NUCLEAR MATRIX OF THE LOWER EUKARYOTE PHYSARUM-POLYCEPHALUM AND THE MAMMALIAN EPITHELIAL LLC-PK1 CELL-LINE - A COMPREHENSIVE INVESTIGATION OF DIFFERENT PREPARATION PROCEDURES

Agarose-encapsulated nuclear matrix preparations of the lower eukaryote Physarum polycephalum and the mammalian renal epithelial LLC-PK1 cell line were analyzed after various experimental protocols with respect to the protein composition. The effect of the mode of deproteinization (2 M NaCl, 0.25 M ammonium sulfate or 25 mM lithium diiodosalicylate), presence of 2-mercaptoethanol, Ca2+, Cu2+, chelating agents, the sequence of protein extraction and nuclease digestion, the use of RNase, the temperature at which the experimental manipulations were performed and the use of hypotonic or isotonic conditions was investigated. No significant differences in the final nuclear matrix composition could be observed, regardless of the experimental procedure applied. In Physarum, the major nuclear matrix proteins range over 12-70 kDa with prominent bands at 24, 31, 37 and 45 kDa; the proteins of the matrix in LLC-PK1 cells extend predominantly over 40-80 kDa. Furthermore, no essential differences in the protein composition could be observed when type I and type II nuclear matrices from the highly differentiated LLC-PK1 cell line were compared. The same was found for analogous matrix preparations of Physarum. Therefore, in both systems a distinction between type I/II matrix is questionable. Immunoblotting of the matrix preparations with a variety of antibodies against intermediate filament proteins and with antinuclear autoantibodies revealed the presence of intermediate filament proteins as components of the nuclear matrix. We conclude that the nuclear matrix represents a much more stable and reproducible structure than has been proposed so far, largely independent of changes in the preparation protocol.