PLANT PHYSIOLOGY , Vol 103, Issue 3 943-948, Copyright © 1993 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Identification of Distinct Internal and External Isozymes of Carbonic Anhydrase in Chlorella saccharophila
T. G. Williams and B. Colman
Department of Biology, York University, 4700 Keele Street, North York, Ontario, Canada M3J 1P3
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.
