Production and characterization of a monoclonal antibody to vacuolar H+ATPase of renal epithelia.
A monoclonal antibody to vacuolar H+ATPase isolated from bovine kidney medulla was produced and characterized by immunoprecipitation and immunocytochemistry. The antibody, immobilized on beads, specifically immunoprecipitated both solubilized N-ethylmaleimide-sensitive ATPase activity and proton-transporting vesicles from renal microsomes; control experiments with an "irrelevant" monoclonal antibody showed no immunoprecipitated activity. By fluorescent immunocytochemistry, the antibody stained the membranes of intracellular vacuolar compartments in LLC-PK1 cells. Immunocytochemical staining showed that the monoclonal antibody colocalized partially with N-(3-[2,4-dinitrophenyl)amino)propyl)-N-(3-amino-propyl)methylamine, a probe for acidic compartments, with the endocytic markers dextran and transferrin, with the lysosomal probe alpha 2-macroglobulin, and with clathrin. The anti-vacuolar H+ATPase antibody showed no colocalization with staining for mitochondrial H+ATPase. The anti-vacuolar H+ATPase antibody should serve as a specific probe for examining the distribution and dynamics of the vacuolar proton pump in renal epithelial cells.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002460:Cell Line; D004848:Epithelium / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D007668:Kidney / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000276:immunology* / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism; D014617:Vacuoles / Q000201:enzymology; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D000911:Antibodies, Monoclonal; D014168:Transferrin; D001619:beta-N-Acetylhexosaminidases; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide