Sodium-Dependent „transport” Reactions in the Cell Nucleus and Their Role in Protein and Nucleic Acid Synthesis

Abstract

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...

Effects of Na+ on Sugar and Amino Acid Transport in Striated Muscle*

“…The recent demonstration by Skou (19) (24) and the fact that active amino acid transport in the human reticulocyte diminishes considerably or disappears concomitant with the loss of subcellular organelles known to contain Na+-and K+-dependent ATPase activity (e.g., nucleus and endoplasmic reticulum) (25,26) are findings consistent with the suggestion that the ATPase complex must be structurally coupled to a specific carrier system. On the other hand, the ability to markedly depress Na+ transport with small quantities of strophanthin K (1 x 10-5 Ml) without a concomitant inhibition of AI13 penetration is consistent with the possibility that there are multiple specific forms of Na+-and K+-activated ATPase.…”

Electron-Histochemical Investigation of the Enzyme Activity of Nuclear Ribonucleoproteins

“…These findings are in a good accordance with numerous biochemical (1,14,17,25,35,36) and histochemical (5,45) data.…”

Evidence for an Amino Acid Transport System in Nuclei Isolated from Embryonic Heart

“…The lack of ouabain inhibition of ATP hydrolysis stimulated by Nai-or K+ in the absence of amino acid supports the assumption that the effect of these cations on the enzyme is not related to their transport across the nuclear membrane per se. In this respect, qualified results using thymus nuclei [22] do not support the latter possibility, and other work on isolated rat liver nuclei give no evidence for active transport of Na+ [8,28]. Even though Na+ is concentrated in the nucleus about I1 : 1 compared to the cytoplasm.…”

“…It was first proposed by Allfrey and colleagues [22,27] using isolated calf thymus nuclei that there is a mechanism, very probably enzymatic, which governs the entry of amino acids into the nucleus. The uptake of certain amino acids into the "free amino acid pool" was shown to be a Na+-dependent process separate from the subsequent activation and utilization of the amino acid in protein synthesis, which do not require Na+.…”