Identification and characterization of a proton pump on lysosomes by fluorescein-isothiocyanate-dextran fluorescence.

TLDR

Fluorescein‑isothiocyanate‑dextran was delivered into hepatic lysosomes via intraperitoneal injection in rats. The lysosomal pH (~5) could be acidified by MgATP, a process requiring chloride and sensitive to N‑ethylmaleimide and quercetin but not to oligomycin, with Mn²⁺, Co²⁺, and Fe²⁺ substituting for Mg²⁺ while Ca²⁺ could not; Cu²⁺, Zn²⁺, and Cd²⁺ were inhibitory, and differences between normal lysosomes and tritosomes were observed, providing evidence for an electrogenic MgATP‑driven H⁺‑ATPase on lysosomes.

Abstract

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.