This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Web of Science (13) Citing Articles via Google Scholar Google Scholar Articles by Yamanishi, H. Articles by Kasamo, K. Search for Related Content PubMed PubMed Citation Articles by Yamanishi, H. Articles by Kasamo, K. Agricola Articles by Yamanishi, H. Articles by Kasamo, K.

Membranes and Bioenergetics

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 ¹

Molecular Function Laboratory, 2-1-2 Kannondai, Tsukuba Science City, Ibaraki, 305, Japan, Biological Function Division, National Food Research Institute, 2-1-2 Kannondai, Tsukuba Science City, Ibaraki, 305, Japan

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-Cl). This inactivation was reversed by addition of dithiothreitol (DTT). Inactivation by NBD-Cl was prevented by Mg-ADP, a competitive inhibitor of ATPase. [¹⁴C]NBD-Cl predominantly modified the 68-kilodalton subunit and the degree of ¹⁴C 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 [¹⁴C]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.

This article has been cited by other articles:

				M. Yamaguchi and K. Kasamo Modulation of Proton Pumping across Proteoliposome Membranes Reconstituted with Tonoplast H+-ATPase from Cultured Rice (Oryza sativa L. var. Boro) Cells by Acyl Steryl Glucoside and Steryl Glucoside Plant Cell Physiol., July 15, 2002; 43(7): 816 - 822. [Abstract] [Full Text] [PDF]

				M. Yamaguchi and K. Kasamo Modulation in the Activity of Purified Tonoplast H+-ATPase by Tonoplast Glycolipids Prepared from Cultured Rice (Oryza sativa L. var. Boro) Cells Plant Cell Physiol., May 1, 2001; 42(5): 516 - 523. [Abstract] [Full Text] [PDF]