An electrophoretic comparison of non-histone proteins from rat liver total chromatin and chromatin depleted of 0.35 M NaCl soluble proteins

• 1.1. A procedure of isolation of non-histone proteins from rat liver chromatin in mild conditions provided 3 groups of these proteins, i.e. NHCP1, NHCP2 and NHCP3.
• 2.2. The investigated proteins are devoid of DNA and revealed various influences on RNA synthesis in vitro.
• 3.3. The extraction of rat liver chromatin with 0.35 M NaCl (pH 7.5) removed about 30% of examined proteins. Electrophoretic patterns of 3 groups of non-histone proteins from total chromatin and chromatin depleted of 0.35 M NaCl soluble proteins are compared.

     

Similarity of the 0.35 M NaCl soluble nuclear proteins and the nonhistone chromosomal proteins

Nuclear proteins which are extractible with 0. 35 M NaCl and the nonhistone chromosomal proteins which are not soluble at this salt concentration separate on analytical polyacrylamide gel electrophoresis into the same 11 main fractions. Only one fraction (less than 7% of the total proteins) is specific for the nonhistone chromosomal proteins and is not found among the proteins soluble in 0. 35 M NaCl.     

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.     

Demonstration of GTP-binding proteins and ADP-ribosylated proteins in rat liver Golgi fraction

A Golgi-rich fraction isolated from rat liver was found to contain GTP-binding proteins with 20-25 kDa, which were tightly bound to the Golgi membrane. The Golgi fraction also contained two species of proteins which were ADP-ribosylated by bacterial toxins. Protein(s) which was ADP-ribosylated by botulinum toxin had a similar molecular mass as those with GTP-binding activity but was easily released from the membrane. Another protein with 46 kDa which was ADP-ribosylated by pertussis toxin was tightly bound to the membrane but had no significant GTP-binding activity under conditions tested here. These proteins were much less or negligible in the plasma membrane and the endoplasmic reticulum.     

A modified procedure for the isolation of a pore complex-lamina fraction from rat liver nuclei

A modified procedure for the isolation of a nuclear pore complex-lamina fraction from rat liver nuclei is described. Evidence is provided that the isolated lamina, a 150-A thick, proteinaceous structure, apposes the inner nuclear envelope membrane, connecting nuclear pore complexes and surrounding the entire nucleus.     

Protein phosphatase from rat liver nuclei

Summary Protein phosphatase, active on non-histone phosphoprotein substrate, was partially purified from rat liver cell nuclei by means of salt extraction, ammoniumsulfate precipitation, DEAE cellulose chromatography, gel filtration and preparative isoelectrofocusing.Rat liver nuclei contain a heterogenous population of different protein phosphatases. All the enzyme fractions eluted from DEAE cellulose are of low molecular weight between 12,000–31,000. The pH 5.5 peak fraction of preparative isoelectrofocusing was characterized in detail. It has a pH optimum of 6.8 using nuclear phosphoprotein substrate. It is inhibited by Na⁺ at 80mm, and to a lesser extent by K⁺, activated by Mg²⁺(5mm) and Mn²⁺ (1mm). However, the latter is inhibitory at 6mm.The nuclear protein phosphatase is also active on labelled F1 and F2b histones and casein, however, its V is lower on histones and it contains component(s) active specifically on nuclear phosphoprotein substrate but not on casein.Abbreviations PP-ase protein phosphat Part of this work was presented at the XI^th FEBS Meeting, Copenhagen 1977.     

Phosphorylation of nuclear matrix proteins in isolated regenerating rat liver nuclei

The phosphorylation of nuclear matrix proteins from normal and regenerating rat liver nuclei was examined using an $\text{in vitro}$ system of isolated nuclei and γ-³²P-ATP. Phosphorylation of the nuclear matrix proteins was 2–3 fold higher than that of the total nuclear proteins in normal nuclei. The level of phosphorylation of the matrix proteins was enhanced an additional three fold at a period in liver regeneration (12 hours) just preceding the onset of DNA synthesis.     

Fractionation of the nucleotides of the acid-soluble liver fraction of rats

A method for separation of rat liver acid-soluble nucleotides was developed including ion exchange polyethyleneimine cellulose chromatography, followed by rechromatography of the separate fractions on Dowex 1 and Aminex MS resins. It is simple, reproducible and does not require expensive reagents and devices. The sensitivity of the method in respect to orotate is 100 pM in a sample. Data on the content of liver basic 5'-ribonucleotides and their derivatives were obtained by the proposed method.     

An ammonium sulphate fraction from rabbit reticulocytes increases the release of proteins from rat liver mitochondria

Incubation of [35S]methionine labeled mitochondria from rat liver with rabbit reticulocyte lysate under the same conditions as those used in the import of mitochondrial protein precursors results in the release of mitochondrial proteins to the medium. Fractionation of the lysates with ammonium sulphate yields a fraction, essentially free of haemoglobin, which exhibits higher activity for the release of mitochondrial proteins than the starting lysate. The fraction has a molecular mass of greater than 10 kDa and is heat-sensitive. The release is insensitive to inhibitors of reticulocyte lipoxygenase.