Assuntos
Adrenoleucodistrofia/genética , Substituição de Aminoácidos/genética , Mutação de Sentido Incorreto/genética , Proteínas/análise , Proteínas/genética , Subfamília D de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Insuficiência Adrenal/genética , Adulto , Arginina/genética , Cisteína/genética , Humanos , Isoleucina/genética , Serina/genéticaRESUMO
Nuclear matrix proteins have been defined as insoluble residual proteins resulting from treatment of isolated nuclei with nucleases, detergents and high ionic strength buffers. They are considered as in part representing the proteins constituting the three-dimensional framework of the interphase nucleus. Though cell-specific nuclear matrix proteins have been differentiated from ubiquitously occurring (common) nuclear matrix proteins, the number and types of common nuclear matrix proteins have not yet been unequivocally established. In the present study nuclear matrix proteins were prepared from isolated nuclei of rat kidney, liver, lung, spleen and testes. The matrix proteins were separated by two-dimensional (2-D) electrophoresis and silver stained. Then the spot patterns were compared by computer-assisted image analysis. Composite images were derived for nuclear matrix proteins of individual tissues. Finding between 396-483 spots per tissue, a total of 964 individual spots were registered. Of these, 102 were common nuclear matrix proteins, as appearing in each of the tissue-characteristic images. The apparent molecular mass and pI data may serve for further identification of these nuclear proteins.
Assuntos
Núcleo Celular/química , Proteínas Nucleares/análise , Animais , Antígenos Nucleares , Eletroforese em Gel Bidimensional , Processamento de Imagem Assistida por Computador , Ponto Isoelétrico , Rim/ultraestrutura , Fígado/ultraestrutura , Pulmão/ultraestrutura , Masculino , Peso Molecular , Proteínas Nucleares/química , Ratos , Ratos Wistar , Coloração pela Prata , Baço/ultraestrutura , Testículo/ultraestruturaRESUMO
Nuclear-matrix proteins were prepared from different rat and human cells and separated by two-dimensional gel electrophoresis. By computer-assisted analysis of the images, two of the proteins were identified as ubiquitously occurring (common) nuclear-matrix proteins, which appeared in tissue-dependent concentrations. The two proteins that originated from human blood mononuclear cells were analyzed further. Tryptic digests of the blotted proteins were analyzed by partial peptide sequencing and matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. The two human common nuclear-matrix proteins were identified as heterogeneous nuclear ribonucleoproteins (hnRNP) H and H' or their variants. Furthermore, mass analysis revealed details on the N terminus of hnRNP H.
Assuntos
Matriz Nuclear/química , Proteínas Nucleares/isolamento & purificação , Ribonucleoproteínas/isolamento & purificação , Sequência de Aminoácidos , Animais , Antígenos Nucleares , Eletroforese em Gel Bidimensional , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Masculino , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ratos , Ratos Wistar , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
By systematic comparison of two-dimensional electrophoretic patterns of nuclear matrix proteins an ubiquitously occurring (common) nuclear matrix protein, termed NMP 238, was detected. Localization of the protein in isolated nuclear matrices and in nuclear and cytoplasmic regions of cells was determined by confocal immunofluorescence microscopy. N-terminal protein sequencing, mass spectrometry, and sequencing of a human EST cDNA clone showed identity of the protein with a nuclear protein, termed TIP49, of as yet uncertain function. Expression of the corresponding gene in diverse human and rat cells was confirmed by Northern blotting. The protein displays two nuclear localization signals. Sequence homologies indicate evolutionary related proteins in nematodes, yeast, and archaebacteria. Similarities to the AAA family of proteins and to a subgroup of chaperones suggest that the nuclear matrix protein may play a role in the assembly and ATP-dependent anchorage of proteins.