RESUMEN
An association between the R990G polymorphism of the CaSR gene, coding for calcium-sensing receptor, and primary hypercalciuria was found in kidney stone formers. To confirm this relationship, we investigated hypercalciuric women without stones and studied the effect of CaSR gene in human embryonic kidney cells (HEK-293). We genotyped for CaSR A986S, R990G, and Q1011E polymorphisms, 119 normocalciuric and 124 hypercalciuric women with negative history of kidney stones. Homozygous (n=2) or heterozygous (n=21) women for the 990G allele considered as one group had an increased risk to be hypercalciuric (odds ratio=5.2; P=0.001) and higher calcium excretion (P=0.005) in comparison with homozygous women for the 990R allele (n=220). HEK-293 cells were transfected with the variant allele at the three CaSR gene polymorphisms and with the most common allele with no variants. The transient increment of intracellular calcium caused by the stepwise increase of extracellular calcium was evaluated in stable transfected cells loaded with fura-2 AM. The extracellular calcium concentration producing the half-maximal intracellular calcium response was lower in HEK-293 cells transfected with the 990G allele than in those transfected with the wild-type allele (P=0.0001). Our findings indicate that R990G polymorphism results in a gain-of-function of the calcium-sensing receptor and increased susceptibility to primary hypercalciuria.
Asunto(s)
Predisposición Genética a la Enfermedad , Hipercalciuria/genética , Polimorfismo Genético , Receptores Sensibles al Calcio/genética , Alelos , Sustitución de Aminoácidos , Western Blotting , Estudios de Casos y Controles , Línea Celular , Codón , Electroforesis en Gel de Poliacrilamida , Exones , Femenino , Colorantes Fluorescentes , Fura-2/análogos & derivados , Frecuencia de los Genes , Glicina/metabolismo , Haplotipos , Heterocigoto , Homocigoto , Humanos , Desequilibrio de Ligamiento , Persona de Mediana Edad , Oportunidad Relativa , Polimorfismo de Nucleótido Simple , Estructura Secundaria de Proteína , Factores de Riesgo , TransfecciónRESUMEN
An investigation was carried out into the possible effect of sphingosine (Sph) on nitric oxide (NO) production in living neurons. Differentiated granule cells were used in a dynamic videoimaging analysis of single cells labeled, simultaneously, with FURA-2 and the NO indicator 4,5-diaminofluorescein. The results demonstrate that Sph exerts a potent inhibitory effect on the Ca2+-dependent production of NO, without modifying the [Ca2+]i. The effect appears to be specific as neither ceramide nor Sph-1-phosphate had any effect on the NO and [Ca2+]i levels. The data demonstrate that Ca2+-dependent NO production is a specific Sph target in living granule cells, suggesting that this bioactive sphingoid plays a relevant role in neuronal NO signaling.
Asunto(s)
Microscopía Fluorescente/métodos , Neuronas/metabolismo , Óxido Nítrico/biosíntesis , Esfingosina/fisiología , Animales , Fluoresceína , Fura-2 , Ratas , Ratas Sprague-DawleyRESUMEN
We investigated the direct effects of casein phosphopeptides (CPP), which are formed by the proteolytic degradation of alpha- and beta-caseins, on calcium uptake by human HT-29 intestinal tumor cells, which undergo an enterocytically oriented differentiation in culture. A commercial preparation containing a mixture of purified CPP and an individual CPP of 25 amino acids, both containing the characteristic Ca(2+) binding motif, ser(P)-ser(P)-ser(P)-glu-glu, were employed. The study was performed at the single-cell level and on a cell population and measured the changes in cytosolic calcium concentration before and after CPP addition. In the presence of 2 mmol/L extracellular calcium, both CPP preparations induced a transient rise of free intracellular calcium ions, which did not influence ATP-induced release of calcium from intracellular stores, and which disappeared completely in the absence of extracellular calcium. Pretreatment of these cells with thapsigargin, which completely empties the intracellular calcium stores, did not abolish the cell responses to CPP. Repetitive stimulation of HT-29 cells with CPP always elicited a transient calcium rise, suggesting a lack of desensitization. The CPP-stimulated cytosolic calcium rise was dependent on CPP dose, in a seemingly nonsaturating mode, and on cell numbers. All of this is consistent with the hypothesis that CPP do not influence membrane-bound receptors or ion channels, but may act as calcium ionophores or calcium carriers across the membrane. The reported findings provide a new basis on which to assess the possibility that CPP enhance calcium absorption and bioavailability in animals.
Asunto(s)
Calcio/metabolismo , Caseínas/farmacología , Fosfopéptidos/farmacología , Cationes Bivalentes , Membrana Celular/metabolismo , Citosol/metabolismo , Colorantes Fluorescentes , Fura-2 , Células HT29 , Humanos , Espectrometría de FluorescenciaRESUMEN
After incubation of intact living cultured rat cerebellar granule cells at 37 degrees C with a new GM1 ganglioside analog, carrying a diazirine group and labeled with (125)I in the ceramide moiety, followed by photoactivation, a relatively small number of radiolabeled proteins were detected in a membrane-enriched fraction. A protein of about 55 kDa with a pI of about 5 carried a large portion of the radioactivity even if incubation and cross-linking were performed at 4 degrees C and in the presence of inhibitors of endocytosis, suggesting that it is cross-linked at the plasma membrane. Immunoprecipitation and Western blotting experiments showed the positivity of this protein for tubulin. Trypsin treatment of intact cells ruled out the involvement of a plasma membrane surface tubulin. Release of radioactivity from cross-linked tubulin after KOH treatment (but not hydroxylamine treatment) suggested that the photoactivated ganglioside reacts with an ester-linked fatty acid anchor of tubulin. Low buoyancy, detergent-resistant membrane fractions, isolated from cells after incubation with the GM1 analogue and photoactivation, proved their enrichment in endogenous and radioactive GM1 ganglioside, sphingomyelin, cholesterol, signal transduction proteins, and tubulin. It is noteworthy that radioactive tubulin was also detected in this fraction, indicating the presence of tubulin molecules carrying a fatty acid anchor in detergent-resistant, ganglioside-enriched domains of the plasma membrane. Parallel experiments carried out with a phosphatidylcholine analogue, also carrying a diazirine group and labeled with (125)I in the fatty acid moiety, showed the specificity of tubulin interaction with GM1. Taken together, these results indicate that some tubulin molecules are associated with a lipid anchor to detergent-resistant glycolipid-enriched domains of the plasma membrane. This novel feature of membrane domains can provide a key for a better understanding of their biological role.
Asunto(s)
Membrana Celular/metabolismo , Cerebelo/metabolismo , Gangliósido G(M1)/metabolismo , Glucolípidos/metabolismo , Lípidos de la Membrana/metabolismo , Neuronas/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Membrana Celular/química , Células Cultivadas , Cerebelo/citología , Cromatografía en Capa Delgada , Reactivos de Enlaces Cruzados , Detergentes , Gangliósido G(M1)/química , Glucolípidos/química , Lípidos de la Membrana/química , Lípidos de la Membrana/aislamiento & purificación , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Tripsina , Tubulina (Proteína)/química , Tubulina (Proteína)/aislamiento & purificaciónRESUMEN
A new photoactivable, radioactive derivative of ganglioside GM1 has been utilized to assess lipid distribution in the caveolae bilayer, taking advantage of the ability of the glycolipid, endogenous or exogenously added, to concentrate within this membrane compartment and to crosslink neighboring molecules upon illumination. After insertion into A431 plasma membrane and photoactivation, a membrane-enriched and a detergent-resistant fraction, enriched in gangliosides, sphingomyelin and cholesterol, were isolated. While a few radioactive proteins were detected in the membrane-enriched fraction, only radioactive caveolin was detected in the detergent-resistant fraction, indicating at the same time the enrichment of this fraction in caveolae and the presence of ganglioside within this compartment. Among lipids, crosslinked phosphatidylcholine, sphingomyelin and cholesterol were detected in the membrane-enriched fraction, while only crosslinked sphingomyelin was detected in the detergent-resistant fraction. These results suggest the enrichment in sphingomyelin - along with ganglioside - within the outer leaflet, and the preferential localization of cholesterol within the endoplasmic leaflet, of the caveolae bilayer.
Asunto(s)
Caveolas/química , Membrana Dobles de Lípidos/química , Fotoquímica/métodos , Caveolas/metabolismo , Caveolinas/metabolismo , Línea Celular , Membrana Celular/química , Membrana Celular/metabolismo , Colesterol/química , Colesterol/metabolismo , Reactivos de Enlaces Cruzados/química , Detergentes/química , Gangliósido G(M1)/análogos & derivados , Gangliósido G(M1)/química , Humanos , Metabolismo de los Lípidos , Lípidos/análisis , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Proteínas/análisis , Proteínas/química , Esfingomielinas/química , Esfingomielinas/metabolismoRESUMEN
Dynamic changes of glycolipid domains within the plasma membranes of cultured rat cerebellar granule cells have been investigated. For this purpose, a pyrene-labelled derivative of G(M1) ganglioside has been incorporated in the cell plasma membrane, and the rate of excimer formation, directly related to the formation of domains, has been studied by a fluorescence imaging technique (excimer-formation imaging). Fluorescence imaging showed that upon addition of 100 microM glutamate, indirectly inducing the activation of protein kinase C (PKC), glycolipid concentration within domains increases in cell bodies. Comparable effects were exerted by the addition of PMA, directly inducing the activation of PKC. On the contrary, the phorbol ester was not effective in the presence of the specific PKC inhibitor, bisindolylmaleimide. These results suggest that glycolipid-enriched domains are dynamic supramolecular structures affected by membrane-associated events, such as PKC activation. Dynamic changes of domains could be important in modulating their postulated participation in a series of functions, including signal transduction and lipid/protein sorting.
Asunto(s)
Glucolípidos/química , Lípidos de la Membrana/química , Neuronas/química , Animales , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Cerebelo/química , Cerebelo/citología , Cerebelo/metabolismo , Endocitosis , Activación Enzimática/efectos de los fármacos , Polarización de Fluorescencia , Colorantes Fluorescentes , Glucolípidos/metabolismo , Fluidez de la Membrana , Lípidos de la Membrana/metabolismo , Microscopía Fluorescente , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteína Quinasa C/metabolismo , Ratas , Espectrometría de Fluorescencia , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
We studied the possibility of supplementing human keratinocytes with exogenous lipids (phospholipids, sphingolipids and cholesterol) and evaluated their influence on cell proliferation, using cells cultured in vitro. Experiments carried out with liposomes composed of cholesterol/GM1 ganglioside and different phospholipids (5:1.5:10, M/M/M), showed that liposomes associated with cells more efficiently when they contained soya lecithin. The treatment with liposomes made of the ternary mixture did not modify the rate of cell proliferation, as assessed by the incorporation of [3H]-thymidine. In contrast, the proliferation rate strongly decreased (65% with respect to the control) using the same liposomes without GM1. Experiments carried out with GM1 alone showed a strong stimulation of the proliferation rate (144% with respect to the control). Fluorescence dequenching experiments, carried out with the probe octadecyl rhodamine B chloride, showed that fusion was the main mechanism of liposome-cell interaction. Metabolic studies established that exogenously administered GM1--either embedded in liposomes or as a pure glycolipid dispersion--led to the production of several products, including ceramide. Altogether, these results show that different, opposing effects can be exerted on cell proliferation by the administration of lipids, separately or in mixtures, to human keratinocytes, and indicate the importance of a correct formulation for supplementing human keratinocytes with exogenous lipids.
Asunto(s)
Queratinocitos/efectos de los fármacos , Liposomas/farmacología , Células Cultivadas , Colesterol/metabolismo , Colesterol/farmacología , Gangliósido G(M1)/metabolismo , Gangliósido G(M1)/farmacología , Humanos , Queratinocitos/química , Queratinocitos/metabolismo , Liposomas/química , Liposomas/metabolismo , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Fosfolípidos/metabolismo , Fosfolípidos/farmacología , Timidina/metabolismo , Factores de Tiempo , TritioRESUMEN
Specialized domains, displaying a peculiar lipid and protein composition, are present within the plasma membrane of mammalian cells and play a pivotal role in fundamental membrane-associated events. Among lipids, sphingolipids (in particular glycolipids and sphingomyelin) are characteristically enriched within such domains. Moreover, a series of functionally related proteins is present, suggesting the involvement of these membrane structures in the mechanism of signal transduction and lipid/protein sorting. An increasing body of evidence suggests that domains are dynamic structures, and that their dynamic fluctuations can modulate the activity of domain-associated proteins through changes of glycolipid protein interaction. Even if a large body of experimental investigation has been carried out on eukaryotic cells, only little attention has been paid to the neuron. The purpose of the present review is to summarize the observations implying a functional role of glycolipid-enriched domains in cultured rat cerebellar granule cells.
Asunto(s)
Glucolípidos/fisiología , Proteínas de la Membrana/fisiología , Factores de Crecimiento Nervioso/fisiología , Neuronas/fisiología , Transducción de Señal , Animales , Membrana Celular/química , Membrana Celular/fisiología , Células Cultivadas , Cerebelo/citología , Cerebelo/fisiología , Glucolípidos/química , Proteínas de la Membrana/química , Estructura Terciaria de Proteína , RatasRESUMEN
We verified the hypothesis that changes in the endogenous GM1 ganglioside density in the environment of TrkB, receptor of brain-derived neurotrophic factor, can affect receptor activity, and focused on rat cerebellar granule cells expressing both GM1 and TrkB. Changes of the amount of GM1 associated to immunoprecipitated TrkB and of receptor tyrosine phosphorylation were evaluated after treatment with phorbol-12-myristate-13-acetate (1 microM, 7 min), reported to affect the plasma membrane distribution of endogenous gangliosides in the same cells. After treatment, the amount of GM1 associated to receptor and TrkB phosphorylation decreased by about 40%. The amount of associated GM1 decreased by about 33% also after concomitant treatment with phorbol ester and brain-derived neurotrophic factor, but in this case the neurotrophin was unable to enhance receptor tyrosine phosphorylation. These results for the first time suggest that changes in the amount of endogenous GM1 in the environment of TrkB can modulate receptor activity, and offer new clues for a better understanding of physiological and pathological events of the nervous system.
Asunto(s)
Gangliósido G(M1)/metabolismo , Neuronas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Animales , Células Cultivadas , Fosforilación , Pruebas de Precipitina , Ratas , Ratas Sprague-Dawley , Receptor de Factor Neurotrófico Ciliar , Tirosina/metabolismoRESUMEN
An increasing body of evidence suggests that glycolipid domains are present on the plasma membrane surface of mammalian cells and play a key role in signal transduction. We have investigated the modulation of glycolipid-protein interaction consequent to a specific event occurring at the plasma membrane. For this purpose, a new photoactivable, radioactive derivative of GM1 ganglioside, carrying a phenyldiazirine fatty acid labelled with 125I, has been used with rat cerebellar granule cells in culture. Upon incubation of photoactivable GM1 with the cells followed by illumination, several proteins become radioactive and were detectable on the two dimensional-electrophoresis, which points to their interaction with the ganglioside. Upon addition of cytotoxic doses of glutamate, known to induce indirectly the activation of protein kinase C (PKC), one of the proteins crosslinked by photoactivable GM1 in control cells of molecular mass about 92 kDa and pI about 4, was not anymore detectable; this suggests its exclusion from the glycolipid domains. On the contrary, another protein, of about 15 kDa and pI 6.5, previously not crosslinked, was interacting with the ganglioside derivative after glutamate treatment. Comparable effects were exerted by phorbol-2-myristate-3-acetate, which directly induces the activation of PKC. These results show that PKC activation, a key step of inbound trans-membrane signalling, affects the interaction between glycolipids and proteins at the plasma membrane surface, possibly within a mixed domain. The dynamic modulation of ganglioside-protein interaction may affect the involvement of glycolipid domains in membrane-located events such as signal transmission and lipid/protein sorting.
Asunto(s)
Cerebelo/metabolismo , Gangliósido G(M1)/metabolismo , Transducción de Señal , Animales , Glucolípidos/metabolismo , Radioisótopos de Yodo , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
While the mechanism of signal transduction across the plasma membrane from the exo- to the endoplasmic side has been extensively investigated, the possible return of messages back to the outer layer is less known. We studied the effect of protein kinase C activation on the ganglioside accessibility at the exoplasmic face of intact rat cerebellar granule cells in culture, using the enzyme sialidase as the probing molecule. Under the experimental conditions (1 milliunit/mL enzyme, 2 min incubation at 37 degreesC), only GT1b and GD1a gangliosides were partially affected by the enzyme (28.6 and 25.7% hydrolysis, respectively). After cell treatment with phorbol 12-myristate 13-acetate, inducing protein kinase C activation, GT1b and GD1a ganglioside susceptibility to sialidase was strongly decreased (8.6 and 15.9% hydrolysis, respectively). A reduction of ganglioside hydrolysis was also observed when protein kinase C activation was induced by cell treatment for 15 min with 100 microM glutamate. On the contrary, accessibility did not vary when protein kinase C translocation was not effective (either in the absence of Ca2+ in the medium or using 1 microM glutamate) or when the kinase activity was inhibited by staurosporine. These data suggest that following PKC activation, a key step of inbound transmembrane signaling, cell may dispatch outbound messages to the plasma membrane outer layer, changing the selective recognition and crypticity of glycolipids at the cell surface, possibly through a modulation of their segregation state.
Asunto(s)
Gangliósidos/metabolismo , Neuronas/metabolismo , Animales , Membrana Celular/metabolismo , Células Cultivadas , Cromatografía Líquida de Alta Presión , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Ácido Glutámico/farmacología , Neuraminidasa/metabolismo , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Estaurosporina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Vibrio cholerae/enzimologíaRESUMEN
The ganglioside composition of the cerebral hemispheres of young and adult rats of either sex has been herein assessed for the first time. In females, the total ganglioside content at any age, the content of GM1, GD1a, and GD1b at 8 days, and the content of GM1, GD1b, GT1b, and GQ1b at 60 days were higher in the right than in the left hemisphere. In males, no difference was observed. Concerning the ceramide moiety, a difference was displayed by C18:1 long-chain base in GD1a, whose proportion was higher in the left than in the right hemisphere of females aged 8 days. The comparison between homolateral hemispheres of rats of different sex revealed several differences. On average, in 8-day-old animals, the content of gangliosides was higher in females than in males. At 60 days the amount of gangliosides was on average lower in females than in males, even if with some exception. The data obtained with the current investigation show the existence of a ganglioside lateralization in rat brain, exclusively in females, and almost entirely at charge of the oligosaccharide portion. Moreover, age-dependent changes of ganglioside pattern and content show a dependence on brain lateralization.
Asunto(s)
Química Encefálica/fisiología , Lateralidad Funcional/fisiología , Gangliósidos/análisis , Caracteres Sexuales , Envejecimiento/metabolismo , Animales , Femenino , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The thermotropic behavior of palmitoylsphingomyelin vesicles containing GM1 ganglioside and cholesterol has been investigated by high-sensitivity differential scanning calorimetry. The thermograms exhibited by binary palmitoylsphingomyelin/GM1 mixtures are resolvable into two components. The relative contribution of the minor component, undetectable in the absence of ganglioside, to the total enthalpy and its transition temperature (>40 degrees C) increase with the concentration of the glycolipid embedded in the vesicles. These data suggest the occurrence of lateral phase separation and that more ordered, higher melting GM1 ganglioside-enriched domains are present within the sphingomyelin bilayer. Studies on binary sphingomyelin/cholesterol mixtures confirmed the known tendency of the sterol to decrease the total enthalpy of sphingomyelin, forming cholesterol-enriched domains. The thermograms exhibited by ternary sphingomyelin/ganglioside/cholesterol mixtures in variable proportions (up to 20% molar GM1 or Chol) displayed, on increasing the content of either the sterol or the ganglioside, features addressable to sphingomyelin/cholesterol (peaks centered at temperature 40 degrees C), respectively. This trend was confirmed by deconvolution analysis, showing that the thermograms are resolvable into components addressable to GM1-enriched and to cholesterol-enriched domains. Taken all together, the results show that the architectural features of sphingomyelin bilayers are strongly dependent on the presence of GM1 ganglioside and cholesterol, whose presence is leading to the formation of separate, GM1-enriched and cholesterol-enriched distinct domains. Ganglioside-sphingomyelin and sphingomyelin-cholesterol, together with mutual ganglioside-ganglioside, interactions could contribute to maintain a network of bonds extending to proteins, forming specialized membrane domains, such as caveolae, or others, whose experimental clues are the glycolipid-enriched detergent-insoluble fractions that can be isolated from cell membranes.
Asunto(s)
Colesterol/química , Gangliósido G(M1)/química , Membrana Dobles de Lípidos/química , Esfingomielinas/química , Termodinámica , Animales , Rastreo Diferencial de Calorimetría , Bovinos , LiposomasRESUMEN
We describe a liposome formulation characterized by sensitivity to concurrent and small temperature and pH changes. Liposome permeability was assessed by monitoring the release of entrapped carboxyfluorescein (CF), using a fluorescence dequenching technique. The thermotropic behavior of the liposomes was investigated by differential scanning calorimetry. After 2 h at 37 degrees C in fetal calf serum, liposomes composed of a mixture of dipalmitoyl phosphatidylcholine/cholesterol/GM1 ganglioside/biotinoyl-dipalmitoyl phosphatidylethanolamine (100:20:6:0.25 molar ratio) released 8% CF at pH 7.4 and 12% CF at pH 6.7. At 41 degrees C the leakage was 72% at pH 7.4 and almost complete (99%) at pH 6.7. The pH and temperature sensitivity, with maximal release when the two circumstances occurred simultaneously, was confirmed by entrapping calcein or [14C]glucose. The reasons for the bilayer sensitivity and the conditions for in vivo drug delivery are discussed.
Asunto(s)
Antineoplásicos/administración & dosificación , Biotina/química , Sistemas de Liberación de Medicamentos , Liposomas/química , Rastreo Diferencial de Calorimetría , Fenómenos Químicos , Química Farmacéutica , Química Física , Concentración de Iones de Hidrógeno , Permeabilidad , Sensibilidad y Especificidad , TemperaturaRESUMEN
Liposomes containing monosialoganglioside (GM1) or polyethylene glycol (PEG) lipid derivatives have prolonged circulation in the blood. This favours liposome extravasation to tumour sites. In this report it is shown that inclusion of GM1, PEG550-DPPE or PEG2000-DPPE in liposomes containing biotin-DPPE significantly diminished the ability of vesicles to bind to streptavidin in vitro. Steric inhibition due to the bulky head group of these lipids was least for biotin-DPPE liposomes containing GM1. Biodistribution studies in C26 tumour-bearing mice showed that GM1-liposomes containing small amounts of biotin-DPPE have long circulation life-times in the blood. Using fluorescent microscopic techniques, liposomes containing both GM1 and biotin-DPPE were detected within extra-vascular spaces in tumours. In addition it was shown that biotin-DPPE in GM1-liposomes bound streptavidin in situ. These results suggest that GM1-liposomes containing biotin-DPPE have potential use as diagnostic or therapeutic reagents in pre-targeting applications dependent on the high-affinity interaction of biotin with streptavidin.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biotina/metabolismo , Liposomas , Animales , Sitios de Unión , Reactivos de Enlaces Cruzados , Portadores de Fármacos , Femenino , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/tratamiento farmacológico , Fosfatidiletanolaminas/metabolismo , Espectrometría de Fluorescencia , EstreptavidinaRESUMEN
Aldose reductase has been shown to be expressed in large amount by human embryonic epithelial cells (EUE) in response to osmotic stress. This conclusion is the result of studies undertaken following the purification to homogeneity of two forms of a 35-kDa protein overexpressed in EUE cells grown in hypertonic saline culture medium as compared to EUE cells grown in isoosmotic medium. Amino-acid composition, molecular weight and partial internal amino-acid sequence showed that the above proteins are two different forms of aldose reductase. These findings were confirmed by the observation that aldose reductase activity increased about 150-fold in adapted cells and returned to basal levels in de-adapted cells.
Asunto(s)
Aldehído Reductasa/aislamiento & purificación , Adaptación Fisiológica , Aldehído Reductasa/química , Aldehído Reductasa/metabolismo , Secuencia de Aminoácidos , Aminoácidos/análisis , Línea Celular/enzimología , Humanos , Datos de Secuencia Molecular , Ósmosis , Solución Salina HipertónicaRESUMEN
A cell line derived from human embryonic epithelium (EUE cells) shows an enhanced expression of a 33 kDa protein when adapted to grow in a hypertonic medium containing 0.246 M NaCl (1.8 x the isotonic concentration). The maximum amount of this protein, followed by SDS-PAGE electrophoresis, was found after 4 days of adaptation; thereafter, the protein band remained fairly constant up to 30 days. When the cells were transferred back to a medium containing 0.137 M NaCl (isotonic medium), the protein pattern reverted to that of control cells. This protein is mainly localized in the cytosol, although a small part is associated with the 150,000 g pellet and needs detergents to be extracted. The molecular weight and the cellular location suggest a possible analogy with the so-called amphitropic proteins, that are known to interact with both the epidermal growth factor receptor and hydrophobic structures, such as the membrane phospholipids and the cytoskeletal components.