Similarity between nuclear matrix proteins of various cells revealed by an improved isolation method.
Comparative analysis of nuclear matrix proteins by two-dimensional electrophoresis may be greatly impaired by copurifying cytoskeletal proteins. The present data show that the bulk of adhering cytofilaments may mechanically be removed by shearing of nuclei pretreated with vanadyl ribonucleoside complexes. Potential mechanisms of action not based on ribonuclease inhibition are discussed. To individually preserve the integrity of nuclear structures, we developed protocols for the preparation of nuclear matrices from three categories of cells, namely leukocytes, cultured cells, and tissue cells. As exemplified with material from human lymphocytes, cultured amniotic cells, and liver tissue cells, the resulting patterns of nuclear matrix proteins appeared quite similar. Approximately 300 spots were shared among the cell types. Forty-nine of these were identified, 21 comprising heterogeneous nuclear ribonucleoproteins. Heterogeneous nuclear ribonucleoproteins L and nuclear lamin B2 isoforms were identified by amino acid sequencing and mass spectrometry. However, individually expressed proteins, such as the proliferating cell nuclear antigen, also pertained following application of the protocols. Thus, enhanced resolution and comparability of proteins improve systematic analyses of nuclear matrix proteins from various cellular sources.
Gerner
C
C
Institute of Tumor Biology-Cancer Research, University of Vienna, Austria. Christopher.Gerner@univie.ac.at
Holzmann
K
K
Grimm
R
R
Sauermann
G
G
D000653:Amniotic Fluid / Q000166:cytology; D034961:Antigens, Nuclear; D015153:Blotting, Western; D002458:Cell Fractionation / Q000379:methods*; D002467:Cell Nucleus / Q000737:chemistry / Q000187:drug effects; D002478:Cells, Cultured; D002648:Child; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D034921:Lamin Type B; D034882:Lamins; D007962:Leukocytes; D008099:Liver / Q000166:cytology; D013058:Mass Spectrometry; D015530:Nuclear Matrix / Q000737:chemistry* / Q000187:drug effects; D009687:Nuclear Proteins / Q000032:analysis*; D014066:Palatine Tonsil / Q000166:cytology; D010455:Peptides / Q000032:analysis; D020033:Protein Isoforms / Q000032:analysis; D012261:Ribonucleoproteins / Q000032:analysis; D012263:Ribonucleosides / Q000494:pharmacology*; D017421:Sequence Analysis; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology*
D034961:Antigens, Nuclear; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins; D010455:Peptides; D020033:Protein Isoforms; D012261:Ribonucleoproteins; D012263:Ribonucleosides; C057724:vanadyl ribonucleoside complex; D014638:Vanadates