RESUMEN

The quantitative assay of protein S can help in rapidly identifying carriers of abnormal protein S molecules through a simple procedure (by determining the total protein S mass, total protein S activity, and protein S-specific activity in blood), without genetic testing. To clarify the relationship between venous thromboembolism (VTE) and protein S-specific activity, and its role in the diagnosis of thrombosis in Japanese persons, the protein S-specific activity was measured and compared between patients with thrombosis and healthy individuals. The protein S-specific activity of each participant was calculated from the ratio of total protein S activity to total protein S antigen level. Plasma samples were collected from 133 healthy individuals, 57 patients with venous thrombosis, 118 patients with arterial thrombosis, and 185 non-thrombotic patients. The protein S-specific activity of one-third of the patients with VTE was below the line of Y = 0.85X (-2 S.D.). Most protein S activities in the plasma of non-thrombotic patients were near the Y = X line, as observed in healthy individuals. In conclusion, it was clearly shown that monitoring protein S activity and protein S-specific activity in blood is useful for predicting the onset and preventing venous thrombosis in at least the Japanese population.

Asunto(s)

Proteína S/metabolismo , Tromboembolia Venosa/etiología , Femenino , Humanos , Japón , Masculino , Factores de Riesgo , Tromboembolia Venosa/fisiopatología

RESUMEN

Thrombophilia is a serious unpredictable complication caused by gene mutations, resulting in anticoagulant deficiencies. We herein report a single-family case series of protein C (PC) deficiency. Case 1 involved a Japanese man whose PC deficiency resulted in severe systemic thrombosis. The patients in cases 2 and 3 were his daughters who were diagnosed with PC deficiency via carrier screening in 2001 and later both became pregnant. Owing to appropriate treatments during pregnancy, they did not develop thrombosis and safely gave birth to healthy infants. This family case series suggests that appropriate knowledge concerning thrombophilia helps prevent future emergencies.

Asunto(s)

Anticoagulantes/uso terapéutico , Deficiencia de Proteína C/complicaciones , Deficiencia de Proteína C/genética , Trombofilia/tratamiento farmacológico , Trombofilia/etiología , Trombofilia/genética , Trombosis/tratamiento farmacológico , Trombosis/etiología , Adulto , Resultado Fatal , Femenino , Predisposición Genética a la Enfermedad , Humanos , Japón , Masculino , Embarazo , Trombosis/genética , Resultado del Tratamiento , Adulto Joven

RESUMEN

Band 3 mediates the Cl- and HCO3- exchange across the red blood cell membrane and plays a pivotal role for delivering oxygen appropriately to metabolically active tissues. For understanding molecular mechanisms, it is essential to know the structure and function relationship. In terrestrial environments, however, nobody could make good quality crystals of Band 3 for the X-ray crystallographic study. In this study, we purified the transmembrane domain of Band 3 from human red blood cells and crystallized the purified Band 3 without the Fab fragment at the International Space Station "KIBO" under microgravity environments.

Asunto(s)

Proteína 1 de Intercambio de Anión de Eritrocito/química , Proteína 1 de Intercambio de Anión de Eritrocito/aislamiento & purificación , Cristalización/métodos , Nave Espacial , Ingravidez , Cristalografía por Rayos X/métodos , Membrana Eritrocítica/química , Humanos

Asunto(s)

Anticoagulantes/uso terapéutico , Proteína S/metabolismo , Administración Oral , Humanos

RESUMEN

A 67-year-old woman who underwent left breast mastectomy and right breast partial mastectomy under the diagnosis of left breast cancer and suspected right breast cancer 10 years earlier was admitted because of dyspnea. Chest computed tomography revealed pericardial fluid accumulation. The patient was treated with pericardial drainage; thereby, 800 mL of bloody fluid was removed. The cytological diagnosis was malignancy. After receiving mitomycin C instillation, she underwent systemic chemotherapy and endocrine therapy. At the last follow-up more than 5 years after the recurrence, no reaccumulation of the pericardial fluid was observed, and she remained alive in good condition.

Asunto(s)

Neoplasias de la Mama/tratamiento farmacológico , Mitomicina/uso terapéutico , Pericarditis/tratamiento farmacológico , Pericarditis/etiología , Anciano , Neoplasias de la Mama/complicaciones , Neoplasias de la Mama/patología , Drenaje , Femenino , Humanos , Derrame Pericárdico/etiología , Recurrencia , Factores de Tiempo

RESUMEN

BACKGROUND: The Japanese Committee for Clinical Laboratory Standards (JCCLS) has developed a multianalyte conventional reference material (MacRM) for nationwide standardization of laboratory measurements. METHODS: To prepare the MacRM, pooled sera were obtained from healthy Japanese individuals. Target values of the pooled sera for 30 analytes were assigned on the basis of the measurement results of 45 certified clinical laboratories whose calibration was verified by measuring certified reference materials (CRMs) provided by the National Institute of Standards and Technology, the Institute for Reference Materials and Measurements, and JCCLS. Commutability of MacRM was assessed by comparison with results for 150 individual inpatients at Fukuoka University Chikushi Hospital. Survey samples were prepared by essentially the same method for MacRM but without target values. The survey samples were used to assess agreement among 165 laboratories that used various assay kits and platforms calibrated with the MacRM. RESULTS: The commutability of MacRM was confirmed for 30 analytes with sera from 150 individual patients. The imprecision (CV) of measurements of survey samples (high and low concentrations) among the 165 laboratories was 0.4%-10.0%. Twenty-six of 30 analytes were within the goals for interinstitutional allowable bias. An aliquot of MacRM stored frozen at -80 °C remained stable for ≥4 years. CONCLUSIONS: The MacRM was successfully applied as a calibrator to achieve nationwide standardization for 30 analytes measured by 165 laboratories that used various methods from different manufacturers.

Asunto(s)

Análisis Químico de la Sangre/normas , Técnicas de Laboratorio Clínico/normas , Adolescente , Adulto , Anciano , Femenino , Congelación , Humanos , Japón , Masculino , Persona de Mediana Edad , Estándares de Referencia , Adulto Joven

RESUMEN

Anion exchanger 1 (AE1), also known as band 3 or SLC4A1, plays a key role in the removal of carbon dioxide from tissues by facilitating the exchange of chloride and bicarbonate across the plasma membrane of erythrocytes. An isoform of AE1 is also present in the kidney. Specific mutations in human AE1 cause several types of hereditary hemolytic anemias and/or distal renal tubular acidosis. Here we report the crystal structure of the band 3 anion exchanger domain (AE1(CTD)) at 3.5 angstroms. The structure is locked in an outward-facing open conformation by an inhibitor. Comparing this structure with a substrate-bound structure of the uracil transporter UraA in an inward-facing conformation allowed us to identify the anion-binding position in the AE1(CTD), and to propose a possible transport mechanism that could explain why selected mutations lead to disease.

Asunto(s)

Proteína 1 de Intercambio de Anión de Eritrocito/química , Proteína 1 de Intercambio de Anión de Eritrocito/genética , Cristalografía por Rayos X , Enfermedad/genética , Proteínas de Escherichia coli/química , Humanos , Proteínas de Transporte de Membrana/química , Mutación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína

RESUMEN

Approximately 50% of Japanese individuals who develop venous thrombosis have reduced activities of protein S, the major component of the Activated Protein C (APC) anticoagulant system. The significance of the measurement of protein S-specific activity and its practical use will be discussed.

Asunto(s)

Proteína S/metabolismo , Trombofilia/metabolismo , Biomarcadores/sangre , Pruebas de Coagulación Sanguínea/métodos , Humanos , Japón , Proteína C/metabolismo , Trombosis de la Vena/diagnóstico , Trombosis de la Vena/etiología

RESUMEN

Protein S (ProS) is a physiological inhibitor of coagulation with an important function in the down-regulation of thrombin generation. ProS deficiency is a major risk factor for venous thrombosis. This study enrolled 40 ProS-deficient probands to investigate the molecular basis of hereditary ProS deficiency in Chinese patients. A mutation analysis was performed by resequencing the PROS1 gene. Large deletions were identified by multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 20 different mutations, including 15 novel mutations, were identified in 21 of the 40 index probands. Small mutations were detected in 18 (45.0%) probands, and large deletions were found in 3 (7.5%) probands, leaving 19 (47.5%) patients without causative variants. To evaluate the functional consequences of 2 novel missense variants, ex vivo thrombin-generation assays, bioinformatics tools, and in vitro expression studies were employed. The p.Asn365Lys ProS variant was found to have moderately impaired secretion and reduced activated protein C cofactor activity. In contrast, the p.Pro410His mutant appeared to have severely impaired secretion but full anticoagulant activity. This study is the largest investigation of ProS deficiency in China and the first investigation of the influence of Type I ProS missense mutations on the global level of coagulation function. The p.K196E mutation, which is common in the neighboring Japanese population, was not found in our Chinese population, and null mutations were common in our Chinese population but not common in Japan. Further genetic analysis is warranted to understand the causes of ProS deficiency in patients without a genetic explanation.

Asunto(s)

Proteínas Sanguíneas/genética , Mutación Missense , Deficiencia de Proteína S/genética , Adulto , Sustitución de Aminoácidos , Pueblo Asiatico , Proteínas Sanguíneas/metabolismo , China , Análisis Mutacional de ADN , Femenino , Células HEK293 , Humanos , Japón , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa Multiplex , Proteína C/genética , Proteína C/metabolismo , Proteína S , Deficiencia de Proteína S/etnología , Deficiencia de Proteína S/metabolismo

RESUMEN

OBJECTIVES: The aim of this study was to examine the effect of phosphoenolpyruvate (PEP), a glycolytic intermediate, on organ damage during cold preservation of liver. METHODS: An ex-vivo mouse liver cold-preservation model and an in-vitro liver injury model induced by hydrogen peroxide in HepG2 cells were leveraged. KEY FINDINGS: PEP attenuated the elevation of aminotransferases and lactate dehydrogenase leakage during organ preservation, histological changes and changes in oxidative stress parameters (measured as thiobarbituric acid reactive substance and glutathione content) induced by 72 h of cold preservation of the liver. The effects were comparable with the University of Wisconsin solution, a gold standard organ preservation agent. The decrease in ATP content in liver during the cold preservation was attenuated by PEP treatment. PEP prevented the cellular injury and increases in intracellular reactive oxygen species in HepG2 cells. In addition, PEP scavenged hydroxyl radicals, but had no effect on superoxide anion as evaluated by an electron paramagnetic resonance spin-trapping technique. CONCLUSIONS: PEP significantly attenuated the injury, oxidative stress and ATP depletion in liver during cold preservation. The antioxidative potential of PEP was confirmed by in-vitro examination. We suggest that PEP acts as a glycolytic intermediate and antioxidant, and is particularly useful as an organ preservation agent in clinical transplantation.

Asunto(s)

Antioxidantes , Criopreservación/métodos , Crioprotectores , Hígado , Preservación de Órganos/métodos , Fosfoenolpiruvato , Adenosina Trifosfato/metabolismo , Animales , Línea Celular Tumoral , Glucólisis , Células Hep G2 , Humanos , Peróxido de Hidrógeno/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismo , Transaminasas/metabolismo

RESUMEN

Thrombophilia that is common among Caucasians is caused by genetic polymorphisms of coagulation factor V Leiden (R506Q) and prothrombin G20210A. Unlike that in Caucasians, thrombophilia that is common in the Japanese and Chinese involve dysfunction of the activated protein C (APC) anticoagulant system caused by abnormal protein S and protein C molecules. Approximately 50% of Japanese and Chinese individuals who develop venous thrombosis have reduced activities of protein S. The abnormal sites causing the protein S molecule abnormalities are distributed throughout the protein S gene, PROS1. One of the most common abnormalities is protein S Tokushima (K155E), which accounts for about 30% of the protein S molecule abnormalities in the Japanese. Whether APC dysfunction occurs in other Asian countries is an important aspect of mapping thrombophilia among Asians. International surveys using an accurate assay system are needed to determine this.

Asunto(s)

Proteína C/metabolismo , Trombofilia/etiología , Pueblo Asiatico , Coagulación Sanguínea , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Humanos , Proteína C/genética , Proteína S/química , Proteína S/genética , Proteína S/metabolismo , Trombofilia/epidemiología , Trombosis de la Vena/etiología , Trombosis de la Vena/genética

RESUMEN

We evaluated the usefulness of phosphoenolpyruvate (PEP), a glycolytic intermediate with antioxidative and energy supplementation potentials, as an organ preservation agent. Using ex vivo mouse liver and kidney of a static cold storage model, we compared the effects of PEP against organ damage and oxidative stress during cold preservation with those of glucose or N-acetylcysteine (NAC). Lactate dehydrogenase (LDH) leakage, histological changes, and oxidative stress parameters (measured as thiobarbituric acid reactive substance and glutathione content) were determined. PEP (100 mM) significantly prevented an increase in LDH leakage, histological changes, such as tubulonecrosis and vacuolization, and changes in oxidative stress parameters during 72 h of cold preservation in mouse liver. Although glucose (100 mM) partly prevented LDH leakage and histological changes, no effects against oxidative stress were observed. By contrast, NAC inhibited oxidative stress in the liver and did not prevent LDH leakage or histological changes. PEP also significantly prevented kidney damage during cold preservation in a dose-dependent manner, and the protective effects were superior to those of glucose and NAC. We suggest that PEP, a functional carbohydrate with organ protective and antioxidative activities, may be useful as an organ preservation agent in clinical transplantation.

Asunto(s)

Ácido Eicosapentaenoico/farmacología , Fibrinolíticos/farmacología , Infarto/prevención & control , Cola (estructura animal)/irrigación sanguínea , Tromboembolia Venosa/prevención & control , Administración Oral , Animales , Carragenina , Modelos Animales de Enfermedad , Ácido Eicosapentaenoico/administración & dosificación , Fibrinolíticos/administración & dosificación , Infarto/sangre , Infarto/inducido químicamente , Ratas , Tromboembolia Venosa/sangre , Tromboembolia Venosa/inducido químicamente

RESUMEN

This study examined the cytoprotective and anti-oxidative properties of phosphoenolpyruvic acid (PEP), a glycolysis metabolite with a high-energy phosphate group. PEP (0.1-10 mM) significantly attenuated the decrease in cell viability induced by hydrogen peroxide (H(2)O(2)) in HeLa cells in a dose-dependent manner. PEP also inhibited the decrease in calcein-acetomethoxy-stained cells and the increase in propidium iodide-stained cells that were induced by H(2)O(2). The H(2)O(2)-stimulated increase in intracellular reactive oxygen species was significantly reduced by PEP. PEP also demonstrated scavenging potential against hydroxyl radicals, as assessed by the electron paramagnetic resonance method. In addition, PEP demonstrated scavenging potential against the 1,1-diphenyl-2-picrylhydrazyl radical, a representative artificial radical, although the potential is very weak. PEP (10 mM) slightly inhibited the decrease in cellular ATP content induced by H(2)O(2), but did not show any effects at low doses (0.1, 1 mM). PEP (0.1-10 mM) also attenuated the cell injury but not the decrease in intracellular ATP content, induced by 2-deoxy-D-glucose, a glycolysis inhibitor. These results indicate that PEP exerts cytoprotective effects and has anti-oxidative potential, although the precise cytoprotective mechanisms are not fully elucidated. We suggest that PEP is a functional carbohydrate metabolite with cytoprotective and anti-oxidative activity, and is potentially useful as a therapeutic agent against diseases that involve the oxidative stress.

Asunto(s)

Antioxidantes/farmacología , Citoprotección/efectos de los fármacos , Fosfoenolpiruvato/análogos & derivados , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Supervivencia Celular/efectos de los fármacos , Desoxiglucosa/farmacología , Glucólisis , Células HeLa , Humanos , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacología , Fosfoenolpiruvato/farmacología

RESUMEN

Venous thromboembolism (VTE) incidence is rising rapidly in Japan with lifestyle westernization and aging. Deficiency of protein S, an important blood coagulation regulator, is a risk factor for VTE. Protein S deficiency prevalence in Asians is approximately 10 times that in Caucasians and that of protein S type II deficiency, associated with the protein S Tokushima mutation (K155E), is quite high in Japan. However, currently available methods for measuring protein S are not precise enough for detection of this deficiency. We developed a novel assay system for precise simultaneous determinations of total protein S activity and total protein S antigen level, using a general-purpose automated analyzer, allowing protein S-specific activity (ratio of total protein S activity to total protein S antigen level) to be calculated. Mean specific activity was 0.99 for samples from healthy individuals but 0.69 or less (mean-3SD) in protein S type II-deficient and warfarin-treated samples, but was 1.0 in an estrogen-treated sample with significantly decreased protein S antigen. Protein S gene analyses in healthy individuals with specific activity 0.69 or less revealed the K155E mutation in all three. These results show our new assay system to be an effective screening tool for protein S type II deficiency. This system can also be used in an automated analyzer, facilitating numerous sample measurements, and is, thus, applicable to regular medical checkups and diagnosing VTE. Such applications would potentially contribute to early detection of protein S type II deficiency, and, thereby, to thrombosis prevention.

Asunto(s)

Deficiencia de Proteína S/sangre , Proteína S/análisis , Estudios de Casos y Controles , Colorimetría/métodos , Femenino , Humanos , Incidencia , Masculino , Tamizaje Masivo , Deficiencia de Proteína S/diagnóstico , Factores de Riesgo , Sensibilidad y Especificidad , Trombosis de la Vena/sangre

RESUMEN

In our previous paper, we demonstrated that Arg 901 in the C-terminal tail of human AE1 (band 3, anion exchanger 1) had a functional role in conformational change during anion exchange. To further examine how Arg 901 is involved in conformational change, we expressed various Arg 901 mutants and alanine mutants of the C-terminal tail (from Leu 886 to Val 911) on the plasma membrane of Saccharomyces cerevisiae and evaluated the kinetic parameters of sulfate ion transport. As a result, Vmax decreased as the hydrophobicities of the 901st and peripheral hydrophilic residues increased, indicating that the hydrophobicity of the C-terminal residue is involved in the conformational change. We also found the alkali and protease resistance of the C-terminal region after Arg 901 modification with hydroxyphenylglyoxal (HPG) or phenylglyoxal (PG), a hydrophobic reagent. These results suggested that the increased hydrophobicity of the C-terminal region around Arg 901 leads to inefficient conformational change by the newly produced hydrophobic interaction.

Asunto(s)

Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Arginina/metabolismo , Membrana Celular/metabolismo , Proteína 1 de Intercambio de Anión de Eritrocito/química , Proteína 1 de Intercambio de Anión de Eritrocito/genética , Arginina/química , Arginina/genética , Membrana Celular/química , Membrana Celular/genética , Expresión Génica , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Transporte Iónico/fisiología , Cinética , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae

RESUMEN

We recently published the three-dimensional structure of the membrane domain of human erythrocyte anion exchanger 1 (AE1) at 7.5 Å resolution, solved by electron crystallography. The structure exhibited distinctive anti-parallel V-shaped motifs, which protrude from the membrane bilayer on both sides. Similar motifs exist in the previously reported structure of a bacterial chloride channel (ClC)-type protein. Here, we propose two topology models of AE1 that reflect the anti-parallel V-shaped structural motifs. One is assumed to have structural similarity with the ClC protein and the other is only assumed to have internal repeats, as is often the case with transporters. Both models are consistent with most topological results reported thus far for AE1, each having advantages and disadvantages.

Asunto(s)

Proteína 1 de Intercambio de Anión de Eritrocito/ultraestructura , Cristalografía/métodos , Microscopía Electrónica/métodos , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Proteína 1 de Intercambio de Anión de Eritrocito/genética , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Reproducibilidad de los Resultados

RESUMEN

UNLABELLED: INTRODUATION: Resveratrol, a phytoestrogen present at a high concentration in red wine, has been reported to possess many health benefit effects that are protective against age-related diseases. Protein S (PS), an important anticoagulant factor in the protein C (PC) anticoagulant pathway, is mainly synthesized by hepatocytes, and its plasma level is decreased in high-estrogen conditions such as pregnancy and oral contraceptive use. The aim of this study was to investigate whether resveratrol affects PS expression in HepG2 cells. MATERIALS AND METHODS: The secreted and intracellular levels of PS were determined by an enzyme-linked ligandsorbent assay and Western blotting. The mRNA expressions of PS, PC and ß chain of C4b-binding protein (C4BP-ß) were analyzed by reverse transcription-polymerase chain reaction. The PS gene promotor activities in HepG2 cells transiently expressing estrogen receptor (ER) α were examined by a luciferase reporter assay. RESULTS: Resveratrol dose- and time-dependently down-regulated the PS expression in HepG2 cells at a transcriptional level, resulting in a significant decrease in secreted PS; however, the PC and C4BP-ß mRNA expressions were not affected. This action of resveratrol was not mediated through either the ER signaling or those of mitogen-activated protein kinases and protein kinase C. Piceatannol, a hydroxylated metabolite of resveratrol, and genistein, an isoflavone found in soy products, also down-regulated the PS expression. CONCLUSIONS: Resveratrol down-regulates the PS expression in HepG2 cells in an ER-independent manner, and the two phenolic hydroxyls at carbon-3 and -5 of resveratrol may be involved in this function.

Asunto(s)

Proteínas Sanguíneas/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Fitoestrógenos/farmacología , Proteína S/metabolismo , Estilbenos/farmacología , Vino , Proteínas Sanguíneas/genética , Western Blotting , Carcinoma Hepatocelular/genética , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Receptor alfa de Estrógeno/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genisteína/farmacología , Células Hep G2 , Antígenos de Histocompatibilidad/metabolismo , Humanos , Neoplasias Hepáticas/genética , Estructura Molecular , Fitoestrógenos/química , Regiones Promotoras Genéticas/efectos de los fármacos , Proteína C/metabolismo , Proteína S/genética , ARN Mensajero/metabolismo , Resveratrol , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estilbenos/química , Relación Estructura-Actividad , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Transfección

RESUMEN

BACKGROUND: Immunoassays are one main detection system used in the field of clinical chemistry. Recent developments of a new detection method utilizing a magnetic marker and magnetic sensor have enabled rapid and sensitive immunoassay without the need for bound/free (BF) separation. METHODS: Newly-synthesized conjugated avidin was used as the magnetic marker for quantitative analysis of human interleukin-8 (hIL-8) and immunoglobulin E (hIgE) in several media. A superconducting quantum interference device sensor detected the magnetic fields from markers fixed to antigens by the sandwich method. Magnetic signals from unbound markers were nearly zero due to Brownian rotation. RESULTS: Our magnetic immunoassay could detect four attomoles of model proteins (hIL-8, hIgE) in phosphate buffer without BF separation. Using our standard curve, the range of protein detected ranged from 40 femtomoles to 4 attomoles, and we observed a strong association between protein amounts and magnetic signals from the bound markers. The homogeneous immunoassay could also quantify three hundred cells from the fungus Candida albicans in phosphate buffer. CONCLUSIONS: The present study demonstrates the ability of magnetic markers for measuring biological targets without BF separation. This detection system has great potential for use as the next generation's analytical system.

Asunto(s)

Inmunoensayo/métodos , Inmunoensayo/normas , Magnetismo , Animales , Avidina/química , Candida albicans/aislamiento & purificación , Humanos , Inmunoensayo/instrumentación , Inmunoglobulina E/análisis , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Interleucina-8/análisis , Interleucina-8/sangre , Interleucina-8/inmunología , Nanoconjugados/química , Fosfatos/química , Estándares de Referencia , Saccharomyces cerevisiae/aislamiento & purificación , Temperatura

RESUMEN

Anion exchanger 1 (AE1 or band 3) is responsible for Cl(-)-HCO3(-) exchange on erythrocyte membrane. Previously, we showed that band 3 is fixed in an inward-facing conformation by specific modification of His 834 with DEPC, resulting in a strong inhibition of its anion transport activity. To clarify the physiological role of His 834, we evaluated the sulfate transport activities of various band 3 mutants: different mutants at His 834 and alanine mutants of peripheral residues around 834 (Lys 829-Phe 836) in yeast cell membranes. The K(m) values of the His 834 mutants were 4-10 times higher than that of the wild type, while their V(max) values were barely lower than that of wild type. Meanwhile, the K(m) values of the peripheral alanine mutants were only slightly increased. These data suggest that His 834 is critically important for the efficient binding of sulfate anion, but not for the conformational change induced by substrate binding.

Asunto(s)

Proteína 1 de Intercambio de Anión de Eritrocito , Histidina/genética , Mutación , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Proteína 1 de Intercambio de Anión de Eritrocito/química , Proteína 1 de Intercambio de Anión de Eritrocito/genética , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Histidina/metabolismo , Humanos , Datos de Secuencia Molecular , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sulfatos/metabolismo