Add time:07/27/2019         Source:sciencedirect.com

SummarySelective vacuolar accumulation of the fluorescent pH-indicator 2′, 7′-bis (2-Carboxyethyl)-5(6)-carboxyfluorescein (BCECF) may allow to study vacuolar acidification in vivo (Brauer et ai., 1995), which in plant cells is driven 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 in vivo. A necessary precondition for such a study is that both enzymes are not affected by the indicator. This was investigated using tonoplast enriched 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 μmol/L BCECF. The Hill constant (nH = 1.3) for inhibition of the V-ATPase insdicates the presence of two binding sites for BCECF. BCECF was less inhibitory to the vacuolar PPase than to the vacuolar ATPase; half-maximal inhibition was obtained with 500 μmol/L BCECF (nH = 0.5). Moreover, we found that BCECF (2 μmol/L) increased the 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 (acetoxymethyl ester of BCECF) gave similar inhibition of the V-ATPase, indicating that the chromophore fluorescein interacts with the enzyme. Finally, living cells were incubated for 1 h in BCECF-AM (5 μmol/L), a cell-permeable ester derivative of BCECF that is hydrolyzed by cytosolic esterases to yield intracellularly trapped BCECF. Tonoplast vesicles were isolated by sucrose density gradient centrifugation from cells washed free from the loading solution. V-ATPase and V-PPase activity of these tonoplast vesicles showed a decrease of 18 % and 45 %, respectively. Together, these data indicate that the widely used in vivo pH-indicator BCECF alters V-ATPase activity and properties and can therefore interfere with the pH of cellular compartments acidified or alkalized by the V-ATPase, whereas BCECF-AM added to living cells inhibits the V-PPase by an interaction with cellular mechanisms regulating its activity.

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