Metabolic studies of histones and residual protein of rat liver nuclei

Abstract

The total histone isolated from rat liver nuclei has been divided into three principal fractions based on acid-extractability. Subfractionation by isoelectric precipitation with ammonia was also used to obtain ammonia-precipitable and ammoniasoluble fractions. In order to test the hypothesis that the most easily acid-extractable histone fraction might be more metabolically active than the more difficultly extractable fractions, a set of time-course incorporation studies of 14C-leucine into histones extracted from nuclei isolated at pH 3.8, 3.3, and 2.2 were made, and the activity of each fraction was measured by gas-flow counting. In a different type of experiment, ammonia-soluble histones obtained from nuclei isolated at pH 3.8 and 3.3 were fractionated into molecular size classes by the use of sephadex G-200, and the fractions from the sephadex column were counted by the use of a scintillation spectrometer. These studies show that the metabolic activity of a histone fraction is not related to its ease of extractability or its ammonia solubility. Instead the analysis by sephadex chromatography indicates that the metabolic activity of a given histone fraction is correlated to a certain extent with its average molecular weight. Similar time course studies were also conducted on the various residual protein fractions to test the hypothesis that the known high metabolic activity of the residual protein fraction might be due to the presence of a small but highly active subfraction which could be separated from the rest of the residual protein by 0.1 n HCl-8 m urea treatment, leaving behind a metabolically inert structural component of the chromosome. Neither 8 m urea-0.1 n HCl extraction nor digestion with RNA-ase followed by urea-HCl extraction removed any such highly active sub-fraction of residual protein. It is concluded that a high metabolic activity is probably an intrinsic property of the residual protein, and that this high activity may be central to the role and function of residual protein in the mammalian cell nucleus.