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- Eriksson, Mats, 1966-, et al.
(författare)
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Discovery of an algal mitochondrial carbonic anhydrase : molecular cloning and characterization of a low-CO2-induced polypeptide in Chlamydomonas reinhardtii
- 1996
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - 0027-8424 .- 1091-6490. ; 93:21, s. 12031-12034
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Tidskriftsartikel (refereegranskat)abstract
- In green unicellular algae, several polypeptides are induced upon exposure to limiting CO2. We report here on the localization and characterization of one of these, a 22-kDa polypeptide in Chlamydomonas reinhardtii. This nuclear-encoded polypeptide is induced in the mitochondria by a lowering of the partial pressure of CO2 in the growth medium from 5% to air CO2 levels. Sequencing of two different cDNA clones coding for the polypeptide identified it as a 20.7-kDa beta-type carbonic anhydrase (CA; carbonate dehydratase, carbonate hydro-lyase, EC 4.2.1.1). The two clones differ in their nucleotide sequences but code for identical proteins, showing that this CA is encoded by at least two genes. Northern blot hybridization reveals that mRNA transcripts are only present in cells transferred to air CO2 levels. A comparison of the deduced amino acid sequence with those of other beta-CAs shows the largest degree of similarity with CA from the cyanobacterium Synechocystis (50% identity and 66% similarity). To our knowledge, this is the first identification and characterization of a mitochondrial CA from a photosynthetic organism.
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| 2. |
- Karlsson, Jan, 1966-, et al.
(författare)
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Intracellular carbonic anhydrase of Chlamydomonas reinhardtii
- 1995
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 109:2, s. 533-539
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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