Role of anions in the acidification of isolated lysosomes. (A) The pH of isolated RAW macrophage lysosomes bathed in Cl⁻-containing medium was monitored by fluorescence spectrometry as described in the text. The pH gradient across the lysosomal membrane was intentionally dissipated during the course of isolation. Where indicated, lysosomal re-acidification was initiated by addition of ATP-Mg. Finally, nigericin was added to release the accumulated H⁺. (B) Isolated lysosomes were bathed in either Cl⁻-based (black squares) or gluconate-based (black triangles) medium and ATP-Mg was added as in A to initiate re-acidification. Where indicated, CcA was added concomitantly. Data are means ± SE of at least three separate experiments of each kind. Lysosomes had undergone significant acidification at 5 min after the addition of MgATP as compared with CcA controls (P = 0.007 and P = 0.0159 for the Cl⁻- and gluconate-based solutions, respectively). (C) Macrophages were incubated in medium devoid of small, monovalent cations (i.e., K⁺ and Na⁺) for 1 h. The lysosomes were then isolated in either a sucrose-based medium or a sucrose-based medium containing Cl⁻. In both cases, the media were devoid of K⁺ and Na⁺. Re-acidification was initiated as above by addition of ATP-Mg. Data are means ± SE of at least three separate experiments of each kind for panels B and C.