Identification and characterization of a proton pump on lysosomes by fluorescein-isothiocyanate-dextran fluorescence.
Fluorescein isothiocyanate-conjugated dextran was introduced preferentially into hepatic lysosomes by intraperitoneal injection into rats. The pH in isolated lysosomes, measured by fluorescein fluorescence, was approximately 5 and gradually increased in KCl (to 7.0) at 25 degrees C. In the presence of Mg2+, ATP caused acidification of lysosomes that was reversed by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Mn2+, Co2+, and Fe2+ could replace Mg2+ but Ca2+ could not. Cu2+, Zn2+, and Cd2+ were inhibitory. A membrane-permeant anion, in practice chloride, was required for this acidification. ATP analogues, including 5'-adenylyl imidodiphosphate, could not be substituted for ATP. ATP-driven acidification was sensitive to N-ethylmaleimide and quercetin but insensitive to oligomycin, ouabain, and vanadate. There were some differences between "normal" lysosomes and tritosomes; the acidification was resistant to azide and N,N'-dicyclohexylcarbodiimide in normal lysosomes but sensitive to these reagents in tritosomes. These results provide evidence for the presence of an electrogenic proton pump driven by MgATP (H+-ATPase) on lysosomes.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active; D003911:Dextrans; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000502:physiology; D008247:Lysosomes / Q000502:physiology*; D008297:Male; D010539:Permeability; D011522:Protons; D051381:Rats
D003911:Dextrans; D005452:Fluoresceins; D011522:Protons; C015219:fluorescein isothiocyanate dextran; D000255:Adenosine Triphosphate; D016650:Fluorescein-5-isothiocyanate