Characterization Of Nonhistone Proteins Research Articles

12] Methods for isolation and characterization of chromosomal nonhistone proteins: Fractionation of chromatin on hydroxyapatite and characterization of the nonhistone proteins by ion exchange chromatography and polyacrylamide gel electrophoresis

Publisher Summary This chapter provides methods for isolation and characterization of chromosomal nonhistone proteins. The methodology is based on the ability of hydroxyapatite to act as an ion exchange medium for macromolecules in the presence of high ionic strength and urea and consists of a single column procedure, yielding high recoveries of chromatin constituents. The methodology utilizes combination of salt and urea, which has been used as a solvent both for the dissociation and solubilization of chromatin and its specific reconstruction from previously isolated components. The complexity of nonhistone proteins is demonstrated by the presence of polypeptides of differing isoelectric points in sodium dodecyl sulfate–protein complexes that appear as individual components in the one–dimensional sodium dodecyl sulfate system. In addition, the two-dimensional separation procedure indicates that there is a considerable range of rapidly phosphorylated proteins in chromatin. The proteins comprising fractions H2 and H3 are markedly different because the latter possess more acidic isoelectric points. However, some overlap probably does occur between these two fractions because the proteins designated as P1 and P2 appear common to both, the more phosphorylated forms in the liver being present in fraction H3.

Studies on deoxyribonucleoproteins: VIII. Isolation and characterization of nonhistone proteins from euchromatic and heterochromatic deoxyribonucleoprotein of rat liver

Rat liver nuclei were fractionated into euchromatin and heterochromatin. Deoxyribonucleoproteins of these fractions were purified by continuous carrier-free electrophoresis and analyzed for their proteins, mainly the nonhistones. The ratio of nonhistones/DNA was found to be much higher in the purified deoxyribonucleoprotein of euchromatin than in the deoxyribonucleoprotein of heterochromatin. Comparative studies of the nonhistones derived from euchromatin and heterochromatin showed no clear indications for qualitative differences. By gel electrophoresis 13–14 bands were obtained, the molecular weight range was found to be in the range between 13 000 and 150 000. The main fractions of the nonhistones possessed isoelectric points from 4.5–6.5. The biological implications of these observations and the special methodical problems are discussed.

Chromatin-associated proteins of the developing sea urchin embryo: I. Kinetics of synthesis and characterization of non-histone proteins

Chromatin-associated non-histone proteins of the sea urchin embryo are heterogeneous, and undergo qualitative as well as quantitative changes throughout early development. The rate of synthesis of these proteins is fairly constant to the pluteus stage and, in contrast to histone synthesis, does not parallel changes in the rates of synthesis of DNA. Evidence for a pool of chromatin-associated nonhistone proteins is provided by following the kinetics of entry into chromatin of labeled protein in pulse-chase experiments of prolonged duration. This pool is synthesized during cleavage and some non-histone proteins continue to associate with chromatin until gastrulation. In addition, different rates of entry of non-histone proteins into chromatin could be detected at different stages.