title: Similarities and differences between the tonoplast-type and the mitochondrial H+-ATPases of oat roots.
abstract: The native tonoplast and the mitochondrial H+-ATPase from oat roots were compared to determine whether the two enzymes have similar mechanisms. H+ pumping in low-density microsomal vesicles reflected activity from the tonoplast-type ATPase, as ATPase activity and ATP-dependent H+ pumping (quinacrine fluorescence quenching) showed similar sensitivities to inhibition by N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, 4,4'-diisothiocyano-2,2'-stilbene disulfonate, nitrate, quercetin, or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. The tonoplast-type ATPase was stimulated by C1-,Br- greater than HCO3- whereas the mitochondrial ATPase was stimulated by HCO3- much greater than C1-,Br-. Both enzymes hydrolyzed ATP preferentially and were inhibited competitively by AMP or ADP. Apart from resistance to azide, the tonoplast-type ATPase was strikingly similar in its inhibitor sensitivities to the mitochondrial ATPase. The insensitivity to vanadate of both enzymes suggests the reaction mechanisms do not involve a covalent phosphoenzyme. Inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide and protection by ATP suggests tyrosine and cysteine residues are in the catalytic site of the tonoplast ATPase. The mitochondrial ATPase was 100 times more sensitive to N,N'-dicyclohexyl-carbodiimide inhibition than the tonoplast H+-ATPase. These results suggest the tonoplast and the mitochondrial H+-ATPases share common steps in their catalytic and vectorial reaction mechanisms, yet sufficient differences exist to indicate they are two distinct ATPases.
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: H
authors.initials: H
authors.lastname: Sze
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D002458:Cell Fractionation; D002523:Edible Grain / Q000201:enzymology; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology* / Q000648:ultrastructure; D009940:Organoids / Q000201:enzymology* / Q000648:ultrastructure; D010944:Plants / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D006180:Proton-Translocating ATPases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid