Purification and characterization of the carbonic anhydrase enzyme from horse mackerel (Trachurus trachurus) muscle and the impact of some metal ions and pesticides on enzyme activity.

In this paper, the total carbonic anhydrase (CA) enzyme was purified from horse mackerel (Trachurus trachurus) muscle with a specific activity of 23,063.93 EU/mg, purification fold of 551.08, total activity of 1522.22 EU/mL and a yield of 18.50% using sulfanilamide affinity column chromatography. For obtaining the subunit molecular mass and enzyme purity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for this part was performed and a single band was clearly recorded. The molecular mass of this enzyme was found approximately 35 kDa. The optimum temperature and pH values were obtained from Arrhenius plot. In addition, the inhibitory effects of different heavy metal ions (Fe2+, Cu2+, Co2+, Pb2+ Hg2+ and As3+) and some pesticides (thiram, clofentezine, propineb, deltamethrin, azoxystrobin and thiophanate) on horse mackerel (Trachurus trachurus) muscle tissue CA enzyme activities were investigated by utilizing esterase assay activity. The used metal ions and pesticides had IC50 values in the range of 0.21-13.84 mM and 3.78-70.58 mM, respectively.

Synthesis, biological evaluation and in silico studies of novel N-substituted phthalazine sulfonamide compounds as potent carbonic anhydrase and acetylcholinesterase inhibitors.

The synthesis, characterization and biological evaluation of a series of novel N-substituted phthalazine sulfonamide (5a-l) are disclosed. Phthalazines which are nitrogen-containing heterocyclic compounds are biologically preferential scaffolds, endowed with versatile pharmacological activity, such as anti-inflammatory, cardiotonic vasorelaxant, anticonvulsant, antihypertensive, antibacterial, anti-cancer action. The compounds were investigated for the inhibition against the cytosolic hCA I, II and AChE. Most screened sulfonamides showed high potency in inhibiting hCA II, widely involved in glaucoma, epilepsy, edema, and other pathologies (Kis in the ranging from 6.32 ±â€¯0.06 to 128.93 ±â€¯23.11 nM). hCA I was inhibited with Kis in the range of 6.80 ±â€¯0.10-85.91 ±â€¯7.57 nM, whereas AChE in the range of 60.79 ±â€¯3.51-249.55 ±â€¯7.89 nM. ADME prediction study of the designed N-substituted phthalazine sulfonamides showed that they are not only with carbonic anhydrase and acetylcholinesterase inhibitory activities but also with appropriate pharmacokinetic, physicochemical parameters and drug-likeness properties. Also, in silico docking studies were investigated the binding modes of selected compounds, to hCA I, II, and AChE.

Cryptophane Nanoscale Assemblies Expand 129Xe NMR Biosensing.

Cryptophane-based biosensors are promising agents for the ultrasensitive detection of biomedically relevant targets via 129Xe NMR. Dynamic light scattering revealed that cryptophanes form water-soluble aggregates tens to hundreds of nanometers in size. Acridine orange fluorescence quenching assays allowed quantitation of the aggregation state, with critical concentrations ranging from 200 nM to 600 nM, depending on the cryptophane species in solution. The addition of excess carbonic anhydrase (CA) protein target to a benzenesulfonamide-functionalized cryptophane biosensor (C8B) led to C8B disaggregation and produced the expected 1:1 C8B-CA complex. C8B showed higher affinity at 298 K for the cytoplasmic isozyme CAII than the extracellular CAXII isozyme, which is a biomarker of cancer. Using hyper-CEST NMR, we explored the role of stoichiometry in detecting these two isozymes. Under CA-saturating conditions, we observed that isozyme CAII produces a larger 129Xe NMR chemical shift change (δ = 5.9 ppm, relative to free biosensor) than CAXII (δ = 2.7 ppm), which indicates the strong potential for isozyme-specific detection. However, stoichiometry-dependent chemical shift data indicated that biosensor disaggregation contributes to the observed 129Xe NMR chemical shift change that is normally assigned to biosensor-target binding. Finally, we determined that monomeric cryptophane solutions improve hyper-CEST saturation contrast, which enables ultrasensitive detection of biosensor-protein complexes. These insights into cryptophane-solution behavior support further development of xenon biosensors, but will require reinterpretation of the data previously obtained for many water-soluble cryptophanes.

Biochemical characterization of the native α-carbonic anhydrase purified from the mantle of the Mediterranean mussel, Mytilus galloprovincialis.

A α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified and characterized biochemically from the mollusk Mytilus galloprovincialis. As in most mollusks, this α-CA is involved in the biomineralization processes leading to the precipitation of calcium carbonate in the mussel shell. The new enzyme had a molecular weight of 50 kDa, which is roughly two times higher than that of a monomeric α-class enzyme. Thus, Mytilus galloprovincialis α-CA is either a dimer, or similar to the Tridacna gigas CA described earlier, may have two different CA domains in its polypeptide chain. The Mytilus galloprovincialis α-CA sequence contained the three His residues acting as zinc ligands and the gate-keeper residues present in all α-CAs (Glu106-Thr199), but had a Lys in position 64 and not a His as proton shuttling residue, being thus similar to the human isoform hCA III. This probably explains the relatively low catalytic activity of Mytilus galloprovincialis α-CA, with the following kinetic parameters for the CO2 hydration reaction: kcat = 4.1 × 105 s-1 and kcat/Km of 3.6 × 107 M-1 × s-1. The enzyme activity was poorly inhibited by the sulfonamide acetazolamide, with a KI of 380 nM. This study is one of the few describing in detail the biochemical characterization of a molluskan CA and may be useful for understanding in detail the phylogeny of these enzymes, their role in biocalcification processes and their potential use in the biomimetic capture of the CO2.

Carbonic anhydrase from Apis mellifera: purification and inhibition by pesticides.

Carbonic anhydrase (CA) enzymes have been shown to play an important role in ion transport and in pH regulation in several organisms. Despite this information and the wealth of knowledge regarding the significance of CA enzymes, few studies have been reported about bee CA enzymes and the hazardous effects of chemicals. Using Apis mellifera as a model, this study aimed to determine the risk of pesticides on Apis mellifera Carbonic anhydrase enzyme (Am CA). CA was initially purified from Apis mellifera spermatheca for the first time in the literature. The enzyme was purified with an overall purification of ∼35-fold with a molecular weight of ∼32 kDa. The enzyme was then exposed to pesticides, including tebuconazole, propoxur, carbaryl, carbofuran, simazine and atrazine. The six pesticides dose-dependently inhibited in vitro AmCA activity at low micromolar concentrations. IC50 values for the pesticides were 0.0030, 0.0321, 0.0031, 0.0087, 0.0273 and 0.0165 µM, respectively. The AmCA inhibition mechanism of these compounds is unknown at this moment.

Purification, enzymatic activity and inhibitor discovery for recombinant human carbonic anhydrase XIV.

Human carbonic anhydrase XIV (CA XIV), a transmembrane protein, highly expressed in the central nervous system, is difficult to recombinantly express and purify in large scale for the measurements of inhibitor binding and drug design. CA XIV belongs to the family of twelve catalytically active CA isoforms in the human body. Disorders in the expression of CA XIV cause serious diseases and CA XIV has been described as a possible drug target for the treatment of epilepsy, some retinopathies, and skin tumors. In this study, the effect of different promoters, E. coli strains, and the length of recombinant CA XIV protein construct were analyzed for the production CA XIV in large scale by using affinity purification. Active site titration by inhibitors and the isothermal titration calorimery revealed over 96% purity of the protein. Enzymatic activity of the purified CA XIV was determined by following the CO2 hydration using the stopped-flow technique. Several inhibitors were discovered that exhibited selectivity towards CA XIV over other CA isoforms and could be developed as drugs.

A magnificent enzyme superfamily: carbonic anhydrases, their purification and characterization.

In this paper, we reviewed the purification and characterization methods of the α-carbonic anhydrase (CA, EC 4.2.1.1) class. Six genetic families (α-, ß-, γ-, δ-, ζ- and η-CAs) all know to date, all encoding such enzymes in organisms widely distributed over the phylogenetic tree. Starting from the manuscripts published in the 1930s on the isolation and purification of α-CAs from blood and other tissues, and ending with the recent discovery of the last genetic family in protozoa, the η-CAs, considered for long time an α-CA, we present historically the numerous and different procedures which were employed for obtaining these catalysts in pure form. α-CAs possess important application in medicine (as many human α-CA isoforms are drug targets) as well as biotechnological processes, in which the enzymes are ultimately used for CO2 capture in order to mitigate the global warming effects due to greenhouse gases. Recently, it was discovered an involvement of CAs in cancerogenesis as well as infection caused by pathogenic agents such as bacteria, fungi and protozoa. Inhibition studies of CAs identified in the genome of the aforementioned organisms might lead to the discovery of innovative drugs with a novel mechanism of action.

CARBONIC ANHYDRASE ACTIVITY OF INTEGRAL-FUNCTIONAL COMPLEXES OF THYLAKOID MEMBRANES OF SPINACH CHLOROPLASTS.

Isolated thylakoid membranes were disrupted by treatment with nonionic detergents digitonin or dodecyl maltoside. Solubilized polypeptide complexes were separated by native gel charge shift electrophoresis. The position of ATP-synthase complex and its isolated catalytic part (CF1) within gel was determined using the color reaction for ATPase activity. Due to the presence of cytochromes, the red band in unstained gels corresponded to the cytochrome b6f complex. Localization of the cytochrome b6f complex, ATP synthase and coupling CF1 in the native gel was confirmed by their subunit composition determined after SDS-electrophoretic analysis. Carbonic anhydrase (CA) activity in polypeptide zones of PS II, cytochrome b6f complex, and ATP-synthase CF1 was identified in native gels using indicator bromothymol blue. CA activity of isolated CF1 in solution was determined by infrared gas analysis as the rate of bicarbonate dehydration. The water-soluble acetazolamide, an inhibitor of CA, unlike lipophilic ethoxyzolamide inhibited CA activity of CF1 Thus, it was shown for the first time that ATP-synthase has a component which is capable of catalyzing the interconversion of forms of carbonic acid associated with proton exchange. The data obtained suggest the presence of multiple forms of carbonic anhydrase in the thylakoid membranes of spinach chloroplasts and confirm their involvement in the proton transfer to the ATP synthase.

Proteomic analysis of carbon concentrating chemolithotrophic bacteria Serratia sp. for sequestration of carbon dioxide.

A chemolithotrophic bacterium enriched in the chemostat in presence of sodium bicarbonate as sole carbon source was identified as Serratia sp. by 16S rRNA sequencing. Carbon dioxide sequestering capacity of bacterium was detected by carbonic anhydrase enzyme and ribulose-1, 5- bisphosphate carboxylase/oxygenase (RuBisCO). The purified carbonic anhydrase showed molecular weight of 29 kDa. Molecular weight of RuBisCO was 550 kDa as determined by fast protein liquid chromatography (FPLC), however, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed presence of two subunits whose molecular weights were 56 and 14 kDa. The Western blot analysis of the crude protein and purified sample cross reacted with RuBisCO large-subunit polypeptides antibodies showed strong band pattern at molecular weight around 56 kDa regions. Whole cell soluble proteins of Serratia sp. grown under autotrophic and heterotrophic conditions were resolved by two-dimensional gel electrophoresis and MALDI-TOF/MS for differential expression of proteins. In proteomic analysis of 63 protein spots, 48 spots were significantly up-regulated in the autotrophically grown cells; seven enzymes showed its utilization in autotrophic carbon fixation pathways and other metabolic activities of bacterium including lipid metabolisms indicated sequestration potency of carbon dioxide and production of biomaterials.

Isolation and sequencing of swine carbonic anhydrase VI, an enzyme expressed in the swine kidney.

BACKGROUND: Carbonic anhydrase VI (CA-VI) is produced by the salivary gland and is secreted into the saliva. Although CA-VI is found in the epithelial cells of distal straight tubule of swine kidneys, the exact function of CA-VI in the kidneys remains unclear. RESULTS: CA-VI was located in the epithelial cells of distal straight tubule of swine kidneys.A full-length cDNA clone of CA-VI was generated from the swine parotid gland by reverse transcription polymerase chain reaction, using degenerate primers designed based on conserved regions of the same locus in human and bovine tissues. The cDNA sequence was 1348 base pairs long and was predicted to encode a 317 amino acid polypeptide with a putative signal peptide of 17 amino acids. The deduced amino acid sequence of mature CA-VI was most similar (77.4%) to that of human CA-VI. CA-VI expression was confirmed in both normal and nephritic kidneys, as well as parotid. As the primers used in this study spanned two exons, the influence of genomic DNA was not detected. The expression of CA-VI was demonstrated in both normal and nephritic kidneys, and mRNA of CA-VI in the normal kidneys which was the normalised to an endogenous ß-actin was 0.098 ± 0.047, while it was significantly lower in the diseased kidneys (0.012 ± 0.007). The level of CA-VI mRNA in normal kidneys was 19-fold lower than that of the parotid gland (1.887). CONCLUSIONS: The localisation of CA-VI indicates that it may play a specialised role in the kidney.

Biochemical characterization of the γ-carbonic anhydrase from the oral pathogen Porphyromonas gingivalis, PgiCA.

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. CAs are present in many pathogenic species and are involved in the bicarbonate metabolism/biosynthetic reactions involving this ion. Ubiquity of these enzymes suggests a pivotal role in microbial virulence and pathogenicity. Porphyromonas gingivalis is an anaerobic bacterium, which colonizes the oral cavity, being involved in the pathogenesis of periodontitis, an inflammatory disease leading to tooth loss. Recently, we reported an anion inhibitory study on the γ-CA (denominated PgiCA) identified in the genome of this Gram-negative bacterium. In this paper we continue our research on PgiCA, and describe the biochemical characterization of the recombinant protein, its thermal stability, the oligomeric state and the enzyme kinetics. PgiCA is a polypeptide chain formed of 192 amino acids and displays an identity of 30-33% when compared with the prototypical γ-CAs, CAM or CAMH (from Methanosarcina thermophila) or CcmM (from Thermosynechococcus elongatus). A subunit molecular mass of 21 kDa was estimated by SDS-PAGE, while HPLC size exclusion chromatography under native conditions gave an estimated molecular mass of 65 kDa suggesting that the recombinant enzyme self-associate in a homotrimer, as all other γ-CAs studied so far. Enzyme kinetic analysis showed that PgiCA is 62 times more effective as a catalyst compared to CAM, the only other γ-CA characterized in detail kinetically. All these features represent an interesting attractive for the drug design of inhibitors/activators of this new enzyme.

Secondary/tertiary benzenesulfonamides with inhibitory action against the cytosolic human carbonic anhydrase isoforms I and II.

Carbonic anhydrase inhibitors of primary sulfonamide type, RSO(2)NH(2), have clinical applications as diuretics, antiglaucoma, antiepileptic, antiobesity and antitumor drugs. Here we investigated inhibition of two human cytosolic isozymes, hCA I and II, with a series of secondary/tertiary sulfonamides, incorporating tosyl moieties (CH(3)C(6)H(4)SO(2)NR1R2). Most compounds inhibited both isoforms in low micromolar range, with inhibition constants between 0.181-6.01 µM against hCA I, and 0.209-0.779 µM against hCA II, respectively. These findings point out that substituted benzenesulfonamides may be used as leads for generating interesting CAIs probably possessing a distinct mechanism of action compared to primary sulfonamides. Indeed, classical RSO(2)NH(2) inhibitors bind in deprotonated form to the Zn(II) ion from the CA active site and participate in many other favorable interactions with amino acid residues lining the cavity. The secondary/tertiary sulfonamides cannot bind to the zinc due to steric hindrance and probably are accommodated at the entrance of the active site, in coumarin binding-site.

Docking, CoMFA and CoMSIA studies of a series of sulfonamides derivatives as carbonic anhydrase I inhibitors.

3D-QSAR methods, CoMFA region focusing (CoMFA-RF) and CoMSIA along with docking studies carried out for investigating 32 carbonic anhydrase I inhibitors. These inhibitors have been studied for the development of antiglaucoma, antitumor, antiobesity or anticonvulsant drugs. Docking analysis by GOLD provide conformations which have been realigned in CoMFA and CoMSIA models. Training set for the CoMFA-RF and CoMSIA models using 24 docked conformations gives q(2)(Loo) values of 0.615 and 0.637 and r(2)(nv) values of 0.701 and 0.713, respectively. The results of CoMFA-RF and CoMSIA with and without docked conformations were compared. The ability of prediction and robustness of the models were evaluated by test set, cross validation (leave-one-out and leave-ten-out), bootstrapping, and progressive scrambling approaches. The all-orientation search (AOS) was used to achieve the best orientation to minimize the effect of initial orientation of the structures. The docking results confirmed CoMFA and CoMSIA contour maps.

More effective dithiocarbamate derivatives inhibiting carbonic anhydrases, generated by QSAR and computational design.

Dithiocarbamates (DTC) are promising compounds with potential applications in antitumoral and glaucoma therapy. Our aim is to understand molecular features affecting DTC interaction with carbonic anhydrases (CAs), zinc-containing enzymes maintaining acid-base balance in blood and other tissues. To this end, we generate QSAR models based on a compound series containing 25 DTC, inhibitors of four human (h) CAs isoforms: hCA I, II, IX and XII. We establish that critical physicochemical parameters for DTC inhibitory activity are: hydrophobic, electronic, steric, topological and shape. The predictive power of our QSAR models is indicated by significant values of statistical coefficients: cross-validated correlation q(2) (0.55-0.73), fitted correlation r(2) (0.75-0.84) and standard error of prediction (0.47-0.23). Based on the established QSAR equations, we analyse 22 new DTC derivatives and identify DTC dicarboxilic acids derivatives and their esters as potentially improved inhibitors of CA I, II, IX and XII.

Inhibition of carbonic anhydrase activity modifies the toxicity of doxorubicin and melphalan in tumour cells in vitro.

Carbonic anhydrase IX (CA IX) is a hypoxia-regulated enzyme, overexpressed in many types of human cancer. CA IX is involved in pH homeostasis, contributing to extracellular acidification and tumourigenesis. Acidification of the extracellular milieu can impact upon cellular uptake of chemotherapeutic drugs by favouring weak acids (e.g. melphalan), but limiting access of weak bases (e.g. doxorubicin). We investigated whether alterations of CA IX activity affected anti-cancer drug uptake and toxicity. CA inhibitor acetazolamide (AZM) enhanced doxorubicin toxicity but reduced melphalan toxicity in cell lines that highly expressed CA IX under anoxic conditions (HT29 and MDA435 CA9/18). The toxicity changes reflected modification of passive drug uptake. AZM did not alter toxicity or uptake in cells with low CA IX activity (HCT116 and MDA435 EV1). AZM lowered intracellular pH in HT29 and MDA435 CA9/18 cells under anoxic conditions. CA IX activity has chemomodulatory properties and is an attractive target for anti-cancer therapy.

One-dimensional and two-dimensional immobilized metal affinity electrophoresis.

Immobilized metal affinity electrophoresis (IMAEP) is a straightforward method in which metal ions are embedded in a polyacrylamide gel strip with a negligible electrophoretic migration. Due to the preferential binding between metal ions and the phosphate group, this method uses immobilized metal ions like iron, manganese, aluminum, or titanium to capture phosphoproteins from a mixture of phosphoprotein and nonphosphoproteins. IMAEP has also been incorporated into a traditional two-dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) system (isoelectric focusing-PAGE) to increase its resolving power. In 2D IMAEP, the metal ions in polyacrylamide gel strip are overlaid on top of the second dimensional polyacrylamide gel to stop electrophoretic migration of phosphoproteins. Data shows that there is no detrimental effect of SDS in IMAEP on the extraction of phosphoproteins from a mixture of proteins. In addition, SDS exposes phosphate groups by unfolding the phosphoproteins to facilitate metal ion-phosphate binding while supplying the protein with negative charges.

A multivariate approach for the determination of isoelectric point of human carbonic anhydrase isoforms by capillary isoelectric focusing.

Human carbonic anhydrase (hCA) IX and XII are isoenzymes which are highly overexpressed in many cancer types. Recently, it has been shown that hCA IX contributes to the acidification of the tumor environment leading to chemoresistance with basic antitumoral drugs. The development of selective hCA inhibitors constitutes a new therapeutic axis. In order to elucidate the specific interactions between hCA and inhibitors, physico-chemical properties of hCA must be evaluated. This work reports the determination of the isoelectric point (pI) of a series of hCA isoforms by capillary isoelectric focusing. First, the method was optimized with synthetic UV-detectable pI markers using a central composite design. The separation was performed in a fused-silica capillary chemically derivatized with hydroxypropylcellulose and using a glycerol-water medium as the anticonvective gel. Three main factors (ampholyte content, focusing time and mobilization pressure) were optimized in order to obtain the best resolution, detection threshold and precision on the pI determination. Then, the model was validated through the analysis of standard proteins mixture having known pI values, before investigating the pI of hCA isoforms.

Carbonic anhydrase inhibitors.

Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread enzymes in all organisms, catalyzing CO2 hydration to bicarbonate and protons. Their inhibition is exploited clinically for decades for various classes of diuretics and systemically acting antiglaucoma agents. In the last years novel applications of CA inhibitors (CAIs) emerged, such as topically acting antiglaucoma, anticonvulsants, antiobesity, antipain, and antitumor agents/diagnostic tools. Such CAIs target diverse isozymes of the 13 catalytically active alpha-CA isoforms present in mammals. CAs belonging to the alpha-, beta-, gamma-, delta-, and zeta-families are found in many organisms all over the phylogenetic tree, and their inhibition was studied ultimately for some pathogenic protozoa (Plasmodium falciparum), fungi (Cryptococcus neoformans, Candida albicans, Candida glabrata, and Saccharomyces cerevisiae), and bacteria (Helicobacter pylori, Mycobacterium tuberculosis, and Brucella suis). Novel interesting chemotypes, in addition to the sulfonamide and sulfamate CAIs, such as coumarins, phenols, and fullerenes, were also reported recently, together with their mechanism of inhibition. This class of enzyme inhibitors shows promise for designing interesting pharmacological agents and understanding in detail protein-drug interactions at molecular level.

An unusual halotolerant alpha-type carbonic anhydrase from the alga Dunaliella salina functionally expressed in Escherichia coli.

A 60-kDa, salt-inducible, internally duplicated alpha-type carbonic anhydrase (Dca) is associated with the plasma membrane of the extremely salt-tolerant, unicellular, green alga Dunaliella salina. Unlike other carbonic anhydrases, Dca remains active over a very broad range of salinities (0-4M NaCl), thus representing a novel type of extremely halotolerant enzyme. To elucidate the structural principles of halotolerance, structure-function investigations of Dca have been initiated. Such studies require considerable amounts of the enzyme, and hence, large-scale algal cultivation. Furthermore, the purified enzyme is often contaminated with other, co-purifying algal carbonic anhydrases. Expression in heterologous systems offers a means to produce, and subsequently purify, sufficiently large amounts of Dca required for activity and structural studies. Attempts to over-express Dca in the Escherichia coli BL21(DE3)pLysS strain, after optimizing various expression parameters, produced soluble, but weakly active protein, composed of fully reduced and variably -S-S- cross-linked chains (each of the Dca repeats contains a pair of cysteine residues, presumably forming a disulfide bond). However, when the E. coli Origami B(DE3)pLysS strain was used as a host, a functionally active enzyme with proper disulfide bonds was formed in good yield. Affinity-purified recombinant Dca resembled the native enzyme from D. salina in activity and salt tolerance. Hence, this expression system offers a means of pursuing detailed studies of this extraordinary protein using biochemical, biophysical, and crystallographic approaches.

Physiological and molecular biological characterization of intracellular carbonic anhydrase from the marine diatom Phaeodactylum tricornutum.

A single intracellular carbonic anhydrase (CA) was detected in air-grown and, at reduced levels, in high CO(2)-grown cells of the marine diatom Phaeodactylum tricornutum (UTEX 642). No external CA activity was detected irrespective of growth CO(2) conditions. Ethoxyzolamide (0.4 mM), a CA-specific inhibitor, severely inhibited high-affinity photosynthesis at low concentrations of dissolved inorganic carbon, whereas 2 mM acetazolamide had little effect on the affinity for dissolved inorganic carbon, suggesting that internal CA is crucial for the operation of a carbon concentrating mechanism in P. tricornutum. Internal CA was purified 36.7-fold of that of cell homogenates by ammonium sulfate precipitation, and two-step column chromatography on diethylaminoethyl-sephacel and p-aminomethylbenzene sulfone amide agarose. The purified CA was shown, by SDS-PAGE, to comprise an electrophoretically single polypeptide of 28 kD under both reduced and nonreduced conditions. The entire sequence of the cDNA of this CA was obtained by the rapid amplification of cDNA ends method and indicated that the cDNA encodes 282 amino acids. Comparison of this putative precursor sequence with the N-terminal amino acid sequence of the purified CA indicated that it included a possible signal sequence of up to 46 amino acids at the N terminus. The mature CA was found to consist of 236 amino acids and the sequence was homologous to beta-type CAs. Even though the zinc-ligand amino acid residues were shown to be completely conserved, the amino acid residues that may constitute a CO(2)-binding site appeared to be unique among the beta-CAs so far reported.