Relationship between medium pH and that of the lysosomal matrix as studied by two independent methods

Abstract

1. 1. The method of estimating the intralysosomal pH by measuring the distribution of [ 14C]methylamine in lysosomes isolated from the livers of Triton WR 1339-treated rats has been critically examined. 2. 2. In lysed lysosomes, methylamine is bound to the membrane fragments, but this binding can be completely suppressed by increasing the concentration of monovalent cations in the medium. 3. 3. In intact lysosomes, the binding of [ 14C]methylamine is only partly inhibited by monovalent cations at 25°C. 4. 4. The accumulation of [ 14C]methylamine in intact lysosomes is progressively inhibited as the concentration of methylamine is increased. A similar inhibition of [ 14C]methylamine accumulation is obtained with NH 4Cl. 5. 5. Similar values for the intralysosomal pH were obtained from measurements of the distribution of methylamine, dimethylamine and trimethylamine, which are accumulated in the lysosomes, and of 5,5-dimethyloxazolidinedione-2,4, which is excluded. 6. 6. The breakdown of endocytosed 125I-labelled bovine serum albumin in intact isolated lysosomes, is much less sensitive to the pH of the medium than the breakdown of added protein by particularly at high medium pH, but have no effect on the breakdown by lysed lysosomes. 7. 7. The intralysosomal pH has been estimated by comparing the rate of breakdown of endocytosed 1 2 5I-labelled albumin in intact lysosomes as a function of medium pH with that of added 1 2 5I-labelled albumin lysed lysosomes at different pH values. The values obtained agree well with those calculated from the distribution of [ 1 4C]methylamine. 8. 8. Methylamine and NH 4CL inhibit the breakdown of 1 2 5I-labelled albumin in intact lysosomes, particularly at high medium pH, but have no effect on the breakdown by lysed lysosomes. 9. 9. it is concluded that a pH difference across the lysosomal membrane (more acidic inside than outside) is maintained by the presence of indiffusible negatively charged groups within hte lysosomes, and by the permeation across the lysosomal membrane of protons together with permeant anoins (or of OH - in exchange for anion).