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.
Highlights
H+- plast of intact cells [1].These results takentogether with the ATPasesfromoatroots were comparedtodeterpresence of ATPase activities and ATP-dependentH+pumpmine whether the two enzymes have similar mecha- ing in isolated vacuoles have established that tonoplast memnisms
The H+-ATPase associated with low-density microsomal vesicles is referred to as a tonoplast-type H+-ATPafsoer the followingreasons
The mitochondrial ATPase activity was measured in a 0.5-ml reaction mixture containing 30 mM BTP
Summary
H+- plast of intact cells [1].These results takentogether with the ATPasesfromoatroots were comparedtodeterpresence of ATPase activities and ATP-dependentH+pumpmine whether the two enzymes have similar mecha- ing in isolated vacuoles have established that tonoplast memnisms. H+pumping inlow-density microsomal vesicles branes possess a H+-pumpingATPase (Ref. 3 and references reflected activity from the tonoplast-type ATPase, as therein). The H+-ATPase associated with low-density microsomal vesicles is referred to as a tonoplast-type H+-ATPafsoer the followingreasons. The mitochondrial ATPase or oxonol absorbance changes [6] This H+-ATPase can be was 100 times more sensitive to N,N‘-dicyclohexyl- clearly distinguished from the H+-pumping ATPase of the carbodiimide inhibition than the tonoplast H*-ATPasmei.tochondria because of its insensitivity to oligomycin or. These results suggest the tonoplast and the mitochon-azide [3, 7, 8]. $ Present address: Biology DepartmentW, uhan University, Wuhan, Hubei, China
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
