Subcellular Particles: A Symposium Held during the Meeting of the Society of General Physiologists at the Marine Biological Laboratory, Woods Hole, Massachusetts, June 9-11, 1958

By Teru Hayashi | Go to book overview

Biochemical Properties of the Isolated Nucleus

V. ALLFREY AND A. E. MIRSKY

The Rockefeller Institute New York, N. Y.

THE EXPERIMENTS ABOUT TO BE DESCRIBED are concerned with synthetic reactions carried out by isolated cell nuclei--with particular emphasis on amino acid uptake into nuclear protein, the incorporation of purine and pyrimidine precursors in nuclear RNA, and nuclear ATP synthesis. Some of the experiments will emphasize the role of deoxyribonucleic acid in these nuclear activities, others will relate the state of the nucleus to methods of its isolation and its chemical environment.1

In earlier work in this laboratory we had been concerned with the chemical composition of cell nuclei isolated in different ways. Nuclei prepared in citric acid, sucrose or saline solutions were compared with those isolated in nonaqueous media by a modified Behrens' procedure (7, 8). The latter type of isolation was selected as a 'standard' because it prevents water-soluble materials from moving between nucleus and cytoplasm during the course of the isolation. Its success depends on rapid freezing followed by lyophilization to remove water from the tissue, which is then ground and fractionated in nonaqueous solvents such as cyclohexane and carbon tetrachloride. The nucleus has a characteristic density range which often permits its separation from other elements of the ground cell suspension by differential centrifugation. The separation of the nuclei is facilitated by selecting a medium of density lower than the nuclear specific gravity but higher than that of possible cytoplasmic contaminants. Centrifugation then yields a nuclear sediment which can often be shown by chemical, immunological and enzymatic tests to be essentially free of cytoplasmic contamination.

Nuclei isolated in nonaqueous media constitute a 'standard' against which nuclei prepared in aqueous media can be compared. For example, liver or kidney nuclei prepared in sucrose or citric acid solutions were shown to have lost much of their protein during isolation simply by comparing them with the corresponding Behrens type nuclei (8, 10).

However, when such comparisons were carried out in the case of calf thymus nuclei, it was found that thymus nuclei isolated in isotonic sucrose solutions were

____________________
1
The following abbreviations are used throughout: RNA, ribonucleic acid; ATP, adenosinetriphosphate; DNA, deoxyribonucleic acid; AMP, adenosinemonophosphate; ADP, adenosinediphosphate; TCA, trichloroacetic acid.

-186-

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Subcellular Particles: A Symposium Held during the Meeting of the Society of General Physiologists at the Marine Biological Laboratory, Woods Hole, Massachusetts, June 9-11, 1958
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