Inhibition of tonoplast ATPase from etiolated mung bean seedlings by fluorescein 5'-isothiocyanate.
Fluorescein 5'-isothiocyanate (FITC) was used to modify the lysine residue in the active site of tonoplast H(+)-ATPase from etiolated mung-bean (Vigna radiata L.) seedlings. FITC caused marked inactivation of the enzyme activities of both membrane-bound and soluble ATPase and its associated H+ translocation. The SDS/PAGE pattern revealed that the FITC-binding site was in the large (A) subunit of ATPase. Inhibition could be substantially prevented by its physiological substrate ATP, pyrophosphate and nucleotides in the decreasing order: ATP greater than pyrophosphate greater than ADP greater than AMP greater than GTP greater than CTP greater than UTP. The mode of inhibition by FITC was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first-order kinetics with a Ki of 0.33 mM, a minimum inactivation half-time of 110 s, and a first-order rate constant of 0.244 s-1. A double-logarithmic plot of apparent rate constant versus FITC concentration gave a slope of 0.913, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labelling studies indicated that the incorporation of approx. 1 mol of FITC/mol of ATPase is sufficient to inhibit ATPase completely. The enhancement and blue shift of emission maxima of FITC after modification of ATPase indicated that the labelled lysine residue was located in a relatively hydrophobic domain.
Tzeng
C M
CM
Institute of Radiation Biology, College of Nuclear Sciences, National Tsing Hua University, Hsin Chu, Taiwan, Republic of China.
Hsu
L H
LH
Pan
R L
RL
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000201:enzymology*; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology*; D005456:Fluorescent Dyes; D007425:Intracellular Membranes / Q000201:enzymology*; D007700:Kinetics; D009711:Nucleotides / Q000494:pharmacology; D010946:Plants, Medicinal; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D009711:Nucleotides; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D016650:Fluorescein-5-isothiocyanate