caa a22 4500 397556519 CHVBK 20180308164803.0 cr unu---uuuuu 161202e199609 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021456 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021456 Properties of H+-ATPase from Rat Liver Lysosomes as Revealed by Reconstitution into Proteoliposomes [Elektronische Daten] [Masayuki Okamoto, Norikazu Hiratani, Kunizo Arai, Shoji Ohkuma] The properties of H+-ATPase from rat liver lysosomes were analyzed by reconstituting proton pump activity from solubilized enzyme and Escherichia coli phospholipids in proteoliposomes devoid of anion-channels. The reconstitution procedure involved solubilization of the ATPase with n-octyl-β-D-thioglucoside in the presence of asolectin, and incorporation of the solubilized enzyme into E. coli phospholipid liposomes by dilution, freeze-thawing, and sonication. Proton pump activity of reconstituted H+-ATPase as detected by the ATP-dependent quenching of acridine orange fluorescence indicated that ATP can be replaced with dATP and to a lesser extent with GTP, but not with any other nucleotide, that Mg2+ can be replaced with Mn2+, but not with Ca2+, Sr2+, or Ba2+, that Zn2+, Pd2+, Cd2+, and Hg2+ were inhibitory, and that the enzyme was sensitive to inhibitors of v-type H+-ATPase, including bafilomycin A1, N-ethylmaleimide, DCCD, DIDS, and tri-n-butyltin. The enzyme showed unique sensitivity to anions and was activated by chloride, fluoride, and bromide from inside, but not from outside the vesicles. It was inhibited by sulfate, sulfite, and thiocyanate from outside the vesicles, and by nitrate from both inside and outside the vesicles. © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence anion-sensitivity nationallicence H+-pump nationallicence lysosome nationallicence reconstitution nationallicence vacuolar H+-ATPase nationallicence Okamoto Masayuki Department of Biochemistry, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Ishikawa 920 aut Hiratani Norikazu Department of Biochemistry, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Ishikawa 920 aut Arai Kunizo Department of Biochemistry, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Ishikawa 920 aut Ohkuma Shoji Department of Biochemistry, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Ishikawa 920 aut The Journal of Biochemistry Oxford University Press 120/3(1996-09), 608-615 0021-924X 120:3<608 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021456 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021456 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Okamoto Masayuki Department of Biochemistry, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Ishikawa 920 aut NATIONALLICENCE 700 1- Hiratani Norikazu Department of Biochemistry, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Ishikawa 920 aut NATIONALLICENCE 700 1- Arai Kunizo Department of Biochemistry, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Ishikawa 920 aut NATIONALLICENCE 700 1- Ohkuma Shoji Department of Biochemistry, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Ishikawa 920 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/3(1996-09), 608-615 0021-924X 120:3<608 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford