PLANT PHYSIOLOGY , Vol 109, Issue 2 533-539, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Intracellular Carbonic Anhydrase of Chlamydomonas reinhardtii
J. Karlsson, T. Hiltonen, H. D. Husic, Z. Ramazanov and G. Samuelsson
Department of Plant Physiology, Umea University, S-901 87 Umea, Sweden (J.K., T.H., Z.R., G.S.)
An intracellular carbonic anhydrase (CA; EC 4.2.1.1) was purified to
homogeneity from a mutant strain of Chlamydomonas reinhardtii (CW 92)
lacking a cell wall. Intact cells were washed to remove periplasmic CA and
were lysed and fractionated into soluble and membrane fractions by
sedimentation. All of the CA activity sedimented with the membrane fraction
and was dissociated by treatment with a buffer containing 200 mM KCl.
Solubilized proteins were fractionated by ammonium sulfate precipitation,
anionic exchange chromatography, and hydrophobic interaction
chromatography. The resulting fraction had a specific activity of 1260
Wilbur-Anderson units/mg protein and was inhibited by acetazolamide (50%
inhibition concentration, 12 nM). Final purification was accomplished by
the specific absorption of the enzyme to a Centricon-10 microconcentrator
filter. A single, 29.5-kD polypeptide was eluted from the filter with
sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer,
and a 1.5 M ammonium sulfate eluate contained CA activity. In comparison
with human CA isoenzyme II, the N-terminal and internal amino acid
sequences from the 29.5-kD polypeptide were 40% identical with the
N-terminal region and 67% identical with an internal conserved region.
Based on this evidence, we postulate that the 29.5-kD polypeptide is an
internal CA in C. reinhardtii and that the enzyme is closely related to the
[alpha]-type CAs observed in animal species.
