Identification of Distinct Internal and External Isozymes of Carbonic Anhydrase in Chlorella saccharophila.

Abstract

External carbonic anhydrase (CA) was detected in whole cells of alkaline-grown Chlorella saccharophila but was suppressed by growth at acid pH or growth on elevated levels of CO2. Internal CA activity was measured potentiometrically as an increase in activity in cell extracts over that of intact cells. Cells grown under all conditions had equal levels of internal CA activity. Two isozymes were identified after electrophoretic separation of soluble proteins on cellulose acetate plates. The fast isozyme was found in cells grown under all conditions, whereas the slow isozyme was found only in cells grown at alkaline pH. Western blot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis using antibodies produced against the periplasmic form of CA from Chlamydomonas reinhardtii revealed a single band at 39 kD, which did not change in intensity between growth conditions and was associated only with proteins eluted from the fast band. The slow isozyme was inactivated by incubation of cell extract at 30[deg]C and by incubation in 10 mM dithiothreitol, whereas the internal form was unaffected. These results indicate that external and internal forms of CA differ in structure and their activities respond differently to environmental conditions.