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Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents.

Proceedings of the National Academy of Sciences of the United States of America, 01 Jul 1978, 75(7):3327-3331
https://doi.org/10.1073/pnas.75.7.3327 PMID: 28524 PMCID: PMC392768

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Abstract 

A quantitative method is described for the measurement of intralysosomal pH in living cells. Fluorescein isothiocyanate-labeled dextran (FD) is endocytized and accumulates in lysosomes where it remains without apparent degradation. The fluorescence spectrum of this compound changes with pH in the range 4-7 and is not seriously affected by FD concentration, ionic strength, or protein concentration. Living cells on coverslips are mounted in a spectrofluorometer cell and can be perfused with various media. The normal pH inside macrophage lysosomes seems to be 4.7-4.8, although it can drop transiently as low as 4.5. Exposure of the cells to various weak bases and to acidic potassium ionophores causes the pH to increase. The changes in pH are much more rapid than is the intralysosomal accumulation of the weak bases. Inhibitors of glycolysis (2-deoxyglucose) and of oxidative phosphorylation (cyanide or azide) added together, but not separately, cause the intralysosomal pH to increase. These results provide evidence for the existence of an active proton accumulation mechanism in the lysosomal membrane and support the theory of lysosomal accumulation of weak bases by proton trapping.

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Proc Natl Acad Sci U S A. 1978 Jul; 75(7): 3327–3331.
PMCID: PMC392768
PMID: 28524

Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents.

S Ohkuma and B Poole
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Abstract

A quantitative method is described for the measurement of intralysosomal pH in living cells. Fluorescein isothiocyanate-labeled dextran (FD) is endocytized and accumulates in lysosomes where it remains without apparent degradation. The fluorescence spectrum of this compound changes with pH in the range 4-7 and is not seriously affected by FD concentration, ionic strength, or protein concentration. Living cells on coverslips are mounted in a spectrofluorometer cell and can be perfused with various media. The normal pH inside macrophage lysosomes seems to be 4.7-4.8, although it can drop transiently as low as 4.5. Exposure of the cells to various weak bases and to acidic potassium ionophores causes the pH to increase. The changes in pH are much more rapid than is the intralysosomal accumulation of the weak bases. Inhibitors of glycolysis (2-deoxyglucose) and of oxidative phosphorylation (cyanide or azide) added together, but not separately, cause the intralysosomal pH to increase. These results provide evidence for the existence of an active proton accumulation mechanism in the lysosomal membrane and support the theory of lysosomal accumulation of weak bases by proton trapping.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Reijngoud DJ, Tager JM. The permeability properties of the lysosomal membrane. Biochim Biophys Acta. 1977 Nov 14;472(3-4):419–449. [Abstract] [Google Scholar]
  • Goldman R, Rottenberg H. Ion distribution in lysosomal suspensions. FEBS Lett. 1973 Jul 1;33(2):233–238. [Abstract] [Google Scholar]
  • Riejngoud DJ, Tager JM. Measurement of intralysosomal pH. Biochim Biophys Acta. 1973 Jan 24;297(1):174–178. [Abstract] [Google Scholar]
  • Henning R. pH gradient across the lysosomal membrane generated by selective cation permeability and Donnan equilibrium. Biochim Biophys Acta. 1975 Aug 20;401(2):307–316. [Abstract] [Google Scholar]
  • Mego JL, Farb RM, Barnes J. An adenosine triphosphate-dependent stabilization of proteolytic activity in heterolysosomes. Evidence for a proton pump. Biochem J. 1972 Jul;128(4):763–769. [Europe PMC free article] [Abstract] [Google Scholar]
  • de Duve C, de Barsy T, Poole B, Trouet A, Tulkens P, Van Hoof F. Commentary. Lysosomotropic agents. Biochem Pharmacol. 1974 Sep 15;23(18):2495–2531. [Abstract] [Google Scholar]
  • Wibo M, Poole B. Protein degradation in cultured cells. II. The uptake of chloroquine by rat fibroblasts and the inhibition of cellular protein degradation and cathepsin B1. J Cell Biol. 1974 Nov;63(2 Pt 1):430–440. [Europe PMC free article] [Abstract] [Google Scholar]
  • Seglen PO, Reith A. Ammonia inhibition of protein degradation in isolated rat hepatocytes. Quantitative ultrastructural alterations in the lysosomal system. Exp Cell Res. 1976 Jul;100(2):276–280. [Abstract] [Google Scholar]
  • Somlyo AP, Garfield RE, Chacko S, Somlyo AV. Golgi organelle response to the antibiotic X537A. J Cell Biol. 1975 Aug;66(2):425–443. [Europe PMC free article] [Abstract] [Google Scholar]
  • COHN ZA, BENSON B. THE DIFFERENTIATION OF MONONUCLEAR PHAGOCYTES. MORPHOLOGY, CYTOCHEMISTRY, AND BIOCHEMISTRY. J Exp Med. 1965 Jan 1;121:153–170. [Europe PMC free article] [Abstract] [Google Scholar]
  • Morton HJ. A survey of commercially available tissue culture media. In Vitro. 1970 Sep-Oct;6(2):89–108. [Abstract] [Google Scholar]
  • Leighton F, Poole B, Beaufay H, Baudhuin P, Coffey JW, Fowler S, De Duve C. The large-scale separation of peroxisomes, mitochondria, and lysosomes from the livers of rats injected with triton WR-1339. Improved isolation procedures, automated analysis, biochemical and morphological properties of fractions. J Cell Biol. 1968 May;37(2):482–513. [Europe PMC free article] [Abstract] [Google Scholar]
  • Reijngoud DJ, Tager JM. Chloroquine accumulation in isolated rat liver lysosomes. FEBS Lett. 1976 Apr 15;64(1):231–235. [Abstract] [Google Scholar]
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