Inhibition of tonoplast ATPase by 2',3'-dialdehyde derivatives of ATP

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 radiata L.). The dial-ATP caused marked inactivation of enzymatic activities of both membrane-bound and...

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Published in:Plant physiology (Bethesda) 1992-01, Vol.98 (1)
Main Authors: Chow, W.M. (National Tsing Hua University, Taiwan, R.O.C.), Tzeng, C.M, Chen, C.S, Kuo, S.Y, Wang, M.Y, Pan, R.L
Format: Article
Language:English
Subjects:
ACTIVIDAD ENZIMATICA
ACTIVITE ENZYMATIQUE
ADENOSINE TRIPHOSPHATE
ADENOSINTRIFOSFATO
HIDROLASAS
HYDROLASE
INHIBIDORES DE ENZIMAS
INHIBITEUR D'ENZYME
MEMBRANAS CELULARES
MEMBRANE CELLULAIRE
PLANTULAS
PLANTULE
VIGNA RADIATA
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Summary: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 radiata 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 dial-ATP 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 Ki 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
ISSN:0032-0889
1532-2548