RESUMEN
Nuclear matrix proteins form the skeleton of the nucleus and participate in the various cellular functions of the nucleus. These proteins have been demonstrated to be tissue-type specific and can potentially reflect changes in the state of differentiation of the cell. Elucidating nuclear matrix protein changes necessitates the use of high-resolution two-dimensional polyacrylamide gel electrophoresis. Separation of this complex mixture into its component parts resolves protein changes when comparing the normal state to a diseased state of a cell. Evidence has been reviewed which shows the potential use of nuclear matrix proteins and antibodies to nuclear matrix proteins as diagnostic tools for various cancers, autoimmune diseases, adenoviral infection, and other diseases. Consequently, the central functions of the nuclear matrix in the cell allow it to have significant potential as a diagnostic agent.
Asunto(s)
Electroforesis en Gel Bidimensional , Matriz Nuclear/química , Proteínas Nucleares/análisis , Electroforesis en Gel Bidimensional/métodos , Humanos , Proteínas Nucleares/aislamiento & purificaciónRESUMEN
An improved two-dimensional gel electrophoresis procedure has been developed utilizing isolated nuclear matrix proteins. The proteins of the cellular nuclear matrix are tissue specific. They are an example of a protein set whose two-dimensional electrophoretic patterns afford much information of clinical significance. However, current two-dimensional gel techniques were not completely satisfactory for the small amounts of protein present in tissue samples. There was a need for a two-dimensional gel procedure which was capable of increased sensitivity and resolution and at the same time was reliable and reproducible. This has been accomplished by implementing several modifications to the current two-dimensional gel procedures. In addition, changes were introduced in the silver staining process of the gels to increase the signal to background ratio. The overall procedure affects a dramatic increase in the resolution and clarity of the proteins visualized on two-dimensional gels and is no more laborious than current techniques.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Proteínas Nucleares/análisis , Plata , Antígenos Nucleares , Geles , Humanos , Coloración y Etiquetado , Células Tumorales CultivadasRESUMEN
We have developed a unilamellar phospholipid vesicle system which contains the N-formyl peptide receptor and GTP binding proteins. Several detergents were investigated but only two, octyl glucoside (35 mM) and deoxycholate (7.5 mM), were capable of extracting N-formyl peptide receptor from neutrophil membranes in a form which remained functionally active upon reconstitution into phospholipid vesicles. Extracted proteins were reconstituted into phosphatidylcholine vesicles by passage over a Sephadex G-50-80 column. The reconstituted formylpeptide receptor could bind [3H]FMLP (3H-labeled fMet-Leu-Phe) and [125I]FMLPL-SASD (125I-labeled N-formylmethionylleucylphenylalanyl-N epsilon-(2-(p-azidosalicylamido)ethyl- 1,3'-dithiopropionyl)lysine) while the endogenous G protein could bind [35S]GTP gamma S. Furthermore, the functional interaction of the two proteins was preserved. Addition of the nonhydrolyzable guanine nucleotide, GTP gamma S, shifted the N-formyl peptide receptor from a high- to a low-affinity binding state for ligand. The development of this in vitro reconstitution system should provide a basis to study the mechanism of interaction of the N-formyl peptide receptor and the G protein.
Asunto(s)
Proteínas de Unión al GTP , N-Formilmetionina Leucil-Fenilalanina/fisiología , Neutrófilos/ultraestructura , Receptores Inmunológicos , Adenosina Difosfato Ribosa/metabolismo , Western Blotting , Membrana Celular/análisis , Detergentes , Proteínas de Unión al GTP/aislamiento & purificación , Proteínas de Unión al GTP/fisiología , Humanos , Microscopía Electrónica , Peso Molecular , Fosfolípidos , Receptores de Formil Péptido , Receptores Inmunológicos/aislamiento & purificación , Receptores Inmunológicos/fisiologíaRESUMEN
A simple and efficient technique for purifying adenoviral chromatin (nucleoprotein cores) with Sephacryl S-1000 is described. This method is significantly faster than previous methods and gives a higher degree of purity with an increased recovery of the nucleoprotein. In addition, the structural integrity of the cores is maintained.
Asunto(s)
Adenovirus Humanos/análisis , Cromatografía en Gel , Proteínas del Núcleo Viral/aislamiento & purificación , Resinas Acrílicas , Electroforesis en Gel de Poliacrilamida , Microscopía Electrónica , Peso MolecularAsunto(s)
Lactoglobulinas , Animales , Bovinos , Disulfuros , Femenino , Cinética , Conformación Proteica , Desnaturalización Proteica , UreaRESUMEN
The conformational stability of globular proteins is remarkably low. Under physiological conditions, the native globular conformation is only from 5 to 15 kcal/mole more stable than unfolded conformations. In addition, small changes in the structure of a protein such as removing one terminal residue or cleaving a single peptide bond frequently lead to a substantial decrease in the stability. Likewise, single substitutions in the amino acid sequence can increase or decrease the stability by several kilocalories per mole. The low conformational stability of globular proteins and the sensitivity to small changes in structure suggest a possible role for conformational stability in the intracellular degradation of proteins. Several lines of evidence from in vivo studies of protein degradation are consistent with this idea.