Protein composition of chromatin fractions differing in their attachment to nuclear structures at low ionic strength

• Published in: Biokhimiia (Moscow, Russia) Vol. 47; no. 6; p. 999
• Main Authors: Lobanenkov, V V, Mironov, N M
• Format: Journal Article
• Language: Russian
• Published: Russia (Federation) 01-06-1982
• Subjects: Animals; Cell Nucleus - ultrastructure; Chromatin - ultrastructure; Chromosomal Proteins, Non-Histone - isolation & purification; Deoxyribonucleoproteins - isolation & purification; Liver - analysis; Liver - ultrastructure; Molecular Weight; Nucleoproteins - isolation & purification; Osmolar Concentration; Rats
• ISSN: 0320-9725

Rat livers were disrupted in the TMS buffer (10 mM Tris-HCl, pH 7.6, 5 mM MgCl2, 0.25 M sucrose +70 microM beta-mercaptoethanol) and the nuclei were purified by sedimentation through 2.2 M sucrose with the same components as in TMS. Then the nuclei were resuspended and washed 3 times in TMS. After that the nuclei were resuspended to 1 mg DNA per ml in TM buffer (10 mM Tris-HCl, pH 7.6, 0.2 mM MgCl2) followed by centrifugation at low speed. About 60% of total nuclear DNP was recovered by this extraction. The protein/DNA ratio in the extracted chromatin fraction (DNPs) was about 1.1. The bulk of the non-extracted in TM residual chromatin fraction was released from the nuclear pellet after treatment with micrococcal nuclease. This matrix-associated chromatin fraction (DNPm) is significantly enriched in non-histone proteins as compared with the DNPs; hence the protein/DNA ratio of DNPm is at least two times higher than that of DNPs. The protein components of DNPs are represented by five histones containing negligible non-histone admixture. One of them was identified as protein A24, another--as non-dissociated from DNA in 0.6 M NaCl acid-soluble protein with m. w. of about 42,500. The possible structural features of these two distinguishable chromatin fractions are discussed.