Plasmodium falciparum carbonic anhydrase is a possible target for malaria chemotherapy

Plasmodiumfalciparum is responsible for the majority of life-threatening cases of human malaria. The global emergence of drug-resistant malarial parasites necessitates identification and characterization of novel drug targets. Carbonic anhydrase (CA) is present at high levels in human red cells and in P. falciparum. Existence of at least three isozymes of the alpha < class was demonstrated in P. falciparum and a rodent malarial parasite Plasmodium berghei. The major isozyme CA1 was purified and partially characterized from P. falciparum (PfCA1). A search of the malarial genome database yielded an open reading frame similar to the alpha-CAs from various organisms, including human. The primary amino acid sequence of the PfCA1 has 60% identity with a rodent parasite Plasmodium yoelii enzyme (PyCA). The single open reading frames encoded 235 and 252 amino acid proteins for PfCA1 and PyCA, respectively. The highly conserved active site residues were also found among organisms having alpha-CAs. The PfCA1 gene was cloned, sequenced and expressed in Escherichia coli. The purified recombinant PfCA1 enzyme was catalytically active. It was sensitive to acetazolamide and sulfanilamide inhibition. Kinetic properties of the recombinant PfCA1 revealed the authenticity to the wild type enzyme purified from P. falciparum in vitro culture. Furthermore, the PfCA1 inhibitors acetazolamide and sulfanilamide showed good antimalarial effect on the in vitro growth of P. falciparum. Our molecular tools developed for the recombinant enzyme expression will be useful for developing potential antimalarials directed at P. falciparum carbonic anhydrase.     

Mitochondrial NADH dehydrogenase from Plasmodium falciparum and Plasmodium berghei

The mitochondrial electron transport system is necessary for growth and survival of malarial parasites in mammalian host cells. NADH dehydrogenase of respiratory complex I was demonstrated in isolated mitochondrial organelles of the human parasite Plasmodium falciparum and the mouse parasite Plasmodium berghei by using the specific inhibitor rotenone on oxygen consumption and enzyme activity. It was partially purified by two sequential steps of fast protein liquid chromatographic techniques from n-octyl glucoside solubilization of the isolated mitochondria of both parasites. In addition, physical and kinetic properties of the malarial enzymes were compared to the host mouse liver mitochondrial respiratory complex I either as intact or as partially purified forms. The malarial enzyme required both NADH and ubiquinone for maximal catalysis. Furthermore, rotenone and plumbagin (ubiquinone analog) showed strong inhibitory effect against the purified malarial enzymes and had antimalarial activity against in vitro growth of P. falciparum. Some unique properties suggest that the enzyme could be exploited as chemotherapeutic target for drug development, and it may have physiological significance in the mitochondrial metabolism of the parasite.     

Comparative studies on dihydrofolate reductases from Plasmodium falciparum and Aotus trivirgatus.

Dihydrofolate reductase (E.C. 1.5.1.3) from Plasmodium falciparum and from its host, the owl monkey (Aotus trivirgatus), were partially purified and characterized. The molecular weight of the parasite enzyme was estimated to be over 10 times as high as that of the host enzyme. The host enzyme had 2 pH optima whereas the parasite enzyme only one. The activity of the host enzyme was greatly stimulated by KCl and urea, while that of the parasite enzyme was inhibited at high concentrations of such chaotropic agents. Km of the parasite enzyme was significantly higher than that of the host enzyme. The parasite enzyme had much lower Ki for pyrimethamine than the host enzyme. Dihydrofolate reductases isolated from pyrimethamine-resistant and pyrimethamine sensitive strains of P. falciparum were found to be similar.     

Import of host delta-aminolevulinate dehydratase into the malarial parasite: identification of a new drug target

The parasite Plasmodium berghei imports the enzyme delta-aminolevulinate dehydratase (ALAD), and perhaps the subsequent enzymes of the pathway from the host red blood cell to sustain heme synthesis. Here we have studied the mechanism of this import. A 65-kDa protein on the P. berghei membrane specifically bound to mouse red blood cell ALAD, and a 93-amino-acid fragment (ALAD-DeltaNC) of the host erythrocyte ALAD was able to compete with the full-length enzyme for binding to the P. berghei membrane. ALAD-DeltaNC was taken up by the infected red blood cell when added to a culture of P. falciparum and this led to a substantial decrease in ALAD protein and enzyme activity and, subsequently, heme synthesis in the parasite, resulting in its death.     

Plasmodium falciparum Carbonic Anhydrase is a Possible Target for Malaria Chemotherapy

Plasmodium falciparum is responsible for the majority of life-threatening cases of human malaria. The global emergence of drug-resistant malarial parasites necessitates identification and characterization of novel drug targets. Carbonic anhydrase (CA) is present at high levels in human red cells and in P. falciparum. Existence of at least three isozymes of the α class was demonstrated in P. falciparum and a rodent malarial parasite Plasmodium berghei. The major isozyme CA1 was purified and partially characterized from P. falciparum (PfCA1). A search of the malarial genome database yielded an open reading frame similar to the α-CAs from various organisms, including human. The primary amino acid sequence of the PfCA1 has 60% identity with a rodent parasite Plasmodium yoelii enzyme (PyCA). The single open reading frames encoded 235 and 252 amino acid proteins for PfCA1 and PyCA, respectively. The highly conserved active site residues were also found among organisms having α-CAs. The PfCA1 gene was cloned, sequenced and expressed in Escherichia coli. The purified recombinant PfCA1 enzyme was catalytically active. It was sensitive to acetazolamide and sulfanilamide inhibition. Kinetic properties of the recombinant PfCA1 revealed the authenticity to the wild type enzyme purified from P. falciparum in vitro culture. Furthermore, the PfCA1 inhibitors acetazolamide and sulfanilamide showed good antimalarial effect on the in vitro growth of P. falciparum. Our molecular tools developed for the recombinant enzyme expression will be useful for developing potential antimalarials directed at P. falciparum carbonic anhydrase.     

Characterization of adenine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum

Because of their inability to synthesize purines de novo, malaria parasites rely on purine phosphoribosyltransferases (PRTases) to convert purine bases salvaged from the host cell (the erythrocyte) into the corresponding purine nucleoside monophosphates. Our studies with late trophozoites of the human malaria parasite, Plasmodium falciparum, showed that virtually all of the purine PRTase activity is accounted for by two distinct enzymes. One enzyme utilizes hypoxanthine, guanine and xanthine (Queen, S.A., Vander Jagt, D. and Reyes, P. (1988) Mol. Biochem. Parasitol. 30, 123-134). The second enzyme utilizes only adenine and is the subject of this paper. This latter enzyme exhibits a biphasic pH-activity profile and is moderately to weakly inhibited by several divalent metal ions. Several of the properties of the P. falciparum enzyme were found to differ significantly from those of human erythrocyte adenine PRTase. (1) The molecular weight (18,000) of the parasite enzyme is smaller than that of the host cell enzyme. (2) The parasite enzyme, unlike the erythrocyte enzyme, is not significantly inhibited by sulfhydryl reagents. (3) 6-Mercaptopurine and 2,6-diaminopurine proved to be competitive inhibitors of the parasite enzyme (Ki 0.70 and 1.0 mM, respectively); on the other hand, the human enzyme is not inhibited by these agents. (4) The Km for adenine (0.80 microM) and 5-phosphoribosyl-1-pyrophosphate (0.70 microM) displayed by the parasite enzyme are significantly smaller than the corresponding Km values shown by the erythrocyte enzyme. These distinctions between the parasite and host enzymes point to the possibility that adenine PRTase of P. falciparum may represent a potential target for chemotherapeutic attack.     

Purification and properties of the carbonic anhydrase of Rhodospirillum rubrum

The carbonic anhydrase (EC 4.2.1.1) of Rhodospirillum rubrum has been purified to apparent homogeneity and some of its properties have been determined. The enzyme was cytoplasmic and was found only in photosynthetically grown cells. It had a molecular weight of about 28,000, and was apparently composed of two equal subunits. The amino acid composition was similar to that of other reported carbonic anhydrases except that the R. rubrum enzyme contained no arginine. The isoelectric point of the enzyme was 6.2 and the pH optimum was 7.5. It required Zn(II) for stability and enzymatic activity. The K _m(CO₂) was 80 mM. Typical carbonic anhydrase inhibition patterns were found with the R. rubrum enzyme. Strong acetazolamide and sulfanilamide inhibition confirmed the importance of Zn(II) for enzymatic activity as did the anionic inhibitors iodide, and azide. Other inhibitors indicated that histidine, sulfhydryl, lysine and serine residues were important for enzymatic activity.Abbreviation CA carbonic anhydrase In memory of R. Y. Stanier     

Characterization of heterologously produced carbonic anhydrase from Methanosarcina thermophila.

The gene encoding carbonic anhydrase from Methanosarcina thermophila was hyperexpressed in Escherichia coli, and the heterologously produced enzyme was purified 14-fold to apparent homogeneity. The enzyme purified from E. coli has properties (specific activity, inhibitor sensitivity, and thermostability) similar to those of the authentic enzyme isolated from M. thermophila; however, a discrepancy in molecular mass suggests that the carbonic anhydrase is posttranslationally modified in either E. coli or M. thermophila. Both the authentic and heterologously produced enzymes were stable to heating at 55 degrees C for 15 min but were inactivated at higher temperatures. No esterase activity was detected with p-nitrophenylacetate as the substrate. Plasma emission spectroscopy revealed approximately 0.6 Zn per subunit. As judged from the estimated native molecular mass, the enzyme is either a trimer or a tetramer. Western blot (immunoblot) analysis of cell extract proteins from M. thermophila indicates that the levels of carbonic anhydrase are regulated in response to the growth substrate, with protein levels higher in acetate than in methanol- or trimethylamine-grown cells.     

Plasmodium falciparum: identification and purification of the phosphoglycerate kinase of the malaria parasite.

Multiplication of the human malaria parasite Plasmodium falciparum within red blood cells is an energy-dependent process and glucose consumption increases dramatically in infected red blood cells (IRBC) versus normal red blood cells (NRBC). The major pathway for glucose metabolism in P. falciparum IRBC is anaerobic glycolysis. Phosphoglycerate kinase (PGK) is one of the key enzymes of this pathway as it generates ATP. We found that the PGK specific activity in P. falciparum IRBC is seven times higher than that in NRBC. The parasitic origin of the increase in PGK activity is confirmed by isoelectric focusing. Indeed, two P. falciparum isoenzymes with neutral isoelectric points were detected. P. falciparum PGK in purified form has a molecular mass of 48 kDa. Antiserum raised against purified P. falciparum PGK specifically recognizes the 48-kDa protein band in P. falciparum and also reacts with P. berghei and P. yoelii IRBC lysates but does not cross-react with PGK associated with NRBC.     

The amino acid substitution and some chemical properties of a variant human erythrocyte carbonic anhydrase: Carbonic anhydrase IdMichigan

Carbonic anhydrase Id_Michigan, an electrophoretic variant of human red cell carbonic anhydrase I, was purified from erythrocytes obtained from an individual heterozygous for the trait. Primary structural analysis indicates that a lysine residue has exchanged for a threonine residue in the variant enzyme. After isolation, there was approximately 1.8 times as much normal as variant enzyme. Thermostability studies demonstrated that carbonic anhydrase Id was more thermolabile than the normal enzyme. The normal and variant enzymes showed no differences in specific carboxylesterase activity or CO₂ hydratase activity. Utilizing the carboxylesterase activity toward -naphthyl acetate, the normal and variant enzymes had similar Michaelis constants, pH profiles, and rates of inhibition by acetazolamide. Immunochemical studies did not demonstrate an antigenic difference for the variant enzyme.Supported in part by Research Grants 2 T1 GM-76, 5 TO1 GM 00071-09, and GM 09252 from U.S. Public Health Service.This report is a portion of a dissertation submitted to the University of Michigan in partial fulfillment of the requirements for the doctor of philosophy degree.     

Carbonic anhydrase activity in the red blood cells of sea level and high altitude natives

Red blood cell carbonic anhydrase (CA) activity has not been studied in high altitude natives. Because CA is an intraerythocytic enzyme and high altitude natives are polycythemic, it is important to know if the activity of CA per red cell volume is different from that of their sea level counterparts. Blood was collected from healthy subjects living in Lima (150m) and from twelve subjects from Cerro de Pasco (4330m), and hematocrit and carbonic anhydrase activity were measured. As expected, the high altitude natives had significantly higher hematocrits than the sea level controls (p = 0.0002). No difference in the CA activity per milliliter of red cells was found between the two populations. There was no correlation between the hematocrit and CA activity.     

Carbonic anhydrase inhibitors. Inhibition of Plasmodium falciparum carbonic anhydrase with aromatic sulfonamides: towards antimalarials with a novel mechanism of action?

The malarial parasite Plasmodium falciparum encodes for an alpha-carbonic anhydrase (CA) enzyme possessing catalytic properties distinct of that of the human host, which was only recently purified. A series of aromatic sulfonamides, most of which were Schiff's bases derived from sulfanilamide/homosulfanilamide/4-aminoethylbenzenesulfonamide and substituted-aromatic aldehydes, or ureido-substituted such sulfonamides, were investigated for in vitro inhibition of the malarial parasite enzyme (pfCA) and the growth of P. falciparum. Several inhibitors with affinity in the micromolar range (K(I)'s in the range of 0.080-1.230 microM) were detected, whereas the most potent such derivatives were the clinically used sulfonamide CA inhibitor acetazolamide, and 4-(3,4-dichlorophenyl-ureidoethyl)-benzenesulfonamide, which showed an inhibition constant of 80 nM against pfCA, being four times more effective an inhibitor as compared to acetazolamide (K(I) of 315 nM). The lipophilic 4-(3,4-dichlorophenylureido-ethyl)-benzenesulfonamide was also an effective in vitro inhibitor for the growth of P. falciparum (IC50 of 2 microM), whereas acetazolamide achieved the same level of inhibition at 20 microM. This is the first study proving that antimalarials possessing a novel mechanism of action can be obtained, by inhibiting a critical enzyme for the life cycle of the parasite. Indeed, by inhibiting pfCA, the synthesis of pyrimidines mediated by carbamoylphosphate synthase is impaired in P. falciparum but not in the human host. Sulfonamide CA inhibitors have the potential for the development of novel antimalarial drugs.     

The purification and properties of carbonic anhydrases from guinea-pig erythrocytes and the mucosae of the gastrointestinal tract

Procedures for isolating carbonic anhydrase (EC 4.2.1.1) enzymes from the erythrocytes and the mucosae of the gastrointestinal tract of guinea pigs are described. From a haemolysate, haemoglobin was removed by the addition of ammonium sulphate, and also by two other methods, namely by gel filtration or by adsorption on DEAE-Sephadex. The crude enzyme thus obtained was resolved into the different isoenzymes by chromatography with DEAE-cellulose. From particle-free supernatants of homogenates of some gastrointestinal tissues, carbonic anhydrases were purified by ammonium sulphate fractionation, gel filtration, and ion-exchange chromatography with DEAE-cellulose. The major isoenzymes from blood, stomach, proximal colonic mucosa and caecal mucosa were homogeneous during ion-exchange chromatography, acrylamide-gel electrophoresis, and centrifugal examination. From these tissues, carbonic anhydrase was isolated as two major isoenzymes. They resemble the pairs of isoenzymes discovered in the bloods of other species. The carbon dioxide hydratase activity of one isoenzyme (;high activity' carbonic anhydrase) was 40 times that of the other isoenzyme (;low activity' carbonic anhydrase), as measured at a single substrate concentration. Two other minor components of the enzyme are also found in guinea-pig erythrocytes. All of the enzymes isolated had molecular weights of nearly 30000 (sedimentation equilibrium). ;High activity' carbonic anhydrases from blood and gastrointestinal tissues were indistinguishable according to some chemical, physical and kinetic measurements; similarly ;low activity' carbonic anhydrases from those tissues were indistinguishable. ;High activity' carbonic anhydrase was markedly different from the ;low activity' carbonic anhydrase with respect to its amino acid composition, chromatographic behaviour and isoelectric pH value. Marked differences were also found in the tissue concentrations of the major isoenzymes. It is suggested that the characteristic and selective distribution of the different forms of carbonic anhydrase in the guinea-pig tissues is related to the specific and different physiological functions of the enzymes.     

Purification and characterization of carbonic anhydrase from the saliva of the rat

Carbonic anhydrase purified from the saliva of the rat had kinetic properties identical with those of carbonic anhydrase II from rat red cells, but its molecular properties were distinctly different from the type II isozyme. Kinetic parameters were measured under steady state conditions by stopped-flow spectrophotometry and under equilibrium conditions by an 18O exchange method. The turnover number kcat for hydration of CO2 was 6.5 X 10(4) s-1 and the Michaelis constant was 4.2 mM at pH 7.5 and 25 degrees C, values which are equal to the steady state constants for red cell carbonic anhydrase II from the rat. Inhibition of the salivary isozyme by sulfanilamide (Ki = 3.7 microM) was nearly as efficient as inhibition of the erythrocyte isozyme II (Ki = 1.1 microM). The molecular weight for the salivary isozyme was 46,000 and the isoelectric point was 5.5. Salivary carbonic anhydrase had high mannose oligosaccharide components as measured by concanavalin A binding. The amino acid composition for the salivary isozyme was not similar to rat type II, but it was similar to that reported for membrane-bound carbonic anhydrase from bovine lung (Whitney, P.L., and Briggle, T.V. (1982) J. Biol. Chem. 257, 12056-12059). These observations suggest to us that salivary carbonic anhydrase is a secretory product.     

Malic Dehydrogenase Heterogeneity in Malaria (Plasmodium lophurae and P. berghei)*

SYNOPSIS. Molecular heterogeneity of malic dehydrogenase (MDH) in malaria was shown by zone electrophoresis in potato starch, starch gel, and by enzymatic activity with analogs of the coenzyme diphosphopyridine nucletide. A single anodal peak of MDH characterized the normal duck red blood cell whereas P. lophurae free of the host cell had a cathodal form of the enzyme. Infected duck erythrocytes had a combination of these electrophoretic forms. The isolated enzymes had different pH optima with oxaloacetate as substrate: pH 7.4 for the duck red cell and 6.4 for the plasmodial enzyme. The Km of each enzyme for oxaloacetate varied with the pH. The Km at pH 7.4 was 4.1 and 4.4 × 10⁵ M for parasite and host, respectively, whereas at 6.4 it was 2.0 × 10⁵ M for P. lophurae and 6.3 × 10⁵M for the duck erythrocyte. At pH 7.4 both enzymes were inhibited by oxaloacetate concentrations greater than 10^?4 M. P. berghei MDH also had a different electrophoretic character from that of the mouse red blood cell. Quantitatively, MDH activity increased with parasitization, and erythrocyte-free P. lophurae contained approximately twice the activity found in the uninfected duck erythrocyte. The quantity of MDH activity of the infected cell was ca. 50% less than the sum of the activities of the parasite and the uninfected cell. It is suggested that these properties of the parasite MDH may give it a physiologic advantage over the red cell under the conditions which prevail intraerythrocytically.     

Purification and characterization of human salivary carbonic anhydrase

A novel carbonic anhydrase was purified from human saliva with inhibitor affinity chromatography followed by ion-exchange chromatography. The molecular weight was determined to be 42,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the human salivary enzyme is larger than the cytosolic isoenzymes CA I, CA II, and CA III (Mr 29,000) from human tissue sources. Each molecule of the salivary enzyme had two N-linked oligosaccharide chains which were cleaved by endo-beta-N-acetylglucosaminidase F but not by endo-beta-N-acetylglucosaminidase H, indicating that the oligosaccharides are complex type. The isoelectric point was determined to be 6.4, but significant charge heterogeneity was found in different preparations. The human salivary isozyme has lower specific activity than the rat salivary isozyme and the human red blood cell isozyme II in the CO2 hydratase reaction. The inhibitory properties of the salivary isozyme resemble those of CA II with iodide, sulfanilamide, and bromopyruvic acid, but the salivary enzyme is less sensitive to acetazolamide and methazolamide than CA II. Antiserum raised in a rabbit against the salivary enzyme cross-reacted with CA II from human erythrocytes, indicating that human salivary carbonic anhydrase and CA II must share at least one antigenic site. CA I and CA III did not crossreact with this antiserum. The amount of salivary carbonic anhydrase in the saliva of the CA II-deficient patients was greatly reduced, indicating that the CA II deficiency mutation directly or indirectly affects the expression of the salivary carbonic anhydrase isozyme. From these results we conclude that the salivary carbonic anhydrase is immunologically and genetically related to CA II, but that it is a novel and distinct isozyme which we tentatively designate CA VI.     

Biochemical characterization and crystallization of recombinant 3-phosphoglycerate kinase of Plasmodium falciparum

Human malaria parasite Plasmodium falciparum depends largely on glycolytic pathway for energy metabolism during the intraerythrocytic life stage. Therefore, enzymes of the glycolytic pathway could offer potential drug targets provided novel biochemical and/or structural features of the parasitic enzymes, which distinguish them from the host counterpart, could be identified. 3-Phosphoglycerate kinase (EC 2.7.2.3) catalyzes an important phosphorylation step leading to the production of ATP in the glycolytic pathway. We have expressed recombinant 3-phosphoglycerate kinase of P. falciparum in Escherichia coli. The recombinant protein purified from the soluble fraction of E. coli is enzymatically active. The apparent K(m) values determined for adenosine triphosphate (ATP) and 3-phosphoglycerate (3-PGA) are 0.63 and 0.52 mM, respectively. The enzyme activity was temperature-sensitive. Suramin was found to inhibit the recombinant enzyme with an IC(50) value of 7 microM. We have crystallized the enzyme form in hexagonal space group P6(1)22 (or its enantiomorphic space group) with unit cell parameters a=b=130.7, c=263.9 A. Native data have been collected at 3.0-A resolution.     

Purification and properties of cyclostome carbonic anhydrase from erythrocytes of hagfish

1. Carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) has been purified from erythrocytes of hagfish (Myxine glutinosa). A single form with low specific CO2 hydration activity was isolated. The purified carbonic anhydrase appeared homogeneous judging from polyacrylamide gel electrophoresis and gel filtration experiments. The protein has a molecular weight of about 29 000, corresponding to about 260 amino acid residues. This molecular weight is in accordance with other vertebrate carbonic anhydrases with the exception of the elasmobranch enzymes, which have Mr 36 000--39 000. 2. The molecular weight obtained for hagfish carbonic anhydrase indicates that a carbonic anhydrase with Mr approx. 29 000 is the ancestral type of the vertebrate enzyme rather than, as in sharks, a heavier carbonic anhydrase molecule. 3. The circular dichroism spectrum may indicate a somewhat different structural arrangement of aromatic amino acid residues in this enzyme than in the mammalian carbonic anhydrases. 4. The enzyme is strongly inhibited by acetazolamide and also to a lesser extent by monovalent anions. 5. Zn2+, which is essential for activity, appears, contrary to other characterized carbonic anhydrases, less strongly bound in the active site of the enzyme.     

Unique properties of Plasmodium falciparum porphobilinogen deaminase

The hybrid pathway for heme biosynthesis in the malarial parasite proposes the involvement of parasite genome-coded enzymes of the pathway localized in different compartments such as apicoplast, mitochondria, and cytosol. However, knowledge on the functionality and localization of many of these enzymes is not available. In this study, we demonstrate that porphobilinogen deaminase encoded by the Plasmodium falciparum genome (PfPBGD) has several unique biochemical properties. Studies carried out with PfPBGD partially purified from parasite membrane fraction, as well as recombinant PfPBGD lacking N-terminal 64 amino acids expressed and purified from Escherichia coli cells (DeltaPfPBGD), indicate that both the proteins are catalytically active. Surprisingly, PfPBGD catalyzes the conversion of porphobilinogen to uroporphyrinogen III (UROGEN III), indicating that it also possesses uroporphyrinogen III synthase (UROS) activity, catalyzing the next step. This obviates the necessity to have a separate gene for UROS that has not been so far annotated in the parasite genome. Interestingly, DeltaPfP-BGD gives rise to UROGEN III even after heat treatment, although UROS from other sources is known to be heat-sensitive. Based on the analysis of active site residues, a DeltaPfPBGDL116K mutant enzyme was created and the specific activity of this recombinant mutant enzyme is 5-fold higher than DeltaPfPBGD. More interestingly, DeltaPfPBGDL116K catalyzes the formation of uroporphyrinogen I (UROGEN I) in addition to UROGEN III, indicating that with increased PBGD activity the UROS activity of PBGD may perhaps become rate-limiting, thus leading to non-enzymatic cyclization of preuroporphyrinogen to UROGEN I. PfPBGD is localized to the apicoplast and is catalytically very inefficient compared with the host red cell enzyme.     

Identification of Intracellular Carbonic Anhydrase in Chlamydomonas reinhardtii which Is Distinct from the Periplasmic Form of the Enzyme

A physiologically significant level of intracellular carbonic anhydrase has been identified in Chlamydomonas reinhardtii after lysis of the cell wall-less mutant, cw15, and two intracellular polypeptides have been identified which bind to anti-carbonic anhydrase antisera. The susceptibility of the intracellular activity to sulfonamide carbonic anhydrase inhibitors is more than three orders-of-magnitude less than that of the periplasmic enzyme, indicating that the intracellular activity was distinct from the periplasmic from of the enzyme. When electrophoretically separated cell extracts or chloroplast stromal fractions were probed with either anti-C. reinhardtii periplasmic carbonic anhydrase antiserum or anti-spinach carbonic anhydrase antiserum, immunoreactive polypeptides of 45 kilodaltons and 110 kilodaltons were observed with both antisera. The strongly immunoreactive 37 kilodalton polypeptide due to the periplasmic carbonic anhydrase was also observed in lysed cells, but neither the 37 kilodalton nor the 110 kilodalton polypeptides were present in the chloroplast stromal fraction. These studies have identified intracellular carbonic anhydrase activity, and putative intracellular carbonic anhydrase polypeptides in Chlamydomonas reinhardtii represented by a 45 kilodalton polypeptide in the chloroplast and a 110 kilodalton form probably in the cytoplasm, which may be associated with an intracellular inorganic carbon concentrating system.