Intracellular Carbonic Anhydrase of Chlamydomonas reinhardtii

Abstract

An intracellular carbonic anhydrase (CA; EC 4.2.1 .I) was purified to homogeneity from a mutant strain of Chlamydomonas reinhardtii (CW 92) lacking a cell wall. lntact 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. l h e resulting fraction had a specific activity of 1260 WilburAnderson 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. I n 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 i s an internal CA in C. reinhardtii and that the enzyme is closely related to the a-type CAs observed i n animal species.

Discovery of an algal mitochondrial carbonic anhydrase: molecular cloning and characterization of a low-CO2-induced polypeptide in Chlamydomonas reinhardtii.

“…1A). [24][25][26][27][28][29][30][31][32][33][34][35][36] (9)(10)(11). Induction of the 22-kDa polypeptide is also demonstrated on autoradiograms of mitochondria isolated from 35S-labeled algae (Fig.…”

“…To obtain N-terminal and internal amino acid sequences, the polypeptide was analyzed as reported by Karlsson et al (24). Using the C. reinhardtii codon usage, two degenerate oligonucleotides were designed and synthesized with parts of two of the internal amino acid sequences as templates (VNNKPHD and VQMELK).…”

Amino Acid Sequence Homology between N- and C-terminal Halves of a Carbonic Anhydrase in Porphyridium purpureum, as Deduced from the Cloned cDNA

“…Using a CA-directed photoaffinity label, intracellular CA of Chlamydomonas was identified as a polypeptide with an apparent molecular mass of 30 kDa (33). However, its partial amino acid sequence exhibited homology to eukaryotic-type CA (34). It will be interesting to examine whether or not the duplicated CA structure is found in other red algae and cyanobacteria.…”

Tansley Review No. 117

“…Moreover, extracellular and intracellular CAs were distinguished by using two specific inhibitors, acetazolamide and ethoxyzolamide respectively, as in numerous other studies in vivo (Badger et al, 1998). However, it is now clear that different CA isoenzymes are very differently sensitive to these two inhibitors (Table 2) (Hiltonen et al, , 1998Karlsson et al, 1995 ;. Earlier conclusions from various CA inhibition studies in vivo of lichens and their bionts must therefore be re-evaluated with the use of more refined methods.…”