Instability of rat liver chromatin and other nuclear non-histone proteins in alkaline solution.

Non-histone proteins from rat liver nuclei and chromatin were shown to be hydrolysed in 0.1M or-1M-NaOH solutions both at 4 degrees and 18 degrees C; 24h in 1M-NaOH at 18 degrees C is sufficient to break down approx. 77% of these proteins to low-molecular-weight peptides. Loss of protein material banding in the region of pH5.5-8.0 has been demonstrated by isoelectric focusing in polyacrylamide gels, and fine high-molecular-weight bands are no longer visible on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The results indicate that care must be taken when analysing non-histone-protein fractions to avoid exposure to alkaline pH conditions.     

In vitro triiodothyronine binding to non-histone proteins from rat liver nuclei

$\text{In vitro}$ incubations of non-histone proteins from rat liver nuclei with labelled L-3, 5, 3′ triiodothyronine demonstrate the existence of high affinity, limited capacity binding sites for the hormone in this protein group; the affinity was found identical for triiodothyroacetic acid and lower for L-thyroxine. Binding ability was highly temperature dependent. At 4°C, the rate constant of association was 0.9 × 10⁷ M^?1 h^?1 and the rate constant of dissociation was 0.015 h^?1. The dissociation constant K_d was calculated from these data or measured by Scatchard analysis and found to be between 1.6 and 5 × 10^?9 M. The maximum binding capacity was 10^?13 moles of L-3, 5, 3′ triiodothyronine per 100 μg non-histone proteins or 6000 hormone molecules per nucleus. Protein binding had a half-life of 20 hours at 4°C, in the absence of hormone, but was found to be very stable in the presence of hormone.     

Acid-soluble, non-histone proteins in rat liver chromatin. Interaction with DNA

Some properties of nonhistone proteins of rat liver chromatin (Mr 40 +/- 1 and 41 +/- 1 KD) are described. These proteins are abundant in monomeric particles formed at the early steps of chromatin fragmentation by Ca2+,Mg2+-DNase. The proteins are not extracted from chromatin by 5% HClO4 and 1 M NaCl, but can be extracted by 0.4 n H2SO4 and 2 M NaCl. Study on proteins binding to DNA demonstrated that in 0.05 M NaCl these proteins are bound both to bovine satellite DNA and to the plasmid pBR 322 DNA.     

Phosphorylation of nuclear proteins in rat regenerating liver.

In studies of the phosphorylated proteins in rat liver and Walker-256, it was established that the ratio of various fractions of P-N linkages to P-O linkages varies from 0.6 to 3.1. In rat regenerating liver nuclei, the ratio of P-N and P-O varies with time after partial hepatectomy. Using [3H]-lysine and 32Pi, it is shown that phosphoryllysine forms in some new and, presumably, some preexisting H1 molecules. Using [3H]histidine and 32Pi, it is shown that phosphohistidine forms exclusively in preexisting H4. The half-life of H4 phosphohistidine appears to be about 2 h.     

Age- and ploidy-related changes in the non-histone proteins of rat liver nuclei

Hepatic nuclei from young, mature and aged rats were separated into 2n, 4n and 8n ploidy classes on sucrose gradients, and characterized in terms of DNA, RNA, histones and non-histone proteins (NHP). The absolute DNA content per nucleus doubled with each increase in ploidy as expected, and the relative proportions of other components remained uniform in all cases. Despite such overall similarity in pattern however, marked electrophoretic heterogeneity was observed among the NHP, both in relation to ploidy and animal age. All nuclei shared a common spectrum of soluble and residual NHP, but discrete variations among several specific proteins were observed. Most prominent was a systematic, age-related decrease in two major polypeptides of about 125 000 and 57 000 mol. wt, respectively. There were no comparable variations in the histone proteins. The results are discussed in terms of possible functional differentiation among hepatocytes of the various ploidy classes.     

Isolation of nuclear acidic proteins from rat tissues. Characterization of acetylated liver nuclear acidic proteins

Nuclear acidic proteins isolated from rat brain, heart, kidney and liver showed similar, complex patterns on electrophoresis in sodium dodecyl sulphate-polyacrylamide gels. The contamination of nuclear acidic proteins by nuclear-membrane acidic proteins was found to the extent of 11%. Incorporation of [(3)H]acetate into the various nuclear acidic proteins in vivo, which were fractionated by polyacrylamide-gel electrophoresis, differed from tissue to tissue. Hydrolysis of these acetylated nuclear acidic proteins with 6m-HCl at 110 degrees C released 70% of the radioactivity, which indicated that labile acetyl groups had been incorporated into these proteins. Analysis of [(3)H]acetate-labelled nuclear acidic proteins revealed two acetylated amino acid residues, N(2)-acetylserine and N(2)-acetyl-lysine. The significance of the role played by nuclear acidic proteins in relation to gene regulation is discussed.