It was observed previously that the rate of incorporation of radioactive amino acids into the proteins of isolated cell nuclei increases sharply when sodium ions are added to the incubation medium.'-' This sodium requirement for amino acid uptake by the isolated nucleus contrasts with the usual observation that potassium is the specific cofactor needed for the incorporation of amino acids by isolated cytoplasmic systems4 and for amino acid transport into intact cells.5Why is protein synthesis in the nucleus sensitive to the presence or absence of sodium ions? This question has now been studied using suspensions of isolated calf thymus nuclei. The experiments to be described will show that there is a mechanism, very probably enzymatic, governing and restricting the entry of amino acids into the cell nucleus; this apparent "transport" reaction is sodiumdependent. Thus, the sodium requirement for nuclear protein synthesis reflects the operation of this mechanism for bringing amino acids to the sites of synthesis.Other synthetic processes in the nucleus may or may not display a similar sodium ion requirement. For example, thymidine-2-C'4 uptake into DNA and adenosine-8-C14 incorporation into RNA both improve when sodium is added to the incubation medium. On the other hand, the utilization of orotic acid for nuclear RNA synthesis-does not show a specific sodium ion effect. Here, too, it will be shown that there is a good correlation between the ionic requirements for uptake and for transport of several isotopic precursors of ribo-and deoxyribonucleic acids. The specific role of sodium in regulating the access of substrates to isolated nuclei suggests its great importance in the regulation of nuclear metabolism in vivo. This correlates well with other observations showing that widely diverse cell types have a high intranuclear sodium concentration.
THE EFFECTS OF MONOVALENT CATIONS ON AMINO ACID INCORPORATION INTO
NUCLEAR PROTEINSIsolated calf thymus nuclei can carry out a sustained incorporation of amino acids into their constituent proteins in vitro.6' 1, 2 The isolation of the nuclei has been described previouslv;2 it involves homogenization of fresh calf thymus tissue in 0.25 M sucrose-0.003 M CaCl2 solution, and subsequent isolation of the nuclei by differential centrifugation in the same isotonic medium. It has been shown that thymus nuclei prepared in this way satisfy many morphological and chemical criteria of nuclear purity.7'-0 Moreover, they retain the capacity to synthesize ATP," activate amino acids,9' 12 and to incorporate a variety of purine and pyrimidine precursors into their nucleic acids.2' [13][14][15][16] When calf thymus nuclei are suspended in a buffered sucrose medium and incubated at 370 in the presence of C14-labeled amino acids, isotope incorporation into protein occurs; but the amount of radioactivity appearing in the nuclear proteins depends on the nature and concentration of the cations in the incubation 907 908 BIOCHEMISTRY: ALLFREY ET AL. PROC. N. A. S.medium. This effect is shown for al...
Bond who synthesized some of the formylhistidine used in this study. 18 In the dialysis of the extracts in some of the later experiments tris-(hydroxyamino)methane buffer, 0.01 M, pH 7.5, was substituted for the triethanolamine buffer, 0.01 M, pH 7.5, described previously4 as this buffer did not require a correction in the protein determinations.
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