2-DIMENSIONAL ELECTROPHORESIS REVEALS A NUCLEAR MATRIX-ASSOCIATED NUCLEOLIN COMPLEX OF BASIC ISOELECTRIC POINT

A monoclonal antibody was raised against a salt-extractable fraction o f nuclear matrix/intermediate filament scaffolds of polarized MDCK cel ls. The antibody recognized an similar to 100 kDa protein in total cel l lysates and nuclear matrices of various human cells and tissues and stained nucleolar structures in immunofluorescence microscopy. By part ial sequencing of five peptides derived from immunoprecipitated protei n, the targeted antigen was found to be homologous to human nucleolin. After two-dimensional electrophoresis of total HeLa cell lysates, imm unoreactive bands were detected at isoelectric point (pi) 5.5-6.1, cha racteristic: for nucleolin, and at pi 8.5-9. Whereas the protein focus ing at acidic pi was found in Triton X-100-soluble cellular fractions, the antigen focusing at basic pI was exclusively contained in the res idual nuclear fraction and was solubilized upon treatment of nuclear m atrices with RNAse. The component solubilized by RNAse treatment was s till detected at basic pI in two-dimensional electrophoresis. However, upon immunoprecipitation of the antigen from the RNAse-released fract ion in the presence of sodium dodecyl sulfate (SDS), the nuclear matri x-derived antigen was positioned at pI 5-6. The present data indicate that the nuclear matrix-bound nucleolin is associated with ribonucleop roteins and a basic component resisting dissociation under conditions of isoelectric focusing.