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Purification and characterisation of an intracellular carbonic anhydrase from the unicellular green alga Coccomyxa

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Abstract

An intracellular carbonic anhydrase (CA; EC 4.2.1.1) was purified and characterised from the unicellular green alga Coccomyxa sp. Initial studies showed that cultured Coccomyxa cells contain an intracellular CA activity around 100 times higher than that measured in high-CO2-grown cells of Chlamydomonas reinhardtii CW 92. Purification of a protein extract containing the CA activity was carried out using ammonium-sulphate precipitation followed by anion-exchange chromatography. Proteins were then separated by native (non-dissociating) polyacrylamide gel electrophoresis, with each individual protein band excised and assayed for CA activity. Measurements revealed CA activity associated with two discrete protein bands with similar molecular masses of 80 +5 kDa. Dissociation by denaturing polyacrylamide gel electrophoresis showed that both proteins contained a single polypeptide of 26 kDa, suggesting that each 80-kDa native protein was a homogeneous trimer. Isoelectric focusing of the 80-kDa proteins also produced a single protein band at a pH of 6.5. Inhibition studies on the purified CA extract showed that 50% inhibition of CA activity was obtained using 1 μM azetazolamide. Polyclonal antibodies against the 26-kDa CA were produced and shown to have a high specific binding to a single polypeptide in soluble protein extracts from Coccomyxa cells. The same antiserum, however, failed to cross-react with soluble proteins isolated from two different species of green algae, Chlamydomonas reinhardtii and Chlorella vulgaris. Correspondingly, antisera directed against pea chloroplastic CA, extracellular CA from C. reinhardtii and human CAII, showed no cross-hybridisation to the 26-kDa polypeptide in Coccomyxa. The 26-kDa protein was confirmed as being a CA by N-terminal sequencing of two internal polypeptide fragments and alignment of these sequences with that of previously identified CA proteins from several different species.

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Abbreviations

CA:

carbonic anhydrase

CCM:

CO2-concentrating mechanism

IEF:

isoelectric focusing

Rubisco:

ribulose-l,5-bisphosphate carboxylase/oxygenase

References

  • Aizawa, K. and Miyachi S. (1986) Carbonic anhydrase and CO2 concentrating mechanisms in microalgae and cyanobacteria. FEMS Microbiol. Rev. 39, 215–233

    Google Scholar 

  • Atkins, C.A., Patterson, B.D., Graham, D. (1972) Plant carbonic anhydrases II. Preparation and some properties of monocotyledon and dicotyledon enzyme types. Plant Physiol. 50, 218–223

    Google Scholar 

  • Badger, M.R. (1987) The CO2 concentrating mechanism in aquatic phototrophs. In: The biochemistry of plants: A comprehensive treatise, vol. 10: Photosynthesis, pp. 219–274, Hatch, M.D., Boardman, N.K., eds. Academic Press, San Diego New York

    Google Scholar 

  • Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–253

    Article  CAS  PubMed  Google Scholar 

  • Burnell, J.N. (1990) Immunological study of carbonic anhydrase in C3 and C4 plants using antibodies to maize cytosolic and spinach chloroplastic carbonic anhydrase. Plant Cell Physiol. 31, 423–427

    Google Scholar 

  • Clarke, A.K., Critchley C (1992) The identification of a heat-shock protein complex in chloroplasts of barley leaves. Plant Physiol. 100, 2081–2089

    Google Scholar 

  • Cowan, I.R. (1986) Economics of carbon fixation in higher plants. In: On the economy of plant form and function, pp. 133–170, Givnish, T.J., ed. Cambridge University Press, Cambridge

    Google Scholar 

  • Fukuzawa, H., Fujiwara, S., Yamamoto, Y, Dionisio-Sese, M.L., Miyachi, S. (1990) cDNA cloning, sequence and expression of carbonic anhydrase in Chlamydomonas reinhardtii: Regulation by environmental CO2 concentration. Proc. Natl. Acad. Sci. USA 87, 4383–4387

    Google Scholar 

  • Graham, D., Reed, M.I., Patterson, B.D., Hockley, D.G., Dwyer, M.R. (1984) Chemical properties, distribution, and physiology of plant and algal carbonic anhydrases. Ann. N.Y. Acad. Sci 429, 222–237

    Google Scholar 

  • Hatch, M.D., Burnell, J.N. (1990) Carbonic anhydrase activity in leaves and its role in the first step of C4 Photosynthesis. Plant Physiol. 93, 825–828

    Google Scholar 

  • Honegger, R. (1991) Functional aspects of the lichen symbiosis. Annu. Rev. Plant. Physiol. Plant Mol. Biol. 42, 553–578

    Google Scholar 

  • Husic, H.D., Kitayama, M., Togasaki, R.K., Moroney, J.V., Morris, K.L., Tolbert, N.E. (1989) Identification of intracellular carbonic anhydrase in Chlamydomonas reinhardtii which is distinct from the periplasmatic form of the enzyme. Plant Physiol. 89, 904–909

    Google Scholar 

  • Husic, H.D., Quigley, E.A. (1990) Salt-induced dissociation of carbonic anhydrase from intact cells of Chlamydomonas reinhardtii. Plant Physiol. 94, 380–383

    Google Scholar 

  • Jahnen-Dechent, W. Simpson, R.J. (1990) A method for preparing proteins and peptides for microsequencing. Plant Mol. Biol. Reporter 8, 92–93

    Google Scholar 

  • Kamo, T., Shimogawara, K., Fukuzawa, H., Muto, S., Miyachi, S. (1990) Subunit constitution of carbonic anhydrase from Chlamydomonas reinhardtii. Eur. J. Biochem. 192, 557–562

    Google Scholar 

  • Kandel, M., Gornall, A.G., Cybulsky, D.L., Kandel, S.I. (1978) Carbonic anhydrase from spinach leaves. Isolation and some chemical properties. J. Biol. Chem. 253, 679–685

    Google Scholar 

  • Kuchitsu, K., Tzusuki, M., Miyachi, S. (1988) Changes in starch localization within the chloroplast induced by changes in CO2 concentration during growth of Chlamydomonas reinhardtii: Independent regulation of pyrenoid starch and stromal starch. Plant Cell Physiol. 29, 1269–1278

    Google Scholar 

  • Kuchitsu, K., Tzusuki, M., Miyachi, S. (1991) Polypeptide composition and enzyme activities of the pyrenoid and its regulation by CO2 concentration in unicellular green algae. Can. J. Bot. 69, 1062–1069

    Google Scholar 

  • Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685

    PubMed  Google Scholar 

  • Majeau, N., Coleman, J.R. (1991) Isolation and characterization of a cDNA coding for pea chloroplastic carbonic anhydrase. Plant Physiol. 95, 264–268

    Google Scholar 

  • Majeau, N., Coleman, J.R. (1992) Nucleotide sequence of a complementary DNA encoding tobacco chloroplastic carbonic anhydrase. Plant Physiol. 100, 1077–1078

    Google Scholar 

  • Makino, A., Sakashita, H., Hidema, J., Tadahiko, M., Kunihiko, O., Osmond, B. (1992) Distinctive responses of ribulose-1,5-bisphosphate carboxylase and carbonic anhydrase in wheat leaves to nitrogen nutrition and their possible relationships to CO2-transfer resistance. Plant Physiol. 100, 1737–1743

    Google Scholar 

  • Moroney, J.V., Husic, H.D., Tolbert, N.E. (1985) Effect of carbonic anhydrase inhibitors on inorganic carbon accumulation by Chlamydomonas reinhardtii. Plant Physiol. 79, 177–183

    Google Scholar 

  • Okabe, K., Yang, S-Y., Tsuzuki, M., Miyachi, S. (1984) Carbonic anhydrase: Its content in spinach leaves and its taxonomic diversity studied with anti-spinach leaf carbonic anhydrase antibody. Plant Sci. lett. 33, 145–153

    Google Scholar 

  • Palmqvist, K. (1993) Photosynthetic CO2-use efficiency in lichens and their isolated photobionts: the possible role of a CO2-concentrating mechanism. Planta 191, 48–56

    Google Scholar 

  • Palmqvist, K., Ögren, E., Lernmark, U. (1994) The CO2-concentration mechanism is absent in the green alga Coccomyxa. A comparative study of photosynthetic CO2 and light responces of Coccomyxa. Chlamydomonas reinhardtii and barley protoplasts. Plant Cell Environ. 17, 65–72

    Google Scholar 

  • Pocker, Y, Ng, J.S.Y. (1973) Plant carbonic anhydrase. Properties and carbon dioxide hydration kinetics. Biochemistry. 12, 5127–5134

    Google Scholar 

  • Porra, R.J., Thompson W.A., Kriederman, P.E. (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta. 975, 384–394

    Google Scholar 

  • Price, G.D., Coleman, J.R., Badger, M.R. (1992) Association of carbonic anhydrase activity with carboxysomes isolated from the cyanobacterium Synechococcus PCC7942. Plant Physiol. 100, 784–793

    Google Scholar 

  • Raines, C.A., Horsnell, P.R., Holder, C., Lloyd, J.C. (1992) Arabidopsis thaliana carbonic anhydrase: cDNA sequence and effect of CO2 on mRNA levels. Plant Mol. Biol. 20, 1143–1148

    Google Scholar 

  • Rawat, M., Moroney, J.W. (1991) Partial characterization of a new isoenzyme of carbonic anhydrase isolated from Chlamydomonas reinhardtii. J. Biol. Chem. 266, 9719–9723

    Google Scholar 

  • Reed, M.L., Graham, D. (1981) Carbonic anhydrase in plants: Distribution, properties and possible physiological roles. In: Progress in phytochemistry, vol. 7, pp. 47–94, Reinhold, L., Harborne, J.B., Swain, T., eds. Pergamon Press, Oxford

    Google Scholar 

  • Rippka, R., Waterbury, J.B., Steiner, R.Y. (1981) Isolation and purification of cyanobacteria: some general principles. In: The procaryotes, pp. 212–220, Staff, M.P., Stolp, H., Trüper, H.G., Balows, A., Schegel, H.G., eds. Springer, Berlin Heidelberg New York

    Google Scholar 

  • Solter, K.M., Gibor, A. (1977) The release of carbohydrates during mating in Chlamydomonas reinhardtii. Plant Sci. Lett. 8, 227–231

    Google Scholar 

  • Surzycki, S.J. (1971) Synchronously grown cultures of Chlamydomonas reinhardtii. Methods Enzymol. 23, 67–73

    Google Scholar 

  • Sültemeyer, D., Schmidt, C., Fock, H.P. (1993) Carbonic anhydrases in higher plants and aquatic microorganisms. Physiol. Plant. 88, 179–190

    Google Scholar 

  • Utsunomiya, E., Muto, S. (1993) Carbonic anhydrase in the plasma membrane from leaves of C3 and C4 plants. Physiol. Plant. 88, 413–419

    Google Scholar 

  • Wilbur, K.M., Anderson, N.G. (1948) Electrometric and colorimetric determination of carbonic anhydrase. J. Biol. Chem. 176 147–154

    Google Scholar 

  • Yagawa, Y, Muto, S., Miyachi, S. (1987a) Carbonic anhydrase of a unicellular red alga Porphyridium cruentum R-1. I. Purification and properties of the enzyme. Plant Cell Physiol. 28, 1253–1262

    Google Scholar 

  • Yagawa, Y, Muto, S., Miyachi, S. (1987b) Carbonic anhydrase of a unicellular red alga Porphyridium cruentum R-1. II. Distribution and role in photosynthesis. Plant Cell Physiol. 28, 1509–1516

    Google Scholar 

  • Yamada, H., Moriya, H., Tsugita, A. (1991) Development of an acid hydrolysis method with high recoveries of tryptophan and cysteine for microquantities of protein. Anal. Biochem. 198, 1–5

    Google Scholar 

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Authors and Affiliations

  1. Department of Plant Physiology, University of Umeå, Sweden

    Thomas Hiltonen, Jan Karlsson, Kristin Palmqvist, Adrian K. Clarke & Göran Samuelsson

Authors
  1. Thomas Hiltonen
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  2. Jan Karlsson
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  3. Kristin Palmqvist
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  4. Adrian K. Clarke
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  5. Göran Samuelsson
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Additional information

We would like to thank Drs. Cecilia Forsman, Inga-Maj Johansson and Nalle Jonsson for their valuable advice concerning the isolation of CA. This work was supported by the Swedish Natural Research Council and Seth M. Kempes Memorial foundation.

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Hiltonen, T., Karlsson, J., Palmqvist, K. et al. Purification and characterisation of an intracellular carbonic anhydrase from the unicellular green alga Coccomyxa . Planta 195, 345–351 (1995). https://doi.org/10.1007/BF00202591

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