin. Affinity-chromatography results indicated that proteins with subunit masses of 36 kDa and 20 kDa were possibly radiolabelled at an early stage. The purified glycated proteins following incubation with both glucose and fructose, which corresponded to 20 kDa and 36 kDa bands on SDS/polyacrylamide gels, were sequenced by Edman degradation. N-terminal sequences of both 20 kDa bands were Gly-Lys-Ile-Thr, characteristic of gamma-crystallins, but the N-termini of both 36 kDa bands were blocked. Further sequencing after digestion of 36 kDa bands with trypsin and running on HPLC revealed that the glucose sample gave the peptide sequences as Gly-Glu-Tyr-Pro-Asp-Tyr-Gln-Gln and Tyr-Glu-Leu-Pro-Asn-Tyr-Arg, which match with bovine gammaIIIb-crystallin. The peptide sequence Tyr-Glu-Leu-Pro-Asn-Tyr-Arg is only present in the published sequence of bovine gammaIIIb-crystallin and not in any other type of gamma-crystallin. The fructose sample gave the peptide sequences Ile-Thr-Phe-Tyr-Glu-Asp-Arg, Arg-Gly-Asp-Tyr-Pro-Asp-Tyr-Gln-Gln-Trp, Gln-Tyr-Leu-Leu-Arg and Val-Val-Asp-Leu-Tyr, which all matched with bovine gammaIIIa-crystallin. The sequence Val-Val-Asp-Leu-Tyr only appears in the sequence of bovine gammaIIIa-crystallin. gammaIII-Crystallin is the most susceptible lens protein to glycation. The primary target of glucose is gammaIIIb-crystallin, whereas that of fructose is gammaIIIa-crystallin. The early glycation of gammaIII-crystallin by glucose and fructose could result in structural alterations, leading to aggregation of crystallin and eventually cataract formation.
Asunto(s)
Cristalino/metabolismo , gamma-Cristalinas/metabolismo , Secuencia de Aminoácidos , Animales , Radioisótopos de Carbono/metabolismo , Bovinos , Fructosa/química , Glucosa/química , Glicosilación , Cristalino/química , Datos de Secuencia Molecular , Análisis de Secuencia de Proteína/métodos , gamma-Cristalinas/químicaRESUMEN
Alpha-crystallin, a molecular chaperone and lens structural protein protects soluble enzymes against heat-induced aggregation and inactivation by a variety of molecules. In this study we investigated the chaperone function of alpha-crystallin in a more physiological system in which alpha-crystallin was incorporated into red cell 'ghosts'. Its ability to protect the intrinsic membrane protein Na/K-ATPase from external stresses was studied. Red cell ghosts were created by lysing the red cells and removing cytoplasmic contents by size-exclusion chromatography. The resulting ghost cells retain Na/K-ATPase activity. alpha-Crystallin was incorporated in the cells on resealing and the activity of Na/K-ATPase assessed by ouabain-sensitive 86Rb uptake. Incubation with fructose, hydrogen peroxide and methylglyoxal (compounds that have been implicated in diabetes and cataract formation) were used to test inactivation of the Na/K pump. Intracellular alpha-crystallin protected against the decrease in ouabain sensitive 86Rb uptake, and therefore against inactivation induced by all external modifiers, in a dose-dependent manner.
Asunto(s)
Membrana Eritrocítica/fisiología , Estrés Oxidativo/fisiología , ATPasa Intercambiadora de Sodio-Potasio/sangre , alfa-Cristalinas/sangre , Adenosina Trifosfato/sangre , Membrana Eritrocítica/efectos de los fármacos , Glicosilación , Hemólisis , Humanos , Peróxido de Hidrógeno/farmacología , Chaperonas Moleculares/sangre , Ouabaína/farmacología , Rubidio/sangre , Rubidio/farmacocinética , Sacarosa/farmacologíaRESUMEN
The role of alpha-crystallin as a molecular chaperone has been shown in many in vitro studies. In the present paper, we report on the chaperone function of alpha-crystallin within resealed erythrocyte ghosts. Eight enzymes were individually resealed within erythrocyte ghosts and assayed at zero time and at 24 h. The ghost cell suspension was separated into soluble and membrane fractions. Five of the enzymes had significantly greater enzyme activity after 24 h than the control within the soluble fractions. Fructation caused a decrease in enzyme activity (relative to the control). Resealing of alpha-crystallin within the ghost cell alongside the enzymes protected against inactivation by fructose within the soluble fraction.
Asunto(s)
Membrana Eritrocítica/enzimología , Fructosa/metabolismo , alfa-Cristalinas/metabolismo , Aldehído Deshidrogenasa/metabolismo , Sitios de Unión , Catalasa/metabolismo , Membrana Celular/metabolismo , Cromatografía en Gel , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Fumarato Hidratasa/metabolismo , Glutatión Reductasa/metabolismo , Glicerolfosfato Deshidrogenasa/metabolismo , Humanos , L-Lactato Deshidrogenasa/metabolismo , Metabolismo de los Lípidos , Malato Deshidrogenasa/metabolismo , Conformación Proteica , Superóxido Dismutasa/metabolismo , Factores de TiempoRESUMEN
The major lenticular protein alpha-crystallin has chaperone activity. With increasing age this chaperone function is compromised. Diabetes and glucocorticoid therapy are risk factors for cataract and are associated with raised sugar and glucocorticoid levels, respectively. These molecules react with proteins. Long-lived lenticular proteins are particularly susceptible to such attack. To investigate this possibility we carried out incubations of alpha-crystallin with fructose 6-phosphate and prednisolone-21-hemisuccinate and investigated the effect of modification on chaperone ability. Fructose 6-phosphate and prednisolone-21-hemisuccinate compromised chaperone activity as measured by the beta L-crystallin thermal aggregation assay. Tryptophan fluorescence provided evidence that the structure of alpha-crystallin had been modified by both compounds.