The presence of extracellular carbonic anhydrase (CA) in
relation to medium composition was investigated using cultures of the
marine diatom Phaeodactylum tricornutum Bohlin. Large-volume
cultures, with low initial cell inocula were grown on ASP-2 (no
dissolved inorganic carbon (DIC), 550 μM
NO3−), f/2 (2·0 mM
DIC, 880 μM NO3−) and
modified f/2 (2·0 mM DIC, 20 μM
NO3− media. Cells growing on ASP-2 showed
extracellular CA in the early stages of growth, whereas extracellular
CA was not detected until partial depletion of total DIC in the
stationary phase for cultures on f/2 or modified f/2. Both
HCO3− and CO2 were important
in carbon limitation, extracellular CA being present when the
free-CO2 concentration fell below 5 μM, but
the HCO3− concentration needed to be
below 1 mM. When carbon-replete cells were transferred to
carbon-limited conditions, extracellular CA was recorded within
minutes, the process being light-dependent and completely inhibited
by 3,3,4-dichlorophenyl-1,1-dimethylurea (DCMU). The addition of DIC
to carbon-limited cells resulted in a rapid decrease in extracellular
CA activity. The membrane-impermeable inhibitor of carbonic
anhydrase, dextran-bound sulphonamide (DBS) was used to inhibit
extracellular CA activity in relation to photosynthetic rate in
carbon-replete and carbon-limited cells. At the lowest DIC
concentration (0·10 mM), for cells with maximum
external CA activity, DBS gave over 80% inhibition of the
photosynthetic rate, demonstrating the key role of external CA in
maintaining high photosynthetic rate under conditions of carbon
limitation.
It is proposed that the key factor in the regulation of
extracellular CA activity is the total flux of inorganic carbon
(Ci) into the cell. This determines the Ci flux
into the chloroplast and when this is inadequate to support the
photosynthetic rate attained by a carbon-replete chloroplast at
optimum photon flux density, extracellular CA is activated. This
mechanism would explain the observed interaction of CO2
and HCO3− in the regulation of
extracellular CA activity.