The fluorescent pH-indicator BCECF: An inhibitor of the vacuolar ATPase from Chenopodium rubrum

Selective vacuolar accumulation of the fluorescent pH-indicator 2', 7'-bis (2-Carboxyethyl)-5(6)-carboxyfluorescein (BCECF) may allow to study vacuola r acidification in vivo (Brauer et al., 1995), which in plant cells is driv en by two distinct proton pumps: the V-ATPase (EC 3.6.1.3) and the V-PPase (EC 3.6.1.1). The use of BCECF may help to investigate these proton pumps i n vivo. A necessary precondition for such a study is that both enzymes are not affected by the indicator. This was investigated using tonoplast enrich ed membrane vesicles from Chenopodium rubrum L. suspension cells. We found that BCECF was a strong inhibitor of the V-ATPase. Half-maximal inhibition of the V-ATPase was observed with 10 mu mol/L BCECF The Hill constant (n(H) = 1.3) for inhibition of the V-ATPase insdicates the presence of two bindi ng sites for BCECF BCECF was less inhibitory to the vacuolar PPase than to the vacuolar ATPase; half-maximal inhibition was obtained with 500 mu mol/L BCECF (n(H) = 0.5). Moreover, we found that BCECF (2 mu mol/L) increased t he affinity of the V-ATPase for its specific inhibitor bafilomycin, i.e. it decreased the concentration for half-maximal inhibition from 2 nmol/L to 0 .17 nmol/L bafilomycin. We suggest positive cooperation between binding of BCECF and bafilomycin to the V-ATPase. Fluorescein acid and its derivatives 5-carboxyfluorescein, 6-carboxyfluorescein, BCECF and BCECF-AM (acetoxymet hyl ester of BCECF) gave similar inhibition of the V-ATPase, indicating tha t the chromophore fluorescein interacts with the enzyme. Finally, living ce lls were incubated for 1 h in BCECF-AM (5 mu mol/L), a cell-permeable ester derivative of BCECF that is hydrolyzed by cytosolic esterases to yield int racellularly trapped BCECF Tonoplast: vesicles were isolated by sucrose den sity gradient centrifugation from cells washed free from the loading soluti on. V-ATPase and V-PPase activity of these tonoplast vesicles showed a decr ease of 18 % and 45 %, respectively. Together, these data indicate that the widely used in vivo pH-indicator BCECF alters V-ATPase activity and proper ties and can therefore interfere with the pH of cellular compartments acidi fied or alkalized by the V-ATPase, whereas BCECF-AM added to living cells i nhibits the V-PPase by an interaction with cellular mechanisms regulating i ts activity.