2.3 Effect of inhibitors on Ci-uptake and external CA activity
Inhibitors were used in pH-drift experiments to determine their effect
on Ci-uptake. A stock solution of AZ (20 mM) was prepared by dissolving
the solid in 20 mM NaOH and 0.61 or 1.21 mL was injected into the
chamber to produce final concentration of 0.1 or 0.2 mM respectively.
Stock solutions of 30 mM DIDS, were prepared daily by dissolving the
powder in distilled water (Cabantchik & Greger, 1992), and 1.21 mL was
injected into the chamber to produce a final concentration of 0.3 mM.
Both stock solutions were kept in the dark at 4°C.
To check if the inhibitory effect of AZ on
HCO3- uptake was reversible, we
performed three consecutive drifts using the same O. alismoidesleaf cut longitudinally into two halves. The first half was used as a
control (first drift) without AZ. The second half was treated with AZ
(second drift). Subsequently, this leaf and chamber were thoroughly
rinsed with clean medium three times over ten minutes, and finally a
post-control (third drift), was performed without the inhibitor. All the
pH-drifts were started at pH 7.6 and stopped at pH 8.5 and replicated at
least in triplicate.