Acridine orange as a fluorescent probe for lysosomal proton pump

Area of Science:

• Cell Biology
• Biochemistry
• Membrane Transport

Background:

• Lysosomes are key organelles for cellular degradation.
• Understanding lysosomal pH regulation is vital for cellular processes.
• Previous studies suggested the presence of a lysosomal proton pump.

Purpose of the Study:

• To investigate the mechanism of lysosomal acidification.
• To confirm the existence and characteristics of a lysosomal H+-pump.
• To utilize acridine orange fluorescence for pH measurements.

Main Methods:

• In vitro experiments using pure lysosomal particles (tritosomes).
• Acridine orange dye was used to measure the pH gradient across the lysosomal membrane.
• Assessing the effect of Mg-adenosine triphosphate (Mg-ATP) on lysosomal pH.
• Testing the sensitivity of acidification to various inhibitors.

Main Results:

• Acridine orange fluorescence quenching correlated with lysosomal acidity (delta pH).
• Mg-adenosine triphosphate (Mg-ATP) induced lysosomal acidification.
• The acidification process was inhibited by N,N'-dicyclohexylcarbodiimide, N-ethylmaleimide, and azide.
• Oligomycin, ouabain, and vanadate did not affect the acidification.

Conclusions:

• The findings provide strong evidence for an active proton (H+) pump in the lysosomal membrane.
• This pump is responsible for maintaining the acidic environment within lysosomes.
• The results support and extend previous hypotheses regarding lysosomal H+-pump activity.