Polypeptide-synthesizing activity of eukaryotic chromatin. Properties, dependence on poly(ADP-ribose) and connection with the cell cycle

Abstract

Isolated nuclei incubated with [14C]protein hydrolysate are shown to incorporate labelled amino acids into the acid-insoluble fraction. Purified chromatin and the complex of DNA with firmly bound proteins possess similar ability. The optimum pH of the reaction is 6.5-7.0, 2 mM MgCl2 stimulates incorporation, the temperature optimum is 37-40 degrees C. Chloramphenicol depresses incorporation by 70%, puromycin by 40%, cycloheximide does not affect the chromatin activity. Incorporation does not depend on the presence of ATP or GTP, and is substantially inhibited by deoxyribonuclease but not by ribonuclease treatment of chromatin or of the nuclei. Specific activity of firmly bound chromatin non-histone proteins is higher than that of labile bound ones; histones are not labelled. After pronase treatment of proteins radioactivity changes to an acid-soluble state. The molecular weight of isolated labelled polypeptides is about 6000 as shown by gel filtration and the analysis of NH2-terminal amino acids. Labelled polypeptides firmly bound to DNA consist of 7-10 amino acids. Specific activity of proteins firmly bound to DNA increases linearly with the time of incubation of chromatin with [14C]protein hydrolysate, the activity curve of labile bound non-histone proteins has a distinct sygmoid character. The polypeptide-synthesizing activity of rat liver chromatin increases between 9 h and 21 h after partial hepatectomy. Irradiation with 800 rads 30 min before the operation prevents activation of amino acid incorporation. From nine amino acids studied alanine, methionine, lysine, tyrosine and arginine are not incorporated in the system described. Glutamic acid is polymerized most effectively. Glutamine, asparagine and glycine are incorporated 7-8 times less. The data are given indicating that the incorporation is not random when an amino acid mixture is present. Preincubation of chromatin with NAD+ but not with its analogues increases the polypeptide-synthesizing activity of chromatin. The activation is prevented by thymidine and nicotinamide. Storage (18 h at 2-4 degrees C) brings about a complete loss of the polypeptide-synthesizing activity of chromatin. The ability of 'old' chromatin to incorporate amino acids can be restored by preincubating it with NAD+. Storage of chromatin in the presence of 5 mM adenosine 3',5'-monophosphate (cAMP) does not result in decrease of the polypeptide-synthesizing activity. It is assumed that poly-(ADP-ribose) is the energy source for amino acid activation in the system described.