title: ATP-dependent acidification of membrane vesicles isolated from purified rat liver lysosomes. Acidification activity requires phosphate.
abstract: Membrane vesicles were isolated from purified liver lysosomes of rats treated with Triton WR-1339. In order to preserve ATP-dependent acidification activity, proteolysis of membranes was minimized by adding protease inhibitors and by centrifuging to form dilute bands of vesicles rather than highly concentrated pellets. The membrane vesicle fraction represented about 20% of the total lysosomal protein, 80% of the ATPase activity, and 3% of the solute proteins as marked by N-acetylglucosaminidase. About one-half of the membranes were oriented right side out. The space unavailable to [14C]sucrose corresponded to 3 microliters/mg of membrane protein which indicates that the membranes form vesicles about one-tenth the size of lysosomes. Uptake of either [14C]methylamine or [14C]chloroquine by lysosomal membrane vesicles was ATP-dependent, indicating acidification of the intravesicle space. The acidification activity was inhibited when either 1.5 microM carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, 100 microM dicyclohexylcarbodiimide, or millimolar concentrations of such permeant weak bases as ammonium sulfate and dansyl cadaverine were added. Acidification of lysosomal vesicles by ATP occurred electroneutrally. This acidification activity was not dependent on added salts but was inhibited by the anion transport inhibitors pyridoxal phosphate and diisothiocyanostilbene disulfonic acid, thus suggesting co-transport of protons and anions. Results which indicate that phosphate is the transported anion included (a) ATP-dependent uptake of [32P]phosphate by lysosomal membrane vesicles and (b) stimulation of ATP-dependent acidification of these vesicles by added phosphate. These observations provide further evidence that maintenance of the acid intralysosomal pH necessary for activation of lysosomal hydrolases is due to an ATP-driven proton pump located in the lysosomal membrane.
mesh_terms: D000118:Acetylglucosaminidase / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002738:Chloroquine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D008744:Methylamines / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D051381:Rats; D013395:Sucrose / Q000378:metabolism
chemical_list: D008744:Methylamines; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D013395:Sucrose; D002738:Chloroquine; D000255:Adenosine Triphosphate; C027451:methylamine; D000118:Acetylglucosaminidase; D000251:Adenosine Triphosphatases