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1.
The activity and subcellular distribution of carbonic anhydrase in a coccolithophorid alga, CCMP 299, was examined. The enzyme could not be detected in crude cell homogenates but was present at high specific activity (27.5 unit·mg?1 protein) in chloroplasts (density, 1.14 g·cm?3) isolated in a sucrose gradient. The carbonic anhydrase activity was sensitive to known inhibitors. Inhibition at 50% (I50) was obtained with concentrations of 4.60 mM and 2.65 mM for acetazolamide and NaN3, respectively. These levels are more consistent with patterns of inhibition previously observed for chloroplastic (as compared to periplasmic) carbonic anhydrase. In this organism, carbonic anhydrase was localized in the chloroplast stroma. These findings are discussed in terms of the relationship among dissolved inorganic carbon interconversions, photosynthesis, and calcification. 相似文献
2.
Safarian S Bagheri F Moosavi-Movahedi AA Amanlou M Sheibani N 《The protein journal》2007,26(6):371-385
Sulfonamide drugs mediate their main therapeutic effects through modulation of the activity of membrane and cytosolic carbonic
anhydrases. How interactions of sulfonamide drugs impact structural properties and activity of carbonic anhydrases requires
further study. Here the effect of acetazolamide on the structure and function of bovine carbonic anhydrase II (cytosolic form
of the enzyme) was evaluated. The Far-UV CD studies indicated that carbonic anhydrase, for the most part, retains its secondary
structure in the presence of acetazolamide. Fluorescence measurements using iodide ions and ANS, along with ASA calculations,
revealed that in the presence of acetazolamide minimal conformational changes occurred in the carbonic anhydrase structure.
These structural changes, which may involve spatial reorientation of Trp 4 and Trp 190 or some other related aminoacyl residues
near the active site, considerably reduced the catalytic activity of the enzyme while its thermal stability was slightly increased.
Our binding results indicated that binding of acetazolamide to the protein could occur with a 1:1 ratio, one mole of acetazolamide
per one mole of the protein. However, the obtained kinetic results supported the existence of two acetazolamide binding sites
on the protein structure. The occupation of each of these binding sites by acetazolamide completely inactivates the enzyme.
Advanced analysis of the kinetic results revealed that there are two substrate (p-NPA) binding sites whose simultaneous occupation is required for full enzyme activity. Thus, these studies suggest that the
two isoforms of CA II should exist in the medium, each of which contains one substrate binding site (catalytic site) and one
acetazolamide binding site. The acetazolamide binding site is equivalent to the catalytic site, thus, inhibiting enzyme activity
by a competitive mechanism. 相似文献
3.
Alexandr V. Shitov Vasily V. Terentyev Sergey K. Zharmukhamedov Margarita V. Rodionova Mehmet Karacan Nurcan Karacan Vyacheslav V. Klimov Suleyman I. Allakhverdiev 《BBA》2018,1859(4):292-299
It is known, that the multi-subunit complex of photosystem II (PSII) and some of its single proteins exhibit carbonic anhydrase activity. Previously, we have shown that PSII depletion of HCO3?/CO2 as well as the suppression of carbonic anhydrase activity of PSII by a known inhibitor of α?carbonic anhydrases, acetazolamide (AZM), was accompanied by a decrease of electron transport rate on the PSII donor side. It was concluded that carbonic anhydrase activity was required for maximum photosynthetic activity of PSII but it was not excluded that AZM may have two independent mechanisms of action on PSII: specific and nonspecific. To investigate directly the specific influence of carbonic anhydrase inhibition on the photosynthetic activity in PSII we used another known inhibitor of α?carbonic anhydrase, trifluoromethanesulfonamide (TFMSA), which molecular structure and physicochemical properties are quite different from those of AZM. In this work, we show for the first time that TFMSA inhibits PSII carbonic anhydrase activity and decreases rates of both the photo-induced changes of chlorophyll fluorescence yield and the photosynthetic oxygen evolution. The inhibitory effect of TFMSA on PSII photosynthetic activity was revealed only in the medium depleted of HCO3?/CO2. Addition of exogenous HCO3? or PSII electron donors led to disappearance of the TFMSA inhibitory effect on the electron transport in PSII, indicating that TFMSA inhibition site was located on the PSII donor side. These results show the specificity of TFMSA action on carbonic anhydrase and photosynthetic activities of PSII. In this work, we discuss the necessity of carbonic anhydrase activity for the maximum effectiveness of electron transport on the donor side of PSII. 相似文献
4.
Sumathra Manokaran Jayati Banerjee Sanku Mallik D.K. Srivastava 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(10):1965-1973
We synthesized a fluorogenic dansylamide derivative (JB2-48), which fills the entire (15 Å deep) active site pocket of human carbonic anhydrase I, and investigated the contributions of sulfonamide and hydrophobic regions of the ligand structure on the spectral, kinetic, and thermodynamic properties of the enzyme–ligand complex. The steady-state and fluorescence lifetime data revealed that the deprotonation of the sulfonamide moiety of the enzyme bound ligand increases the fluorescence emission intensity as well as the lifetime of the fluorophores. This is manifested via the electrostatic interaction between the active site resident Zn2+ cofactor and the negatively charged sulfonamide group of the ligand, and such interaction contributes to about 2.2 kcal/mol (ΔΔG°) and 0.89 kcal/mol (ΔΔG‡) energy in stabilizing the ground and the putative transition states, respectively. We provide evidence that the anionic and neutral forms of JB2-48 are stabilized by the complementary microscopic/conformational states of the enzyme. The implication of the mechanistic studies presented herein in rationale design of carbonic anhydrase inhibitors is discussed. 相似文献
5.
Modification of carbonic anhydrase activity by antisense and over-expression constructs in transgenic tobacco 总被引:1,自引:0,他引:1
The activity and location of carbonic anhydrase has been modified by transformation of tobacco with antisense and over-expression constructs. Antisense expression resulted in the inhibition of up to 99% of carbonic anhydrase activity but had no significant impact on net CO2 assimilation. Stomatal conductance and susceptibility to water stress appeared to increase in response to the decline in carbonic anhydrase activity. An over-expression construct designed to increase cytosolic carbonic anhydrase abundance resulted in a significant increase in net activity, a small increase in stomatal conductance but little impact on CO2 assimilation. Chloroplastic carbonic anhydrase activity was enhanced by the expression of an additional construct which targeted the polypeptide to the organelle. The increase in chloroplastic carbonic anhydrase appeared to be accompanied by a concomitant increase in Rubisco activity. 相似文献
6.
Carbonic anhydrase activities of pea thylakoids as well as thylakoid fragments enriched either in Photosystem 1 (PS1-membranes)
or Photosystem 2 (PS2-membranes) were studied. The activity of PS1-membranes if calculated on chlorophyll basis was much higher
than the activity of PS2-membranes. Acetazolamide, a non-permeable inhibitor of carbonic anhydrases, increased carbonic anhydrase
activity of PS2-membranes at concentrations lower than 10−6 M and suppressed this activity only at higher concentrations. A lipophilic inhibitor of carbonic anhydrases, ethoxyzolamide,
effectively suppressed the carbonic anhydrase activity of PS2-membranes (I
50 = 10−9 M). Carbonic anhydrase activity of PS1-membranes was suppressed alike by both inhibitors (I
50 = 10−6 M). In the course of the electrophoresis of PS2-membranes treated with n-dodecyl-β-maltoside “high-molecular-mass” carbonic anhydrase activity was revealed in the region corresponding to core-complex
of this photosystem. Besides, carbonic anhydrase activity in the region of low-molecular-mass proteins was discovered in the
course of such an electrophoresis of both PS2-and PS1-membranes. These low-molecular-mass carbonic anhydrases eluted from
corresponding gels differed in sensitivity to specific carbonic anhydrase inhibitors just the same as PS1-membranes versus
PS2-membranes. The results are considered as evidence for the presence in the thylakoid membranes of three carriers of carbonic
anhydrase activity.
Published in Russian in Biokhimiya, 2006, Vol. 71, No. 5, pp. 651–659. 相似文献
7.
The case for chloroplast thylakoid carbonic anhydrase 总被引:8,自引:0,他引:8
Alan J. Stemler 《Physiologia plantarum》1997,99(2):348-353
Washed thylakoid membranes and photosystem II-enriched membrane fragments from cyanobacteria, green algae, and chloroplasts from both C3 and C4 plants possess the ability to reversibly hydrate CO2 . That is, the membranes have an intrinsic carbonic anhydrase activity. The present review outlines the discovery of thylakoid carbonic anhydrase and presents the evidence that it is a unique isozyme, distinct from other cellular carbonic anhydrases. It appears that at least some thylakoid carbonic anhydrase is closely associated with photosystem II and may be required for electron transport. This would explain why all inhibitors of carbonic anhydrase also inhibit photosystem II. Several speculative functions of thylakoid carbonic anhydrase are discussed. These include a possible role in carbon metabolism, in the protonation of plastoquinone, and/or in oxygen evolution. 相似文献
8.
Craig A. Atkins 《Phytochemistry》1974,13(1):93-98
Carbonic anhydrase activity (hydration of CO2 was found in homogenates of leaves (116–500 units.mg?1 protein) and root nodules (27–255 units.mg?1 protein) from 8 legume genera inoculated in each case with a host specific Rhizobium. No enzyme, or only trace amounts (2–7 units.mg?1 protein), were detected in root extracts, The enzymatic activity was inhibited in all cases by azide and acetazolamide. The sizes of nodule and leaf carbonic anhydrases, estimated by gel filtration of partially purified preparations from Phaseolus vulgaris, were around 45 000 and 205 000 respectively. These enzymes also differed in sensitivity to inhibitors. More than 99% of the activity present in Vicia faba nodules was recovered as a soluble enzyme and only a trace was located in the isolated bacteroids. 相似文献
9.
不同理化因子对雨生红球藻CG-11碳酸酐酶活性的影响 总被引:1,自引:0,他引:1
以雨生红球藻CG-11为实验藻株,探讨在不同CO2、HCO3-、Zn2+浓度以及pH和氮磷比例条件下,藻细胞的碳酸酐酶活性对这些理化因子的响应。结果表明,通入空气实验组的碳酸酐酶活性最高,为(75.20±1.53)U·mg-1(Chla),通入5%CO2条件下的碳酸酐酶活性为(9.96±1.43)U·mg-1(Chla);高浓度HCO3-对碳酸酐酶活性亦具有明显抑制作用,培养液中可溶性无机碳的浓度与碳酸酐酶活性呈负相关;在实验设置的pH范围内,pH9.0时碳酸酐酶活性最高,为(62.32±3.25)U·mg-1(Chla);适当的氮磷比与Zn2+浓度显著提高了雨生红球藻CG-11的生长速率,碳酸酐酶的活性亦有明显提高。 相似文献
10.
Rose L. Mikulski David N. Silverman 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(2):422-426
The undisputed role of His64 in proton transfer during catalysis by carbonic anhydrases in the α class has raised questions concerning the details of its mechanism. The highly conserved residues Tyr7, Asn62, and Asn67 in the active-site cavity function to fine tune the properties of proton transfer by human carbonic anhydrase II (HCA II). For example, hydrophobic residues at these positions favor an inward orientation of His64 and a low pKa for its imidazole side chain. It appears that the predominant manner in which this fine tuning is achieved in rate constants for proton transfer is through the difference in pKa between His64 and the zinc-bound solvent molecule. Other properties of the active-site cavity, such as inward and outward conformers of His64, appear associated with the change in ΔpKa; however, there is no strong evidence to date that the inward and outward orientations of His64 are in themselves requirements for facile proton transfer in carbonic anhydrase. 相似文献
11.
Mark V. Darlington H.J. Meyer George Graf Thomas P. Freeman 《Journal of insect physiology》1983,29(2):157-162
The general composition of the calcified puparium of Musca autumnalis was determined. Ash-weight analyses show that 62% of the puparium is inorganic material. The major components of the puparium are calcium, magnesium, phosphate and carbonate. Calcium and magnesium phosphate are the predominant salts in the puparium, with a lesser contribution by their respective carbonates. Scanning electron microscopy revealed that the exocuticle was the site of calcification in the puparium. Inorganic analyses of the insoluble salt isolated from face fly larvae showed it to be compositionally identical with the puparial salt. Major organic components of the puparium are lipid, chitin and protein. The presence of carbonic anhydrase and alkaline phosphatase activity in post-feeding larvae was confirmed. 相似文献
12.
The levels of, zinc, copper, Fe, Zn, Cu, Mn, Mg, K, Na, and Cl and the activity of carbonic anhydrase were determined in lambs
with pneumonia. A significant decrease of p<0.01 level in Zn concentration, in Cu level (p<0.001) and significant increases in K and Na levels (p<0.05) and of the Cu/Zn ratio (p<0.001) were observed in the study group. The carbonic anhydrase activity was decreased in the study group, but the decrease
was not statistically significant (p>0.05). Also, nonsignificant decreases of Fe, Mg, and Cl and increase of the Mn concentration were also observed in the lambs
with pneumonia (p>0.05). Our results suggest that the significant element changes reported here and the Cu/Zn ratio, but not the activity of
carbonic anhydrase, can be used as indicators of pneumococcal infection. 相似文献
13.
S. Kevrešan B. Krstić M. Popović L. Kovačev S. Pajević J. Kandrač đ. Malenčić 《Biologia Plantarum》1997,40(2):245-250
Nine sugar beet lines were grown in a glasshouse on chernozem soil watered to 35, 50 and 65 % of maximal soil water capacity.
After 12 d, plant water potential, net photosynthetic rate, contents of soluble proteins, DNA and RNA, proportion of ribulose-1,5-bisphosphate
carboxylase/oxygenase (RUBISCO) protein, and carbonic anhydrase activity were measured. As soil moisture decreased, the leaf
water potential and net photosynthetic rate decreased. DNA and RNA content and carbonic anhydrase activity decreased under
moderate drought, and increased with severe drought. RUBISCO protein proportion did not change significantly under decreased
soil moisture, while the total soluble protein content decreased.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
14.
15.
Claudia Melis Andrea Angeli Simona Distinto Giulia Bianco Clemente Capasso 《Journal of enzyme inhibition and medicinal chemistry》2017,32(1):68-73
The isatin scaffold is the constitutive fragment of several natural and synthetic bioactive molecules. Albeit several benzene sulphonamide-based carbonic anhydrase inhibitors (CAIs) have been reported, only recently isatin benzene sulphonamides have been studied and proposed as CAIs. In this study we have designed, synthesised, and evaluated the biological activity of a series of differently substituted isatin-based benzene sulphonamides which have been designed for the inhibition of carbonic anhydrase isoforms. The activity of all the synthesised compounds was evaluated towards human carbonic anhydrase I, II, IX, and XII isozymes. Our results indicate that the nature and position of substituents on the isatin ring can modulate both activity and isozyme selectivity. 相似文献
16.
Christine A. Raines Philip R. Horsnell Caroline Holder Julie C. Lloyd 《Plant molecular biology》1992,20(6):1143-1148
A full-length cDNA clone encoding carbonic anhydrase was isolated from an Arabidopsis thaliana (Columbia) leaf library. Comparison of the derived amino acid sequence obtained from this clone with those of pea and spinach reveals a considerable degree of identity. The carbonic anhydrase cDNA was used to probe the level of RNA encoding this protein in the leaves of plants grown in elevated CO2 (660 ppm). We have found that under these conditions the steady-state level of carbonic anhydrase mRNA was increased in comparison with control plants grown in normal atmospheric concentrations of CO2 (330 ppm). This raises the intruiging possibility that there exists in higher plants a mechanism for perceiving and responding to changes in environmental CO2 concentrations at the genetic level. 相似文献
17.
The activity and cellular localization of carbonic anhydrase (CA) in two alkaliphilic anaerobes growing in soda lakes at pH 9–10 were studied. CA activity in the cell extracts of the acetogenic bacterium Natroniella acetigena was comparable to that of neutrophilic acetogens. Hydrogenotrophically grown cells of Desulfonatronum lacustre exhibited higher CA activity compared to the cells grown on medium with formate. High CA activity in the cytoplasmic fraction and the absence of high activity in the extracellular fraction were demonstrated. We propose that the cytoplasmic CA in alkaliphilic sulfate-reducers participates in conversion of bicarbonate to CO2, which is reduced in the cell to acetate via the acetyl-CoA pathway. 相似文献
18.
van Hunnik E Livne A Pogenberg V Spijkerman E van den Ende H Mendoza EG Sültemeyer D de Leeuw JW 《Planta》2001,212(3):454-459
In order to broaden our understanding of the eukaryotic CO2-concentrating mechanism the occurrence and localization of a thylakoid-associated carbonic anhydrase (EC 4.2.1.1) were studied
in the green algae Tetraedron minimum and Chlamydomonas noctigama. Both algae induce a CO2-concentrating mechanism when grown under limiting CO2 conditions. Using mass-spectrometric measurements of 18O exchange from doubly labelled CO2, the presence of a thylakoid-associated carbonic anhydrase was confirmed for both species. From purified thylakoid membranes,
photosystem I (PSI), photosystem II (PSII) and the light-harvesting complex of the photosynthetic apparatus were isolated
by mild detergent gel. The protein fractions were identified by 77 K fluorescence spectroscopy and immunological studies.
A polypeptide was found to immunoreact with an antibody raised against thylakoid carbonic anhydrase (CAH3) from Chlamydomonas reinhardtii. It was found that this polypeptide was mainly associated with PSII, although a certain proportion was also connected to
light harvesting complex II. This was confirmed by activity measurements of carbonic anhydrase in isolated bands extracted
from the mild detergent gel. The thylakoid carbonic anhydrase isolated from T. minimum had an isoelectric point between 5.4 and 4.8. Together the results are consistent with the hypothesis that thylakoid carbonic
anhydrase resides within the lumen where it is associated with the PSII complex.
Received: 13 May 2000 / Accepted: 16 August 2000 相似文献
19.
20.
A cDNA encoding the mature, chloroplast-localized carbonic anhydrase in pea has been expressed inE. coli. The enzyme is fully active and yields of up to 20% of the total soluble protein can be obtained from the bacteria. This expression system was used to monitor the effects of site-directed mutagenesis of seven residues found within conserved regions in the pea carbonic anhydrase amino acid sequence. The effects of these modifications are discussed with respect to the potential of various amino acids to act as sites for zinc coordination or intramolecular proton shuttles. 相似文献