lntracellular carbonic anhydrase of Chlamydomonas reinhardtii

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 KCI. 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.

Title	lntracellular carbonic anhydrase of Chlamydomonas reinhardtii
Creator	Ramazanov, Z.
	Husic, H. David
	Samuelsson, G.
	Hiltonen, T.
	Karlsson, J.
Publisher	Plant Physiology
Academic Department	Chemistry
Division	Natural Sciences
Organization	Lafayette College
Date Issued	1995
Date Available	2013-01-29T20:18:39Z
Type	Article
Language	English
Bibliographic Citation	Karlsson, J., et al. (1995) "lntracellular carbonic anhydrase of Chlamydomonas reinhardtii." Plant Physiology 109 (2): 533-539.
Standard Identifier	DOI 10.1104/pp.109.2.533
Standard Identifier	Handle 10385/1072
Permalink	http://hdl.handle.net/10385/1072
Rights Statement	In Copyright
Rights Holders	American Society of Plant Physiologists