| 1. |
- Hiltonen, Thomas, 1960-, et al.
(författare)
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Intracellular beta-carbonic anhydrase of the unicellular green alga Coccomyxa
- 1998
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Ingår i: Plant Physiology. - 0032-0889 .- 1532-2548. ; 117:4, s. 1341-1349
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Tidskriftsartikel (refereegranskat)abstract
- Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO,, a reaction that is important in many physiological processes. We have cloned and sequenced a full length cDNA encoding an intracellular P-CA from the unicellular green alga Coccomyxa. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids. The predicted polypeptide is similar to beta-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30%. The Coccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically characterized. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO2-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants. Fractionation studies further showed that Coccomyxa CA is extrachloroplastic.
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| 2. |
- Larsson, S, et al.
(författare)
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Molecular cloning and biochemical characterization of carbonic anhydrase from Populus tremula x tremuloides
- 1997
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Ingår i: Plant Molecular Biology. - 0167-4412 .- 1573-5028. ; 34:4, s. 583-592
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Tidskriftsartikel (refereegranskat)abstract
- A leaf cDNA library from hybrid aspen, Populus tremula x tremuloides, was constructed. From this two different cDNA clones, denoted CAla and CAlb, encoding a chloroplastic carbonic anhydrase (CA) were isolated and DNA sequenced. Analysis of the deduced amino acid sequences showed that the isolated CAs belong to the beta-CA family, and have identities around 70% to other dicotyledonous plant CAs. The two hybrid aspen cDNA clones display a high nucleotide sequence identity, only 12 nucleotides differ. Since only one gene copy of this soluble chloroplastic CA is present in the nuclear genome, we postulate that the two isolated cDNA clones are alleles. Northern blot hybridization revealed a CA transcript of ca. 1300 bases, 140 bases shorter than in pea. Western and northern blot hybridizations on crude protein extracts and on total RNA, respectively, isolated from stem and leaves, showed that hybrid aspen CA is expressed specifically in the leaf under the growth conditions used. Based on the deduced amino acid sequence, the mature hybrid aspen CA enzyme subunit has a molecular mass of 24.8 kDa. The enzyme was over-expressed in Escherichia coli, and purified by affinity chromatography. Biochemical characterization showed that the protein structure and the CO2-hydration activity are similar to the pea enzyme. Molecular characterization of a CA from a perennial plant has not previously been performed, and it demonstrates that both the structure and activity of hybrid aspen CA resembles CAs from annual plants.
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