Acid titrations of intact and butanol-treated cells of Streptococcus faecalis revealed that nearly all of the intracellular K+ ions could diffuse into the suspending medium in association with small anions, including ribonucleic acid breakdown products, when the cell membrane was damaged. In contrast, nearly all of the intracellular Mg2+ ions appeared to be firmly bound to stable internal cell components but could be displaced reversibly by hydronium ions. The cell membrane acted as a barrier to ion movements, and Mg2+ displacement from intact cells required more acid conditions, by as much as 2.5 pH units, than did displacement from butanol-damaged cells. Some 15 to 20% of the cell magnesium appeared to be associated with surface structures in that it could be removed at pH 7 with ethylenediaminetetraacetic acid or displaced by Co2+, Ni2+, Sr2+, or La3+. Magnesium could be displaced from isolated cell walls and membranes by hydronium ions in the pH range from 5 to 3, over which carboxyl groups were titrated. Displacement of magnesium from ribosomes also took place between pH 5 and 3, but it was more difficult to identify the magnesium-releasing groups because both protein carboxyl groups and purine and pyrimidine ring nitrogens can become protonated in this pH range. Isolated protoplast membranes remained structurally intact when completely depleted of magnesium. Furthermore, protoplasts isolated from intact cells were found to have greatly enhanced resistance to osmotic shock in acid media, even when solute loss was not extensive. Osmotic resistance was lost when the protoplasts were again placed in neutral media, and this reversibility suggested that acidification caused changes in the physical properties of membranes as well as solute leakage from cells
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