Inhibition of tonoplast ATPase by 2′,3′-dialdehyde derivative of ATP

Wing Ming Chow, Chi Meng Tzeng, Chung Shung Chen, Soong Yu Kuo, May Yun Wang, Rong Long Pan

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

The 2′,3′-dialdehyde derivative of ATP (dial-ATP) has been shown to be an affinity label for the ATP binding site of the H+-ATPase from tonoplast of etiolated mung bean seedlings (Vigna radiate L.). The dial-ATP caused marked inactivation of enzymatic activities of both membrane-bound and soluble ATPase and its associated proton translocation. The inactivation was reversible, but could be stabilized by NaBH4. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern revealed that the dialATP binding site was in the large (A) subunit of ATPase. The inhibition could be substantially protected by its physiological substrate ATP, pyrophosphate, and nucleotides in the decreasing order: ATP > pyrophosphate > ADP = AMP > GTP > CTP = UTP. A Lineweaver-Burk plot showed that the mode of inhibition was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first order kinetics with a K1 of 4.1 millimolar, a minimum inactivation half-time of 20 seconds, and a pseudo-first order rate constant of 0.035 s-1. The double logarithmic plot of apparent rate constant versus dial-ATP concentration gave a slope of 0.927, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labeling studies with [3H]dial-ATP indicate that the incorporation of approximately 1 mole of dial-ATP per mole ATPase is sufficient to completely inhibit the ATPase. A working model of nonequivalent subunits for enzymatic mechanism of vacuolar ATPase is suggested.

Original languageEnglish (US)
Pages (from-to)44-52
Number of pages9
JournalPlant Physiology
Volume98
Issue number1
DOIs
StatePublished - 1992

ASJC Scopus subject areas

  • Physiology
  • Genetics
  • Plant Science

Fingerprint Dive into the research topics of 'Inhibition of tonoplast ATPase by 2′,3′-dialdehyde derivative of ATP'. Together they form a unique fingerprint.

Cite this