BINDING OF 7-CHLORO-4-NITROBENZO-2-OXA-1,3-DIAZOLE TO AN ESSENTIAL CYSTEINE RESIDUE(S) IN THE TONOPLAST H+-ATPASE FROM MUNG BEAN (VIGNA-RADIATA L) HYPOCOTYLS

Vacuolar-type H+-ATPase was solubilized from tonoplasts of mung bean (Vigna radiata L.) and purified on a Mono Q anion-exchange column by fast protein liquid chromatography. The purified enzyme was inactivated by the reactive adenine analog, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-CI). This inactivation was reversed by addition of dithiothreitol (DTT). Inactivation by NBD-Cl was prevented by Mg-ADP, a competitive inhibitor of ATPase. [C-14]NBD-Cl predominantly modified the 68-kilodalton subunit and the degree of C-14 incorporation was decreased in the presence of Mg-ADP or upon subsequent addition of DTT. The loss of activity followed pseudo first-order kinetics with respect to NBD-Cl concentration, and double log plots of pseudo first-order rate constants versus reagent concentration yielded a straight line with a slope of 0.957. The NBD-modified/inactivated enzyme showed an absorbance maximum at 418 nanometers and a fluorescence emission peak at 515 nanometers. The absorption and fluorescence emission spectra of the NBD-modified enzyme were essentially the same as those of the model compound, N-acetyl-S-NBD cysteine. Absorbance by the modified enzyme at 418 nanometers disappeared upon addition of DTT, which coincided with the restoration of ATPase activity and the decrease in bound [C-14]NBD-Cl. These findings show that NBD-Cl modifies an essential cysteine residue(s) at or near the catalytic site in the 68-kilodalton subunit of tonoplast H+-ATPase and that the modification closely correlates with the loss of ATPase activity.