RESUMEN

The correlation between protein molecular weight and the number of lysine or basic amino acid residues was found to be high for broad range molecular weight standards, subunits of Escherichia coli F1F0-ATP synthase and the translated open reading frame of E. coli. A relatively poor correlation between protein molecular weight and the number of cysteine residues was observed in all cases. The ability of amine-reactive, thiol-reactive and basic amino acid-binding fluorophores to detect the eight subunits of F1F0-ATP synthase complex was assessed using 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), monobromobimane (MBB) and SYPRO Ruby protein gel stain, respectively. Though experimentally none of the fluorophores provided accurate estimates of the subunit stoichiometry of this complex, MDPF and SYPRO Ruby protein gel stain were capable of semiquantitative detection of every subunit. MBB, however, failed to detect subunits a, b and c of the hydrophobic F0 complex, as well as subunit epsilon of the F1 complex. All three fluorescent detection procedures permitted subsequent identification of representative subunits by peptide mass profiling using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The use of thiol-reactive fluorophores for the global analysis of protein expression profiles does not appear to be advisable as a significant number of proteins have few or no cysteine residues, thus escaping detection.

Asunto(s)

ATPasas de Translocación de Protón Bacterianas/aislamiento & purificación , Escherichia coli/enzimología , Colorantes Fluorescentes , Secuencia de Aminoácidos , ATPasas de Translocación de Protón Bacterianas/química , ATPasas de Translocación de Protón Bacterianas/genética , Compuestos Bicíclicos con Puentes , Cisteína/química , Escherichia coli/genética , Furanos , Genoma Bacteriano , Datos de Secuencia Molecular , Peso Molecular , Subunidades de Proteína , Espectrometría de Fluorescencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

The fluorescent hydrazide, Pro-Q Emerald 300 dye, may be conjugated to glycoproteins by a periodic acid Schiff's (PAS) mechanism. The glycols present in glycoproteins are initially oxidized to aldehydes using periodic acid. The dye then reacts with the aldehydes to generate a highly fluorescent conjugate. Reduction with sodium metabisulfite or sodium borohydride is not required to stabilize the conjugate. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and glycoproteins may be visualized within 2-4 h of electrophoresis. This is substantially more rapid than PAS labeling with digoxigenin hydrazide followed by detection with an antidigoxigenin antibody conjugate of alkaline phosphatase, or PAS labeling with biotin hydrazide followed by detection with horseradish peroxidase or alkaline phosphatase conjugates of streptavidin, which require more than eight hours to complete. Pro-Q Emerald 300 dye-labeled gels and blots may be poststained with SYPRO Ruby dye, allowing sequential two-color detection of glycosylated and nonglycosylated proteins. Both fluorophores are excited with mid-range UV illumination. Pro-Q Emerald 300 dye maximally emits at 530 nm (green) while SYPRO Ruby dye maximally emits at 610 nm (red). As little as 300 pg of alpha 1-acid glycoprotein (40% carbohydrate) and 1 ng of glucose oxidase (12% carbohydrate) or avidin (7% carbohydrate) are detectable in gels after staining with Pro-Q Emerald 300 dye. Besides glycoproteins, as little as 2-4 ng of lipopolysaccharide is detectable in gels using Pro-Q Emerald 300 dye while 250-1000 ng is required for detection with conventional silver staining. Detection of glycoproteins may be achieved in sodium dodecyl sulfate-polyacrylamide gels, two-dimensional gels and on polyvinylidene difluoride membranes.

Asunto(s)

Electroforesis en Gel de Poliacrilamida/métodos , Glicoproteínas/aislamiento & purificación , Animales , Avidina/aislamiento & purificación , Electroforesis en Gel Bidimensional/métodos , Colorantes Fluorescentes , Glucosa Oxidasa/aislamiento & purificación , Glicoconjugados/aislamiento & purificación , Glicosilación , Focalización Isoeléctrica/métodos , Nanotecnología , Orosomucoide/aislamiento & purificación , Proteoma/aislamiento & purificación

RESUMEN

SYPRO Rose Plus protein blot stain is an improved europium-based metal chelate stain for the detection of proteins on nitrocellulose and poly(vinylidene difluoride) (PVDF) membranes. Staining is achieved without covalently modifying the proteins. The stain may be excited with a 254 nm (UV-C), 302 nm (UV-B), or 365 nm (UV-A) light source and displays a sharp emission maximum at 612 nm. The emission peak has a full width at half-maximum of only 8 nm. The stain exhibits exceptional photostability, allowing long exposure times for maximum sensitivity. Since the dye is composed of a europium complex, it has a long emission lifetime, potentially allowing time-resolved detection, greatly reducing background fluorescence. Proteins immobilized to a nitrocellulose or PVDF membrane by electroblotting, dot-blotting, or vacuum slot-blotting are incubated with SYPRO Rose Plus protein blot stain for 15-30 min. Membranes are rinsed briefly, visualized with UV epi-illumination and the luminescence of the europium dye is measured using a 490 nm long-pass or 625 +/- 15 nm band-pass filter in combination with a conventional photographic or charge-coupled device (CCD) camera system. Alternatively, the dye may be visualized using a xenon-arc illumination source. The stain is readily removed from proteins by incubating membranes at mildly alkaline pH. The reversibility of the protein staining procedure allows for subsequent biochemical analyses, such as immunoblotting and biotin-streptavidin detection using colorimetric, direct fluorescence or fluorogenic visualization methods.

Asunto(s)

Colodión , Colorantes , Europio , Polivinilos , Proteínas/análisis , Western Blotting , Electroforesis/métodos , Immunoblotting , Mediciones Luminiscentes , Sensibilidad y Especificidad , Espectrometría de Fluorescencia , Coloración y Etiquetado/métodos

RESUMEN

A two-color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY FL-X to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl(DDAO)-phosphate in combination with alkaline-phosphatase-conjugated reporter molecules. This results in all proteins in the profile being visualized as green signal while those detected specifically with the alkaline-phosphatase conjugate appear as red signal. The dichromatic detection system is broadly compatible with a wide range of analytical imaging devices including UV epi- or transilluminators combined with photographic or charge-coupled device (CCD) cameras, xenon-arc sources equipped with appropriate excitation/emission filters, and dual laser gel scanners outfitted with a 473 nm second-harmonic generation or 488 nm argon-ion laser as well as a 633 nm helium-neon or 635 nm diode laser. The dichromatic detection method permits detection of low nanogram amounts of protein and allows for unambiguous identification of target proteins relative to the entire protein profile on a single electroblot, obviating the need to run replicate gels that would otherwise require visualization of total proteins by silver staining and subsequent alignment with chemiluminescent or colorimetric signals generated on electroblots.

Asunto(s)

Western Blotting/métodos , Colorantes Fluorescentes , Proteínas/análisis , Espectrometría de Fluorescencia , Acridinas/química , Fosfatasa Alcalina , Animales , Compuestos de Boro/química , Química Encefálica , Ácidos Carboxílicos , Bovinos , Proteínas del Huevo/análisis , Electroforesis/métodos , Fibroblastos/química , Glicoproteínas/análisis , Indicadores y Reactivos , Fosfatos , Polivinilos , Ratas , Tubulina (Proteína)/análisis

RESUMEN

As mitochondria play critical roles in both cell life and cell death, there is great interest in obtaining a human mitochondrial proteome map. Such a map could potentially be useful in diagnosing diseases, identifying targets for drug therapy, and in screening for unwanted drug side effects. In this paper, we present a novel approach to obtaining a human mitochondrial proteome map that combines sucrose gradient centrifugation with standard two-dimensional gel electrophoresis. The resulting three-dimensional separation of proteins allows us to address some of the problems encountered during previous attempts to obtain mitochondrial proteome maps such as resolution of proteins and solubility of hydrophobic proteins during isoelectric focusing. In addition, we show that this new approach provides functional information about protein complexes within the organelle that is not obtained with two-dimensional gel electrophoresis of whole mitochondria.

Asunto(s)

Centrifugación por Gradiente de Densidad , Electroforesis en Gel Bidimensional/métodos , Mitocondrias/química , Proteínas/análisis , Adenosina Trifosfato/metabolismo , Animales , Western Blotting , Encéfalo/ultraestructura , Bovinos , Fraccionamiento Celular , Línea Celular , Creatina Quinasa/metabolismo , Fibroblastos/ultraestructura , Humanos , Hidrólisis , Mitocondrias Cardíacas/química , Mapeo Peptídico , Proteínas/aislamiento & purificación

RESUMEN

A dichromatic method for measuring the specific activity of beta-glucuronidase from complex cell homogenates or partially purified protein fractions is presented. Dual fluorescence is achieved by using the green emitting fluorogenic substrate ELF 97 beta-D-glucuronide to detect beta-glucuronidase activity, followed by the red emitting SYPRO Ruby protein gel stain or SYPRO Ruby IEF gel stain to detect the remaining proteins in the electrophoretic profile. Both ELF 97 alcohol, the highly fluorescent hydrolytic product generated from the enzyme substrate, and the SYPRO Ruby total protein stains are maximally excited by ultraviolet illumination. ELF 97 alcohol emits maximally at 525 nm while the SYPRO Ruby dyes emit maximally at 610 nm. Since ELF 97 beta-glucuronide is a precipitating substrate, it allows precise localization of beta-glucuronidase activity with minimal band diffusion. The staining method is simple and direct, without the requirement for ancillary coupling reactions. Dichromatic protein detection is demonstrated after sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis, carrier ampholyte-mediated isoelectric focusing or two-dimensional gel electrophoresis.

Asunto(s)

Electroforesis/métodos , Colorantes Fluorescentes , Glucuronidasa/análisis , Proteínas/análisis , Espectrometría de Fluorescencia/métodos , Electroforesis en Gel Bidimensional/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Glucuronatos/metabolismo , Focalización Isoeléctrica/métodos

RESUMEN

Protein distribution profiles may be used to characterize both physiological and pathophysiological cellular changes, but rigorous biochemical assays for measuring such movements are lacking. This paper reports on a protein redistribution assay that combines reversible metal chelate-based total protein detection with a four-fraction subcellular detergent fractionation procedure. TNF-alpha stimulated cultured human omental microvessel endothelial cells are fractionated into cytosol, membrane/organelle, nuclear (envelope and associated), and cytoskeletal/DNA compartments. Protein fractions are separated electrophoretically and electroblotted or slot-blotted onto PVDF membranes without electrophoretic separation. A key feature is that total protein is measured and analyzed directly on the resultant PVDF membrane, using a Ferrozine/ferrous metal-chelate stain, without the added step of a prior solution-phase protein assay. As a result, factors that may adversely affect NFkappaB quantification, such as saturation of the solid-support membrane, are rigorously evaluated and controlled. Following removal of the Ferrozine/ferrous total protein stain, NFkappaB distribution is determined via standard immunodetection procedures. This assay reveals a new level of complexity regarding NFkappaB distribution and translocation. NFkappaB is shown to translocate from the cytosol to the membrane/organelle and cytoskeletal/DNA fractions, whereas trace levels of NFkappaB are observed in the nuclear (envelope and associated) fraction. Dose-curve analysis reveals that the response is initiated at 10 U/ml of TNF-alpha, plateaus at approximately 1000 U/ml, and remains essentially constant up to 2000 U/ml. Time-course analysis demonstrates a measurable response as early as 5 min and a peak response at approximately 30 min, after which the distribution begins to return to baseline. The assay should provide a valuable tool for rapid evaluation and mechanistic studies of NFkappaB redistribution.

Asunto(s)

Transporte Activo de Núcleo Celular , Endotelio Vascular/metabolismo , Immunoblotting/métodos , FN-kappa B/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Western Blotting , Fraccionamiento Celular , Células Cultivadas , Detergentes , Relación Dosis-Respuesta a Droga , Electroforesis , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Ferrozina/metabolismo , Humanos , FN-kappa B/análisis , Epiplón/citología , Epiplón/efectos de los fármacos , Epiplón/metabolismo , Fracciones Subcelulares/química , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Factores de Tiempo , Factor de Necrosis Tumoral alfa/farmacología

RESUMEN

SYPRO Ruby dye is a permanent stain comprised of ruthenium as part of an organic complex that interacts noncovalently with proteins. SYPRO Ruby Protein Gel Stain provides a sensitive, gentle, fluorescence-based method for detecting proteins in one-dimensional and two-dimensional sodium dodecyl sulfate-polyacrylamide gels. Proteins are fixed, stained from 3h to overnight and then rinsed in deionized water or dilute methanol/acetic acid solution for 30 min. The stain can be visualized using a wide range of excitation sources commonly used in image analysis systems including a 302 nm UV-B transilluminator, 473 nm second harmonic generation (SHG) laser, 488 nm argon-ion laser, 532 nm yttrium-aluminum-garnet (YAG) laser, xenon arc lamp, blue fluorescent light bulb or blue light-emitting diode (LED). The sensitivity of SYPRO Ruby Protein Gel Stain is superior to colloidal Coomassie Brilliant Blue (CBB) stain or monobromobimane labeling and comparable with the highest sensitivity silver or zinc-imidazole staining procedures available. The linear dynamic range of SYPRO Ruby Protein Gel stain extends over three orders of magnitude, which is vastly superior to silver, zinc-imidazole, monobromobimane and CBB stain. The fluorescent stain does not contain superfluous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. While peptide mass profiles are severely altered in protein samples prelabeled with monobromobimane, successful identification of proteins by peptide mass profiling using matrix-assisted laser desorption/ionization mass spectrometry was easily performed after protein detection with SYPRO Ruby Protein Gel stain.

Asunto(s)

Dextranos , Colorantes Fluorescentes , Proteínas/análisis , Rodaminas , Rutenio , Coloración y Etiquetado/métodos , Electroforesis en Gel Bidimensional/métodos , Geles , Mediciones Luminiscentes , Colorantes de Rosanilina , Sensibilidad y Especificidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos

RESUMEN

Proteomics investigations endeavor to provide a global understanding of gene product synthesis rate, degradation rate, functional competence, posttranslational modification, subcellular distribution and physical interactions with other cell components. Protein expression encompasses an enormous dynamic range. Since rare proteins cannot be amplified by any type of PCR method, sensitive detection is critical to proteome projects. Fluorescence methods deliver streamlined detection protocols, superior detection sensitivity, broad linear dynamic range and excellent compatibility with modern microchemical identification methods such as mass spectrometry. Two general approaches to fluorescence detection of proteins are currently practiced: the covalent derivatization of proteins with fluorophores or noncovalent interaction of fluorophores either via the SDS micelle or through direct electrostatic interaction with proteins. One approach for quantifying fluorescence is to use a photomultiplier tube detector combined with a laser light scanner. In addition, fluorescence imaging is performed using a charge-coupled device camera combined with a UV light or xenon arc source. Fluorescent dyes with bimodal excitation spectra may be broadly implemented on a wide range of analytical imaging devices, permitting their widespread application to proteomics studies and incorporation into semiautomated analysis environments.

Asunto(s)

Colorantes Fluorescentes , Procesamiento de Imagen Asistido por Computador/instrumentación , Biología Molecular/instrumentación , Robótica/instrumentación , Animales , Expresión Génica , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Biología Molecular/métodos , Proteoma/análisis , Proteoma/genética , Robótica/métodos

RESUMEN

As proteomics evolves into a high-throughput technology for the study of global protein regulation, new demands are continually being placed upon protein visualization and quantitation methods. Chief among these are increased detection sensitivity, broad linear dynamic range and compatibility with modern methods of microchemical analyses. The limitations of conventional protein staining techniques are increasingly being encountered as high sensitivity electrophoresis methods are interfaced with automated gel stainers, image analysis workstations, robotic spot excision instruments, protein digestion work stations, and mass spectrometers. Three approaches to fluorescence detection of proteins in two-dimensional (2-D) gels are currently practiced: covalent derivatization of proteins with fluorophores, intercalation of fluorophores into the sodium dodecyl sulfate (SDS) micelle, and direct electrostatic interaction with proteins by a Coomassie Brilliant Blue-type mechanism. This review discusses problems encountered in the analysis of proteins visualized with conventional stains and addresses advances in fluorescence protein detection, including immunoblotting, as well as the use of charge-coupled device (CCD) camera-based and laser-scanner-based image acquisition devices in proteomics.

Asunto(s)

Electroforesis en Gel Bidimensional/métodos , Colorantes Fluorescentes , Proteoma/análisis , Animales , Fluorescencia , Humanos , Mediciones Luminiscentes , Coloración y Etiquetado/métodos

RESUMEN

SYPRO Ruby IEF Protein Gel Stain is an ultrasensitive, luminescent stain optimized for the analysis of protein in isoelectric focusing gels. Proteins are stained in a ruthenium-containing metal complex overnight and then rinsed in distilled water for 2 h. Stained proteins can be excited by ultraviolet light of about 302 nm (UV-B transilluminator) or with visible light of about 470 nm. Fluorescence emission of the dye is maximal at approximately 610 nm. The sensitivity of the SYPRO Ruby IEF protein gel stain is superior to colloidal Coomassie blue stain and the highest sensitivity silver staining procedures available. The SYPRO Ruby IEF protein gel stain is suitable for staining proteins in nondenaturing or denaturing carrier ampholyte isoelectric focusing and immobilized pH gradient gel electrophoresis. The stain is compatible with N,N'-methylenebisacrylamide or piperazine diacylamide cross-linked polyacrylamide gels as well as with agarose gels and high tensile strength Duracryl gels. The stain does not contain extraneous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. Successful identification of stained proteins by peptide mass profiling is demonstrated.

Asunto(s)

Quelantes/química , Focalización Isoeléctrica/métodos , Proteínas/análisis , Rutenio/química , Secuencia de Aminoácidos , Colorantes , Colorantes Fluorescentes , Datos de Secuencia Molecular , Peso Molecular , Mapeo Peptídico , Proteínas/química , Sensibilidad y Especificidad , Espectrometría de Fluorescencia

RESUMEN

SYPRO Tangerine stain is an environmentally benign alternative to conventional protein stains that does not require solvents such as methanol or acetic acid for effective protein visualization. Instead, proteins can be stained in a wide range of buffers, including phosphate-buffered saline or simply 150 mM NaCl using an easy, one-step procedure that does not require destaining. Stained proteins can be excited by ultraviolet light of about 300 nm or with visible light of about 490 nm. The fluorescence emission maximum of the dye is approximately 640 nm. Noncovalent binding of SYPRO Tangerine dye is mediated by sodium dodecyl sulfate (SDS) and to a lesser extent by hydrophobic amino acid residues in proteins. This is in stark contrast to acidic silver nitrate staining, which interacts predominantly with lysine residues or Coomassie Blue R, which in turn interacts primarily with arginine and lysine residues. The sensitivity of SYPRO Tangerine stain is similar to that of the SYPRO Red and SYPRO Orange stains - about 4-10 ng per protein band. This detection sensitivity is comparable to colloidal Coomassie blue staining and rapid silver staining procedures. Since proteins stained with SYPRO Tangerine dye are not fixed, they can easily be eluted from gels or utilized in zymographic assays, provided that SDS does not inactivate the protein of interest. This is demonstrated with in-gel detection of rabbit liver esterase activity using alpha-naphthyl acetate and Fast Blue BB dye as well as Escherichia coli beta-glucuronidase activity using ELF-97 beta-D-glucuronide. The dye is also suitable for staining proteins in gels prior to their transfer to membranes by electroblotting. Gentle staining conditions are expected to improve protein recovery after electroelution and to reduce the potential for artifactual protein modifications such as the alkylation of lysine and esterification of glutamate residues, which complicate interpretation of peptide fragment profiles generated by mass spectrometry.

Asunto(s)

Electroforesis en Gel de Poliacrilamida/métodos , Cloruro de Sodio/química , Dodecil Sulfato de Sodio/química , Espectrometría de Fluorescencia/métodos , Secuencia de Aminoácidos , Animales , Western Blotting , Colorantes Fluorescentes , Datos de Secuencia Molecular , Conejos , Soluciones , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

Proteomic projects are often focused on the discovery of differentially expressed proteins between control and experimental samples. Most laboratories choose the approach of running two-dimensional (2-D) gels, analyzing them and identifying the differentially expressed proteins by in-gel digestion and mass spectrometry. To date, the available stains for visualizing proteins on 2-D gels have been less than ideal for these projects because of poor detection sensitivity (Coomassie blue stain) or poor peptide recovery from in-gel digests and mass spectrometry (silver stain), unless extra destaining and washing steps are included in the protocol. In addition, the limited dynamic range of these stains has made it difficult to rigorously and reliably determine subtle differences in protein quantities. SYPRO Ruby Protein Gel Stain is a novel, ruthenium-based fluorescent dye for the detection of proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels that has properties making it well suited to high-throughput proteomics projects. The advantages of SYPRO Ruby Protein Gel Stain relative to silver stain demonstrated in this study include a broad linear dynamic range and enhanced recovery of peptides from in-gel digests for matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry.

Asunto(s)

Dextranos , Colorantes Fluorescentes , Proteínas/análisis , Rodaminas , Compuestos de Rutenio , Tinción con Nitrato de Plata/métodos , Coloración y Etiquetado/métodos , Resinas Acrílicas , Animales , Electroforesis en Gel Bidimensional/métodos , Mapeo Peptídico/métodos , Péptidos , Ratas , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Tripsina

RESUMEN

SYPRO Ruby protein blot stain provides a sensitive, gentle, fluorescence-based method for detecting proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby dye is a permanent stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Stained proteins can be excited by ultraviolet light of about 302 nm or with visible light of about 470 nm. Fluorescence emission of the dye is approximately 618 nm. The stain can be visualized using a wide range of excitation sources utilized in image analysis systems including a UV-B transilluminator, 488-nm argon-ion laser, 532-nm yttrium-aluminum-garnet (YAG) laser, blue fluorescent light bulb, or blue light-emitting diode (LED). The detection sensitivity of SYPRO Ruby protein blot stain (0.25-1 ng protein/mm(2)) is superior to that of amido black, Coomassie blue, and india ink staining and nearly matches colloidal gold staining. SYPRO Ruby protein blot stain visualizes proteins more rapidly than colloidal gold stain and the linear dynamic range is more extensive. Unlike colloidal gold stain, SYPRO Ruby protein blot stain is fully compatible with subsequent biochemical applications including colorimetric and chemiluminescent immunoblotting, Edman-based sequencing and mass spectrometry.

Asunto(s)

Colorantes , Proteínas/análisis , Rutenio , Colodión , Colorantes Fluorescentes , Immunoblotting , Mediciones Luminiscentes , Espectrometría de Masas , Membranas Artificiales , Polivinilos , Espectrometría de Fluorescencia , Coloración y Etiquetado/métodos

RESUMEN

Vascular pathologies induced by ischemia/reperfusion involve the production of reactive oxygen species (ROS) that in part cause tissue injury. The production of ROS that occurs upon reperfusion activates specific second messenger pathways. In diabetic retinopathy there is a characteristic loss of the microvascular pericyte. Pericytes are more sensitive than endothelial cells to low concentrations of ROS, such as hydrogen peroxide (H(2)O(2)) when tested in vitro. Whether the pericyte loss is due to toxic cell death triggered by the noxious H(2)O(2) or apoptosis, due to activation of specific second messenger pathways, is unknown. During apoptosis, a cell's nucleus and cytoplasm condense, the cell becomes fragmented, and ultimately forms apoptotic bodies. It is generally assumed that apoptosis depends on nuclear signaling, but cytoplasmic morphological processes are not well described. We find that exposing cultured retinal pericytes to 100 microM H(2)O(2) for 30 min leads to myosin heavy chain translocation from the cytosol to the cytoskeleton and a significant decrease in cell surface area. Pericyte death follows within 60-120 min. Exposing cells to 150 mJ/cm(2) ultraviolet radiation, an alternate free radical generating system, also causes pericyte myosin translocation and apoptosis. Proteolytic cleavage of actin is not observed in pericyte apoptosis. 3-aminobenzamide, a pharmacological inhibitor of the cleavage and activation of the DNA-repairing enzyme poly (ADP-ribose) polymerase (PARP) inhibits pericyte apoptosis, and prevents myosin translocation. Deferoxamine, an iron chelator known to interfere with free radical generation, also inhibits pericyte myosin translocation, contractility, and cell death. Myosin translocation to the cytoskeleton may be an early step in assembly of a competent contractile apparatus, which is involved in apoptotic cell condensation. These results suggest that pericyte loss associated with increased free radical production in diabetic retina may be by an apoptotic phenomenon.

Asunto(s)

Apoptosis/efectos de los fármacos , Miosinas/metabolismo , Pericitos/metabolismo , Retina/efectos de los fármacos , Animales , Anexina A5/metabolismo , Benzamidas/farmacología , Bovinos , Células Cultivadas , Deferoxamina/farmacología , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Radicales Libres/farmacología , Peróxido de Hidrógeno/farmacología , Cinética , Microscopía Fluorescente , Fosfatidilserinas/metabolismo , Unión Proteica , Especies Reactivas de Oxígeno/metabolismo , Retina/efectos de la radiación , Rayos Ultravioleta

RESUMEN

While annotated two-dimensional (2D) gel electrophoresis databases contain thousands of proteins, they do not represent the entire genome. High-molecular-mass proteins in particular are conspicuously absent from such databases. Filamin is prototypical of this class of proteins since it is a dimer with relative molecular mass (Mr) of 520000 containing at least 240 potential phosphorylation sites. Filamin is not readily separated by current 2D procedures, and is difficult to study with respect to cycles of phosphorylation-dephosphorylation. Novel technologies are needed to identify biochemical pathways impinging upon such targets. The success of immunofluorescence microscopy as a research tool can be attributed in part to the fact that proteins redistribute in response to a variety of physiological stimuli. Comparable quantitative methods are required in proteome analysis. Three components are necessary for development of an approach that is capable of screening for protein redistribution events: (1) subcellular fractionation, (2) protein labeling and (3) data acquisition. An integrated approach is presented that utilizes differential detergent fractionation combined with reversible, luminescent protein stains and analytical imaging for high-throughput analysis of signal transduction events leading to protein subcellular redistribution. The procedure has been successfully implemented to rapidly define key second messenger pathways leading to endothelial cell junctional permeability and to guide in the design of a new family of peptide-based anti-inflammatory drugs.

Asunto(s)

Proteínas/genética , Fracciones Subcelulares/química , Secuencia de Aminoácidos , Animales , Genoma , Humanos , Datos de Secuencia Molecular , Proteínas/química , Proteínas/aislamiento & purificación

Asunto(s)

Quelantes , Electroforesis en Gel Bidimensional/métodos , Proteínas/análisis , Coloración y Etiquetado/métodos , Humanos , Metales

Asunto(s)

Electroforesis en Gel Bidimensional/métodos , Proteínas/análisis , Humanos , Coloración y Etiquetado/métodos

RESUMEN

Endothelial cell (EC) propagation has been simplified by developing cell-specific selection criteria. Methods commonly used for selectively isolating EC include: (i) differential sieving of disaggregated tissue, (ii) differential plating of cells on extracellular matrices, (iii) lectin affinity isolation of cell populations and (iv) fluorescence-activated cell sorting of cells labeled with a carbocyanine dye of acetylated low-density lipoprotein (DiI-Ac-LDL). Few criteria for selectively propagating pericytes (PC) are currently available. Nonspecific esterases exhibit a high degree of multiplicity when compared with other mammalian isozymes and may be suitable for the identification and selective propagation of cells of the microvasculature. Evaluation of esterase isotype expression in PC and EC by zymography indicates PC contain alpha-naphthyl acetate and alpha-naphthyl butyrate hydrolyzing esterases as well as dipeptidyl peptidase I, while EC only contain alpha-naphthyl acetate esterase. The cytotoxic response of PC and EC to various amino acid esters is assessed by monitoring vital dye uptake and by light microscopy. Several amino acid esters are cytotoxic to both cell types, whereas 50 mM L-leucine methyl ester (L-Leu OMe) is toxic to EC but not to PC. This amino acid ester is also toxic to mesothelial and retinal pigmented epithelial cells, other common contaminants of PC cultures. Analysis of protein composition by two-dimensional gel electrophoresis indicates that L-Leu OMe does not stimulate expression of stress response proteins in PC. Thus, L-Leu OMe can be utilized to cultivate PC selectively from mixed cell populations.

Asunto(s)

Dipéptidos/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Inmunosupresores/farmacología , Pericitos/efectos de los fármacos , Retina/citología , Aminoácidos/farmacología , Animales , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/efectos de los fármacos , Calreticulina , Bovinos , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Proteínas del Citoesqueleto/biosíntesis , Proteínas del Citoesqueleto/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Esterasas/biosíntesis , Esterasas/efectos de los fármacos , Ésteres/farmacología , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/efectos de los fármacos , Humanos , Pericitos/citología , Pericitos/enzimología , Proteína Disulfuro Isomerasas/biosíntesis , Proteína Disulfuro Isomerasas/efectos de los fármacos , Ribonucleoproteínas/biosíntesis , Ribonucleoproteínas/efectos de los fármacos

RESUMEN

Two principal forms of the actin binding protein, filamin, are expressed in mammalian cells: nonmuscle and muscle isotypes (FLN-1 and FLN-2). A protein that copurifies with an alpha-naphthyl acetate hydrolyzing esterase from human omentum microvessel endothelial cells (EC) is isolated by nondenaturing electrophoresis, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and electroblotting. The purified protein is subjected to in situ trypsin cleavage, reversed-phase high performance liquid chromatography (HPLC) and automated Edman degradation. Six peptide fragments from the protein are identified to have 60-66% identity with nonmuscle filamin (ABP-280). Two of these peptides are 100% identical to a previously sequenced human muscle filamin fragment. Polyclonal antibody is produced using a 16-residue synthetic peptide corresponding to a structural beta-sheet region of muscle filamin. Compared with a variety of vascular cells evaluated, retinal pericytes express an abundance of both muscle and non-muscle filamin isotypes. Pericytes contain at least 10 times more muscle filamin than human umbilical vein EC and at least three times the amount expressed in human omentum microvessel and bovine pulmonary artery EC. Differential detergent fractionation indicates that both filamin isotypes are primarily localized in the cytosol and membrane/organelle fractions of pericytes. Another actin crosslinking protein, alpha-actinin, is primarily found in the cytosol and cytoskeletal fractions. The dynamic regulation of actin microfilament organization in pericytes may be controlled in part by the two filamin isotypes, which in turn may contribute to pericyte contractility.

Asunto(s)

Proteínas Contráctiles/metabolismo , Endotelio Vascular/metabolismo , Proteínas de Microfilamentos/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Células Cultivadas , Proteínas Contráctiles/inmunología , Esterasas/aislamiento & purificación , Filaminas , Humanos , Proteínas de Microfilamentos/inmunología , Datos de Secuencia Molecular , Epiplón , Conejos , Retina/metabolismo , Análisis de Secuencia , Fracciones Subcelulares