Phosphorylated nonhistone proteins in different fractions of rat liver nuclei obtained by metrizamide gradient centrifugation.
The distribution of nuclear nonhistone proteins (NHPs) labelled in vivo with 32P was investigated in different subnuclear fractions obtained by metrizamide gradient centrifugation of mildly sonicated rat liver nuclei. The bulk chromatin banding at 1.20-1.22 g to cm3 (fraction C) contained only minor amounts both of non-phosphorylated and 32P-labelled NHPs. Nick-translated DNA sequences were found to sediment at a slightly higher density (1.26 g/cm3) in a minor fraction (A). Fraction A also contained a significant portion of the newly synthesized RNA and was enriched additionally with a broad spectrum of NHPs; some of them were highly 32P-labelled. Two hnRNP fractions (R2 and R1) sedimenting at densities greater than 1.28 g/cm3 were analyzed, too. By SDS-PAGE it was shown that the most abundant 32P-NHPs were found in three groups (fraction A, R1, and R2). Two of them, 32-34 KDa, and 37-40 KDa, are components of the major proteins of the hnRNP particles. A group of four moderately labelled 32P-NHPs of approximately 55 K, 64 K, 70 K, and 90 KDa appeared substantially enriched in fraction A. They were found also in RNP fractions R1 and R2, but to a smaller mount. Therefore, we suggest that in fraction A they exist in a complex with hnRNP. Fraction A was also enriched with numerous 32P-NHPs of very high molecular weight found in other types of experiments nearly exclusively in the nuclear residual structures. The 32P-protein pattern of the nucleoli was shown to be different from that of the other subnuclear fractions. Considering our observations on the accumulation of nick-translated DNA sequences, the presence of newly synthesized RNA, the high content of NHPs, and the association of special 32P-NHPs we suggest that fraction A represents transcriptionally active chromatin.
D000818:Animals; D002466:Cell Nucleolus / Q000032:analysis; D002467:Cell Nucleus / Q000032:analysis*; D002499:Centrifugation, Density Gradient / Q000379:methods; D002843:Chromatin / Q000032:analysis; D002868:Chromosomal Proteins, Non-Histone / Q000302:isolation & purification*; D008099:Liver / Q000032:analysis*; D008793:Metrizamide; D009963:Orotic Acid; D010750:Phosphoproteins / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D010750:Phosphoproteins; D009963:Orotic Acid; D008793:Metrizamide