Platinum complexes inhibit ATP‐driven basic dye uptake in lysosomes from rat liver

Electrogeneicity of the lysosomal proton pump

“…The activities of the following enzymes were determined as markers for various subcellular val , organelles: 5 ' -nucleotidase for plasma membranes [ 141, cytochrome c oxidase for mitochondria [15], glucose-6-phosphatase [ 141 for endoplasmic reticulum, and acid phosphatase [16] with minor modifications [9] for lysosomes. P, was measured as in [17].…”

“…In some trials purified lysosome preparations were obtained by centrifugation of the lysosome-enriched fraction in a Percoll gradient. The heavier density fractions were collected as in [9,18] and subjected to acid phosphatase and 5 ' -nucleotidase assay. Enrichment in specific acid phosphatase activity over the homogenate of the subfraction LZ defined in [13] was as high as 60-70-fold in the best preparations.…”

“…Acridine orange (Merck, purified as in [9]) uptake (or release) was followed spectrophotometritally, by measuring the decrease (or increase) in dye absorbance at 492 nm, with 550 nm as the reference wavelength [21], using a double-wavelength apparatus, constructed in the workshop of the Institute of Physics of the University of Padova. pH changes were measured with a glass electrode and a Radiometer pHM 84 pH-meter connected to a Linseis recorder.…”

“…It has been previously shown that nigericin, a known catalyzer of H+/K+ exchange, may induce lysosome uptake or release of acridine dye, depending on the medium's K+ concentration [9]. When, in addition to the dye technique ( fig.lA), an H+ electrode was used to follow ~ge~cin-induced H+ translocation, it was observed that lysosomal uptake or release of H+, or no H+ movement also depended on the K+ concentration of the medium ( fig.lB).…”

“…The experimental findings in recent years in support of the thesis of a proton 'pump' as the main mechanism for internal lysosomal acidification [l-4] have been based on various techniques: measurement of transmembrane pH gradient by means of methyl~ne distribution [5]; determination of energy-dependent amino acid accumulation in lysosomes (61; use of the fluorescent pH indicator, fluorescein isothiocyanate-conjugated dextran, trapped within lysosomes by endocytosis [7], and spectrophotometric [8,9] or fluorimetric [lo] monitoring of the dye acridine orange, which accumulates in lysosomes as a weak base. It was questioned in [5,7,8] whether the claimed H+-ATPase was electrogenic or electroneutral.…”