Some properties of nuclear protein-synthesizing system of eukaryotic cells
Matinian, K.S.; Umanskiĭ, S.R.
Biokhimiia 43(1): 111-120
1978
ISSN/ISBN: 0320-9725 PMID: 623839 Document Number: 128144
During incubation with 14C-protein hydrolysate isolated nuclei from rat liver or spleen incorporate labeled amino acids into the acid-insoluble fraction. Similar ability is observed with purified chromatin and the complex of DNA with tightly bound proteins. The pH optimum for the reaction is in the range 6.5-7.0; 2 mM MgCl2 enhance the incorporation; the temperature optimum is 37-40.degree. C. Chloramphenicol inhibits the incorporation by 70%, and puromycin by 40%; cycloheximide has little effect on the reaction rate. The incorporation does not depend on the presence of ATP or GTP and is strongly inhibited when chromatin or nuclei are treated with DNase rather than with RNase. The specific activity of tightly bound chromatin nonhistone proteins is higher than that of loosely bound ones; the label is not incorporated into the histones. Gel-filtration and N-terminal amino acid analysis showed that the MW of the labeled polypeptides isolated is about 6000. On the average the labeled polypeptides tightly bound to DNA are composed of 9 amino acids. A linear relationship exists between the increase in radioactivity of proteins tightly bound to DNA and the time of chromatin incubation with 14C-protein hydrolysate. The incorporation of label into the loosely bound nonhistone proteins significantly increases only 30 min after the start of incubation. The mechanism of polypeptide synthesis involved in this system is probably different from the classical mechanism of protein synthesis in the ribosomes.