Abstract
The purified tonoplast H+-ATPase from oat roots (Avena sativa L. var. Lang) consists of at least three different polypeptides with masses 72, 60, and 16 kDa. We have used covalent modifiers (inhibitors) and polyclonal antibodies to identify the catalytic subunit of the H+-pumping ATPase. The inactivation of ATPase activity by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (Nbd-Cl, an adenine analog) was protected by MgATP or MgADP, and showed kinetic properties consistent with active site-directed inhibition. Under similar conditions, [14C]Nbd-Cl preferentially labeled the 72-kDa polypeptide of the purified ATPase. This binding was reduced by MgATP or 2' (3')-)O-(2,4,6-trinitrophenyl) ATP. Nbd-Cl probably modified cysteinyl--SH or tyrosyl--OH groups, as dithiothreitol reversed both ATPase inactivation and [14C]Nbd-Cl binding to the 72-kDa subunit. The finding that N-ethylmaleimide inhibition of ATPase activity was protectable by nucleotides is consistent with the idea of sulfhydryl groups in the ATP-binding site. Polyclonal antibody made to the 72-kDa polypeptide specifically reacted (Western blot) with a 72-kDa polypeptide from both tonoplast-enriched membranes and the purified tonoplast ATPase, but it did not cross-react with the mitochondrial or Escherichia coli F1-ATPase. The antibody inhibited tonoplast ATPase and H+-pumping activities. We conclude from these results that the 72-kDa polypeptide of the tonoplast H+-ATPase contains an ATP- (or nucleotide-) binding site that may constitute the catalytic domain.
Highlights
Thepurified tonoplast H*-ATPase from oat roots C1, and DIDS [10, 11].The tonoplast ATPase is insensitive
Bowman et al [19] found that an adenine ethylmaleimide inhibition of ATPase activity was pro- analog, Nbd-C1, labeled a 70-kDa polypeptide of the vacuolar tectable by nucleotides is consistent with the idea of membrane ATPase from Neurospora crassa
Reacted (Western blot) with a 72-kDa polypeptide from kDa polypeptide constitutes a proton channel [11, 12, 15], both tonoplast-enriched membranes and the purified analogous to theDCCD-binding proteolipid of the mitochontonoplast ATPase, but it did not cross-react with the drial FIFo-ATPase [21]
Summary
ATPase than MgADP (Ki= 0.35 mM; Ref. 10). A Dixon plot shows that TNP-ATPbinds tightlyto thecatalytic siteof the tonoplast ATPase, witha Ki of 40 nM (Fig. 8). No cross-reactivity was seen with a 100-kDa polygroups Consistent with these results, dithiothreitol reduced peptide (mass of the plasma membrane H+-ATPase) [45] in [14C]Nbd-C1binding to the72-kDa polypeptide of the purified a plasma membrane-enriched fraction (not shown). It is clear that a substantial amount (at least 65%) tibody did react with a single polypeptide of approximately of the Nbd-C1 binding was reversed It did not cross- cles was inhibited by anti-72 (Fig. 11).These preliminary reactwith either the mitochondrial or Escherichia coli F,- results show that the anti-72 of the tonoplast ATPase was highly specific, and thaitt inhibited ATPase activitpyrobably by perturbing the catalytic domainof the 72 kDa
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
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 call
