Two-dimensinal gel electrophoresis of rat liver nuclear washes, nuclear matrix, and hnRNA proteins.

Abstract

The proteins of rat liver cytoplasm, nuclear washes, matrix, membrane, heterogeneous nuclear (hn)RNA proteins and chromatin were examined by 2-dimensional gel electrophoresis. The inclusion in the gels of 6 common protein standards of carefully selected MW and isoelectric point allowed the distribution of specific proteins during nuclear extraction to be clearly followed. In the nuclear washes and chromatin, 5 classes of proteins were observed: exclusively cytoplasmic proteins, present in the first saline-EDTA wash but rapidly disappearing from subsequent washes; ubiquitous proteins of 75,000, 68,000, 57,000 and 43,000 MW, the latter being actin, found in the cytoplasm, all nuclear washes and the final chromatin pellet; proteins of 94,000, 25,000, 22,500 and 20,500 MW specific to the nuclear washes; proteins present in the nuclear washes and final chromatin, represented by species at 62,000, 55,000, 54,000 and 48,000 MW, primarily derived from the nuclear matrix; and 2 proteins of 68,000 MW present only in the final chromatin. The major 65,000-75,000 MW proteins seen by 1-dimensional gel electrophoresis of nuclear matrix were very heterogeneous and contained a major acidic, an intermediate, and a basic group. A single 68,000 MW polypeptide constituted the majority of the membrane-lamina fraction, consistent with immunological studies indicating that a distinct subset of matrix proteins occurs, associated with heterochromatin, at the periphery of the nucleus. Actin was the 2nd major nuclear membrane-lamina protein. Two polypeptides at 36,000 and 34,000 MW constituted 60% of the hnRNP. Approximately 80% of the mass of the nonhistone chromosomal proteins (NHP) from unwashed nuclei is contributed by nuclear matrix and hnRNP, and essentially the same patterns were seen with chromatin NHP. The concept of NHP being a distinct set of DNA-bound proteins is unnecessarily limiting. Many are derived from the nuclear matrix or hnRNP particles and vary in the degree to which they share different intracellular compartments.