Properties of H(+)-ATPase from rat liver lysosomes as revealed by reconstitution into proteoliposomes.
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-beta-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.
Okamoto
M
M
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Kanazawa University.
Hiratani
N
N
Arai
K
K
Ohkuma
S
S
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D003902:Detergents; D004926:Escherichia coli; D007700:Kinetics; D008081:Liposomes; D008247:Lysosomes / Q000201:enzymology*; D010713:Phosphatidylcholines; D010743:Phospholipids; D011510:Proteolipids / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D012995:Solubility; D013379:Substrate Specificity; D013863:Thioglucosides / Q000494:pharmacology
D002413:Cations, Divalent; D003902:Detergents; D008081:Liposomes; D010713:Phosphatidylcholines; D010743:Phospholipids; D011510:Proteolipids; D013863:Thioglucosides; C020485:proteoliposomes; C024990:asolectin; C042989:n-octyl-beta-D-thioglucopyranoside; D006180:Proton-Translocating ATPases