Carbonic Anhydrase Enzyme Activities Research Articles

Effects of Some Hormones on Enzyme Activities of Carbonic Anhydrase from Brain in vitro

Effects of Some Hormones on Enzyme Activities of Carbonic Anhydrase from Brain in vitro

Targeting erythrocyte carbonic anhydrase and 18O-isotope of breath CO2 for sorting out type 1 and type 2 diabetes.

The inability to envisage the acute onset and progression of type 1 diabetes (T1D) has been a major clinical stumbling block and an important area of biomedical research over the last few decades. Therefore there is a pressing need to develop a new and an effective strategy for early detection of T1D and to precisely distinguish T1D from type 2 diabetes (T2D). Here we describe the precise role of the enzymatic activity of carbonic anhydrase (CA) in erythrocytes in the pathogenesis of T1D and T2D. We show that CA activities are markedly altered during metabolism of T1D and T2D and this facilitates to the oxygen-18 (18O) isotopic fractionations of breath CO2. In our observations, T1D exhibited considerable depletions of 18O-isotopes of CO2, whereas T2D manifested isotopic enrichments of 18O in breath CO2, thus unveiling a missing link of breath18O-isotopic fractionations in T1D and T2D. Our findings suggest that the alterations in erythrocytes CA activities may be the initial step of altered metabolism of T1D and T2D, and breath 18O-isotope regulated by the CA activity is a potential diagnostic biomarker that can selectively and precisely distinguish T1D from T2D and thus may open a potential unifying strategy for treating these diseases.

Purification and characterization of the carbonic anhydrase enzyme from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney and inhibition effects of some metal ions on enzyme activity.

In this study, the carbonic anhydrase (CA) enzyme was purified from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney with a specific activity of 603.77EU/mg and a yield of 35.5% using Sepharose-4B-l-tyrosine- sulphanilamide affinity column chromatography. For determining the enzyme purity and subunit molecular mass, sodiumdodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed and single band was observed. The molecular mass of subunit was found approximately 29.71kDa. The optimum temperature, activation energy (Ea), activation enthalpy (ΔH) and Q10 values were obtained from Arrhenius plot. Km and Vmax values for p-nitrophenyl acetate of the purified enzyme were calculated from Lineweaver-Burk graphs. In addition, the inhibitory effects of different heavy metal ions (Fe(2+), Pb(2+), Co(2+), Ag(+) and Cu(2+)) on Black Sea trout kidney tissue CA enzyme activities were investigated by using esterase method under in vitro conditions. The heavy metal concentrations inhibiting 50% of enzyme activity (IC50) were obtained. Finally Ki values and inhibition types were calculated from Lineweaver-Burk graphs.

In vivo effects of curcumin on the paraoxonase, carbonic anhydrase, glucose-6-phosphate dehydrogenase and β-glucosidase enzyme activities in dextran sulphate sodium-induced ulcerative colitis mice

Increases in the risk of infections and malignancy due to immune suppressive therapies of inflammatory bowel diseases (IBDs) have led the researchers to focus on more nontoxic and acceptable natural products like curcumin. Here we investigate whether prophylactic and therapeutic application of the curcumin alters the enzyme activities of paraoxonase (PON), carbonic anhydrase (CA), glucose-6-phosphate dehydrogenase (G6PD) and cytosolic β-glucosidase in dextran sulphate sodium (DSS)-induced ulcerative colitis mice. Prophylactic application of curcumin resulted in higher MPO activity, less body weight loss and longer colon lengths compared to therapeutic group indicating preventive role of curcumin in IBDs. DSS-induced decrease in liver and serum PON activities were completely recovered by prophylactic administration of curcumin. DSS-induced reduction in liver cytosolic β-glucosidase activity was not affected by curcumin neither in the prophylactic group nor in the therapeutic group. Erythrocyte CA activity was significantly increased in curcumin groups, however no remarkable change in G6PD activity was observed.

Gene expression and enzyme activities of carbonic anhydrase and glutaminase in rat kidneys induced by chronic systemic hypoxia

Background: Hypoxia can cause acidosis. Kidney plays an essential role in maintaining acid-base balance, which involves the activities of carbonic anhydrase (CA) and glutaminase (GLS). This study is aimed to determine the expression and activities of the CA9 and GLS1 enzymes in relation to hypoxia inducible factor-1α (HIF-1α), a transcription factor protein which is a marker of hypoxia.Methods: This study was an in vivo experimental study with coupled paralel design. used 25 male Sprague-Dawley rats weighing 150-200 g. Rats were divided into 5 groups: the control group (normoxic condition) and 4 treatment groups. The latter were kept in a hypoxic chamber (10% O2: 90% N2) for 1, 3, 5 and 7 days. All rats were euthanized after treatment, kidneys excised, tissues homogenized and investigated for gene expression of CA9, GLS1 and HIF-1α. On protein level, total enzymatic activities of CA and GLS and protein of HIF-1α were also investigated. Data were analyzed statistically using ANOVA for significance, and as its alternative, used Mann-Whitney and Kruskal-Wallis test.Results: Results showed that HIF-1α mRNA increased during hypoxia, but not HIF-1α protein. It seemed that acidosis occurs in kidney tissue, indicated by increased CA9 and GLS1 mRNA expression and specific activity of total CA and GLS1. Expression of CA9 and GLS1 mRNA both showed strong positive correlation with HIF-1α mRNA, but not with HIF-1α protein.Conclusion: It is suggested that during chronic systemic hypoxia, gene expression of CA9 and GLS1 and their enzyme activities were increased as a response to acidosis and related with the expression of HIF-1α mRNA.

Variable responses of temperate calcified and fleshy macroalgae to elevated pCO2 and warming

Abstract Anthropogenic carbon dioxide (CO2) emissions simultaneously increase ocean temperatures and reduce ocean surface pH, a process termed ocean acidification (OA). OA is expected to negatively affect the growth and physiology of many calcified organisms, but the response of non-calcified (fleshy) organisms is less well understood. Rising temperatures and pCO2 can enhance photosynthetic rates (within tolerance limits). Therefore, warming may interact with OA to alter biological responses of macroalgae in complicated ways. Beyond thresholds of physiological tolerance, however, rising temperatures could further exacerbate negative responses to OA. Many studies have investigated the effects of OA or warming independently of each other, but few studies have quantified the interactive effects of OA and warming on marine organisms. We conducted four short-term independent factorial CO2 enrichment and warming experiments on six common species of calcified and fleshy macroalgae from southern California to investigate the independent and interactive effects of CO2 and warming on growth, carbonic anhydrase (CA) enzyme activity, pigment concentrations, and photosynthetic efficiency. There was no effect of elevated pCO2 on CA activity, pigment concentration, and photosynthetic efficiency in the macroalgal species studies. However, we found that calcareous algae suffered reduced growth rates under high pCO2 conditions alone, although the magnitude of the effect varied by species. Fleshy algae had mixed responses of growth rates to high pCO2, indicating that the effects of pCO2 enrichment are inconsistent across species. The combined effects of elevated pCO2 and warming had a significantly negative impact on growth for both fleshy and calcareous algae; calcareous algae experienced five times more weight loss than specimens in ambient control conditions and fleshy growth was reduced by 76%. Our results demonstrate the need to study the interactive effects of multiple stressors associated with global change on marine communities.

Different mechanisms of photosynthetic response to drought stress in tomato and violet orychophragmus

Carbonic anhydrase (CA) catalyzes reversible hydration of CO2 and it can compensate for the lack of H2O and CO2 in plants under stress conditions. Antioxidative enzymes play a key role in scavenging reactive oxygen species and in protecting plant cells against toxic effects. Tomato represents a stress-sensitive plant while violet orychophragmus belongs to adversity-resistant plants. In order to study the drought responses in tomato and violet orychophragmus plants, CA and antioxidative enzyme activities, photosynthetic capacity, and water potential were determined in plants under drought stress. We found that there were similar change trends in CA activity and drought tolerance in violet orychophragmus, and in antioxidative enzymes and drought tolerance in tomato plants. Basic mechanisms of drought resistance should be identified for understanding of breeding measures in plants under stress conditions.

Biosurfactant production by a CO2 sequestering Bacillus sp. strain ISTS2

A chemolithotrophic bacterium, Bacillus sp. strain ISTS2, produced biosurfactant when enriched in the chemostat in presence of sodium bicarbonate as carbon source was evaluated for carbon dioxide (CO2) sequestration and biosurfactant production. CO2 sequestration efficiency of the bacterium was determined by enzymatic activity of carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Biosurfactant production ability at 100mM NHCO3 and 5% CO2 was screened by surface and interfacial tension measurement, emulsification stability test, hydrophobicity test, contact angle measurement, bacterial adhesion to hydrocarbon and purified by silica gel column (60–120mesh). Thin layer chromatography (TLC) and gas chromatography–mass spectrometry (GC–MS) showed that the crude biosurfactant of ISTS2 were composed of lipopeptides and free fatty acids (FA) and its hydrophobic fraction contained five kinds of fatty acids (FA) with chain lengths of C14–C19. Thus Bacillus sp. strain IST2 can be used as a cleaner bioprocess for the utilization of industrial CO2 as alternate substrate.

The effects of some tumor markers on human erythrocyte (HCA-I and HCA-II), bovine erythrocyte (BCA) and bovine lung (CA-IV) carbonic anhydrase enzyme activities in vitro.

The influence of prostatic acid phosphatase (PAP) and human chorionic gonadotropin (HCG), tumor markers have been investigated on human erythrocyte carbonic anhydrase (HCA-I and HCA-II) and bovine erythrocyte (BCA) and bovine lung carbonic anhydrase (CA-IV) in vitro. Tumor markers are substances that can often be detected in higher-than-normal amounts in the blood, urine, or body tissues of some patients with certain types of cancer. Tumor markers are produced either by the tumor itself or by the body in response to the presence of cancer or certain benign (noncancerous) conditions. In addition to their role in cancer diagnosis, some tumor marker levels are measured before treatment to help doctors plan appropriate therapy. All of the tumor markers were determined to have inhibition effect, on human CA-I, CA-II, bovine erythrocyte CA (BCA) and bovine lung CA-IV isoenzymes. The effect of each tumor marker on CA was investigated by Wilbur-Andersen method modified by Rickly et al Inhibition effects of two different tumor markers on human CA-I, CA-II, bovine erythrocyte CA (BCA) and bovine lung CA-IV isoenzymes were determined by using the CO2-Hydratase method by plotting activity % vs (tumor markers). I50 values of tumor markers exhibiting inhibition effects were found by means of these graphs (Tab.1, Fig. 2, Ref. 20).

Inhibition of Carbonic Anhydrase Results in Blood Lactate Accumulation (BLA) in STZ Induced Diabetic Rats

Carbonic anhydrase is a pH regulatory enzyme, whose inhibition leads to metabolic acidosis. We investigated whether carbonic anhydrase inhibition may be associated with blood lactate accumulation in Streptozotocin (STZ) induced diabetic rats. The study aimed to provide a new marker to assist in identifying diabetic individuals at a high risk of developing lactic acidosis. Erythrocyte carbonic anhydrase activity with 4-nitrophenyl acetate as substrates was investigated in (STZ) induced diabetic rats. STZ induced diabetic rats showed significant increase in erythrocyte carbonic anhydrase (CA) activity of 9.7 ± 0.7 μMol/min/μL and blood lactate level of 6.2 ± 1.2 mMol/L when compared with Non Diabetic group CA enzyme activity of 5.5 ± 0.6 μMol/min/μL and blood lactate level of 3.9 ± 0.5 mMol/L. Inhibition of erythrocyte carbonic anhydrase activity with Acetazolamide results in 6.9 fold increase in blood lactate level when compared with Non diabetic group. Therefore inhibition of Carbonic anhydrase in STZ induced diabetic rats’ result in blood lactate accumulation and reduced blood glucose concentration.

In vivo changes in carbonic anhydrase activity and histopathology of gill and liver tissues after acute exposure to chlorpyrifos in rainbow trout.

Chlorpyrifos is an organophosphate pesticide widely used in agriculture and aquaculture. This study investigated its effects on carbonic anhydrase (CA) enzyme activity and histopathology of rainbow trout gill and liver. The fish were exposed to 2.25 (25% of 96 h LC50), 4.5 (50% of 96 h LC50), and 6.75 μg L⁻¹ (75% of 96 h LC50) of chlorpyrifos for 24, 48, 72, and 96 h. CA activity was measured in liver and gills and histopathological changes were examined by light microscopy. The most common liver changes at most of the chlorpyrifos concentrations were hyperaemia and degenerative changes. Gill tissues were characterised by lamellar hyperaemia, lamellar oedemas, clumping, cellular degeneration, hyperplasia, and lamellar atrophy. CA enzyme activity in the gills decreased at all concentrations at 48, 72, and 96 h after exposure to chlorpyrifos (p<0.05). Similarly, there was a time-dependent decrease in CA activity at all of the concentrations in liver tissues (p<0.05). The present study indicated that chlorpyrifos inhibits CA enzyme activity and causes histopathological damage in gill and liver tissues.

Immobilization of Carbonic Anhydrase on Modified Electrospun Poly(Lactic Acid) Membranes: Quest for Optimum Biocatalytic Performance

Electrospun poly (lactic acid) membranes modified with graphene oxide (GO) and nano sized metal organic frameworks (nMOF) were synthesized, characterized and used for the immobilization of carbonic anhydrase (CA). The mechanical strength and young’s modulus of the membrane were slightly decreased when GO and nMOF were introduced, due to irregularity in the dispersion. On the other hand, the incorporation of GO into the membrane increased its thermal degradation temperature. Enzyme loading was substantially improved with GO and nMOF integration (~1.83 and ~1.57 times higher than that of poly (lactic acid)). Thermal stability, storage stability and reusability of immobilized CA, all of which are important for industrial applications, were also found to be enhanced significantly. Improved loading, stability and biocatalytic activity of carbonic anhydrase enzyme on modified electrospun membrane are reported

Activity enhancement of carbonic anhydrase in Chlamydomonas sp. for effective CO2 sequestration

Present study describes the induction of carbonic anhydrase (CA) activity in Chlamydomonas sp. in presence of calcium carbonate. Results indicated that calcium carbonate increased the activity of extracellular CA enzyme by 14-fold compared to culture grown at 3 % CO2, and by fourfold compared to the culture grown at 0.03 % CO2. CA was purified by gel exclusion followed by ion exchange chromatography. Molecular weight of the purified CA was found to be approximately 28 kDa. The enzyme showed optimum stability in the pH range of 8.0–9.0 and temperature of 35 °C. CA activity was inhibited with Hg2+ and Pb2+, while Zn2+ was found to accelerate its activity. Purified CA was employed for carbonation reaction with CO2 and the calcite produced was characterized by scanning electron microscope and X-ray diffraction. The enhancement in CA activity was found to be reliable, convenient, and may be employed for CO2 sequestration using whole cells or immobilized bioreactor systems.

Production and characterization of biodiesel from carbon dioxide concentrating chemolithotrophic bacteria, Serratia sp. ISTD04

A chemolithotrophic bacterium, Serratia sp. ISTD04, enriched in the chemostat in presence of sodium bicarbonate as sole carbon source was evaluated for potential of carbon dioxide (CO2) sequestration and biofuel production. CO2 sequestration efficiency of the bacterium was determined by enzymatic activity of carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Further, Western blot analysis confirmed presence of RuBisCO. The bacterium produced 0.487 and 0.647mgmg(-1) per unit cell dry weight of hydrocarbons and lipids respectively. The hydrocarbons were within the range of C13-C24 making it equivalent to light oil. GC-MS analysis of lipids produced by the bacterium indicated presence of C15-C20 organic compounds that made it potential source of biodiesel after transesterification. GC-MS, FTIR and NMR spectroscopic characterization of the fatty acid methyl esters revealed the presence of 55% and 45% of unsaturated and saturated organic compounds respectively, thus making it a balanced biodiesel composition.

Effects of environmental hypercapnia on hemato-immunological parameters, carbonic anhydrase, and Na+, K+-ATPase enzyme activities in rainbow trout (Oncorhynchus mykiss) tissues

In this study, the effects of environmental hypercapnia on hemato-immunological parameters and the activities of respiratory enzymes such as carbonic anhydrase (CA) and Na+, K+-ATPase were investigated in rainbow trout (Oncorhynchus mykiss) tissues (gill, liver and kidney). Batches of 12 fish were exposed to 4.5 mg L−1 (control) and 14 mg L−1 CO2. No mortalities occurred during the 14 days of the experimental period. Red blood cell (RBC), hemoglobin (Hb), and hematocrit (Ht) levels, and innate immune parameters such as nitro blue tetrazolium (NBT), lysozyme, and myeloperoxidase activities, and the melano-macrophage frequency were negatively affected by elevated CO2 levels. Patterns of change in CA activity differed among the gill, liver, and kidney. Compared with the activities of CA in the control group, the CA enzyme was significantly stimulated at day 7 in the gill tissue, whereas it was stimulated at day 14 of the experiment in the liver tissue of fish exposed to 14 mg L−1 CO2 (P < 0.05). In contrast to the pattern of CA enzyme activities, the Na+, K+-ATPase enzymes were stimulated significantly in the liver after day 7 but inhibited in the kidney and gill (P < 0.05). These results suggest that a subchronic exposure to hypercapnia of rainbow trout tissues may lead to adaptive changes in the respiratory enzymes and negatively affects hemato-immunological parameters.

Carbonic anhydrase activity from the gilthead sea bream (Sparus aurata) liver: The toxicological effects of heavy metals

Many studies have shown that metal ions may lead to oxidative stress in biological systems. Accordingly, DNA damage, protein modification, enzyme inhibition and activation, lipid peroxidation and many other effects may occur in living organisms. Many different formations of metal ions may enter human cells along with water, air, and various foods, and humans are negatively affected by these conditions, either directly or indirectly. These effects may cause irreversible damage to human metabolism. In this study, the toxicological effects of heavy metals on carbonic anhydrase enzyme activity from the gilthead sea bream liver were investigated. The carbonic anhydrase enzyme was purified via affinity chromatography and had a specific activity of 6775.5EUmg−1. The kinetics and characteristic properties, such as optimum pH, stable pH, optimum temperature, activation energy (Ea), activation enthalpy (ΔH), Q10, Km, and Vmax, were determined for the purified enzyme SDS-polyacrylamide gel electrophoresis showed a single band and molecular weight of the subunit was approximately 25kDa. Cd(II), Cu(II), Ni(II) and Ag(I) inhibited the enzyme activity in vitro. The type of inhibition and Ki values for these metals were calculated from Lineweaver–Burk plots as 17.74mM, 36.20mM, 12.85mM and 0.025mM for Cd(II), Cu(II), Ni(II) and Ag(I), respectively. All the metals were noncompetitive inhibitors.

Investigating Catalase and Carbonic Anhydrase Enzyme Activities and Levels of Certain Trace Elements and Heavy Metals in Patients with Primary and Metastatic Hepatic Carcinoma

Hepatocellular carcinoma (HCC) is among most common terminal cancer types in the world. Primary etiological factors include cirrhosis, hepatitis, aflatoxin and alcohol. The current study was conducted to determine cytosolic erythrocyte carbonic anhydrase and catalase enzyme activities and levels of some trace elements. For this purpose, 40 patients with primary and metastatic hepatic cancer and 29 healthy volunteers enrolled to the study. Catalase and carbonic anhydrase enzyme activities and serum trace element levels were measured in patient and control groups. In the current study, serum copper, magnesium, manganese and zinc levels were lower in the primary and metastatic hepatic cancer group in comparison with the control group (P

Effect of levosimendan injection on oxidative stress of rat myocardium

This experiment was designed to investigate the effect of levosimendan injection on lipid peroxidation product malondialdehyde (MDA) and antioxidant glutathione (GSH) levels, and activities of antioxidant enzymes in myocardium of rats. Twenty male Wistar-albino rats were divided randomly into 2 study groups, each consisting of 10 rats. The animals in the first group were not treated with drug and served as control. It was found that the MDA and GSH levels decreased in levosimendan injected group. Superoxide dismutase, glutathione peroxidase, catalase and carbonic anhydrase enzyme activities were lower in levosimendan injected group than controls. It was concluded that lower tissue free radical level caused by levosimendan injection led to a lower antioxidant enzymes synthesis in the body and a decrease in the antioxidant enzyme activity and free radical scavenger level in myocardium of rat.

Inhibitory effects of hypo-osmotic stress on extracellular carbonic anhydrase and photosynthetic efficiency of green alga Dunaliella salina possibly through reactive oxygen species formation

In this study, Dunaliella salina (D. salina) maintained in 30‰ salinity for more than two years was exposed to the salinities of 5‰, 10‰, 20‰, 30‰ (control) in order to investigate oxidative burst and it's possible connection with extracellular carbonic anhydrase (CA) under hypo-osmotic stress (low salinity). The results indicated that intracellular ROS contents increased significantly when cells were exposed to salinity of 5 and 10‰, and the increase also occurred at 20‰ salinity. The activity of extracellular CA and its gene (P60) expression decreased significantly when cells were exposed to salinity of 5-20‰. Data from H₂O₂ treatments hinted that ROS production was possibly one of the factors affecting CA, including enzyme activity and gene expression levels. Significant inhibition of effective quantum efficiency of PSII and photosynthetic oxygen evolution rate were observed with the increase of ROS production and decline of CA activities. Taken together, hypo-osmotic stresses could induce ROS production in D. salina, and CA enzyme activities and expression levels were consequently inhibited. As a result, algal photosynthesis and oxygen evolution were inhibited.

Dual signal amplification for bioassays using ion release from nanolabels and ion-activated enzyme kinetics

A dual signal amplification technique was developed for bioassays. The technique consists of zinc-ion release from ZnS nanoparticle labels and enzyme kinetics activated by the released zinc ions as cofactors. In the ion release process, each ZnS nanoparticle label liberates a high number of zinc ions by acidic dissolution. After the ion release, at appropriate pH levels, the released zinc ions are used as cofactors to trigger the enzymatic activity of carbonic anhydrase. The fluorescence produced from the activated enzyme kinetics is measured for bioassay signal quantification. A model bioassay on mouse IgG adopting this technique presents a detection limit around 0.5 pM and a detection range over at least two orders of magnitude. This technique was also successfully applied to the detection of human cardiac troponin I (cTnI) in human serum samples to demonstrate a clinical diagnosis application. The developed immunoassay is capable of distinguishing clinically critical levels of cTnI. This technique possesses a high detection resolution and offers the advantage of straightforward operation (simple preparation of ZnS nanoparticles and no enzyme immobilization).