• Korean Journal of Clinical Laboratory Science
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• v.43 no.2
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• pp.33-42
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• 2011

Zinc plays a key role in genetic expression, cell division, and cell growth and is essential for the functions of more than 450 metalloenzyme. There are high concentrations of zinc in pigment epithelium in bullfrog eye. Zinc deficiency causes night blindness and abnormal dark adaptation. The purpose of this study was to identify ERG (electroretinogram) b-wave sensitivity during light and dark adaptation in bullfrog retina after zinc and zinc chelators treatment such as histidine and TSQ (N-(6-methoxy-8-qunolyl)-p-toluenesulfon amide). Especially, we focused whether histidine act as a zinc chelator in the Muller cell. The results of our study are summarized as follows: 1) Both zinc and histidine elevated ERG b-wave amplitude and threshold in Muller cells by accelerating rhodopsin regeneration time and increased a-peak absorbance during light adaptation. 2) TSQ reduced those by prolonging rhodopsin regeneration time and decrement of a-peak absorbance during light adaptation. 3) Zinc shortened rhodopsin regeneration time and prolonged a-peak absorbance. These results suggested that histidine may act as a zinc-mediated transporter in presynaptic Muller cell membrane rather than zinc chelator and acts as a GABA-receptor inhibitor which blocks $Cl^-$ influx to the postsynapse.

• Molecules and Cells
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• v.42 no.6
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• pp.460-469
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• 2019

Bacterial ${\alpha}-type$ carbonic anhydrase (${\alpha}-CA$) is a zinc metalloenzyme that catalyzes the reversible and extremely rapid interconversion of carbon dioxide to bicarbonate. In this study, we report the first crystal structure of a hyperthermostable ${\alpha}-CA$ from Persephonella marina EX-H1 (pmCA) in the absence and presence of competitive inhibitor, acetazolamide. The structure reveals a compactly folded pmCA homodimer in which each monomer consists of a 10-stranded ${\beta}-sheet$ in the center. The catalytic zinc ion is coordinated by three highly conserved histidine residues with an exchangeable fourth ligand (a water molecule, a bicarbonate anion, or the sulfonamide group of acetazolamide). Together with an intramolecular disulfide bond, extensive interfacial networks of hydrogen bonds, ionic and hydrophobic interactions stabilize the dimeric structure and are likely responsible for the high thermal stability. We also identified novel binding sites for calcium ions at the crystallographic interface, which serve as molecular glue linking negatively charged and otherwise repulsive surfaces. Furthermore, this large negatively charged patch appears to further increase the thermostability at alkaline pH range via favorable charge-charge interactions between pmCA and solvent molecules. These findings may assist development of novel ${\alpha}-CAs$ with improved thermal and/or alkaline stability for applications such as $CO_2$ capture and sequestration.

• Journal of Life Science
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• v.9 no.2
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• pp.44-48
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• 1999

The ionization of tyrosine residue is known to be involved in the stabilization of transition-state in catalysis of astacin based upon the astacin-transition state analog structure. Two tyrosine residues, Tyr-216 and Tyr-219, are conserved in all MMPs related with astacin family, We replaced Tyr-216 and Tyr-219 into phenylalanine, respectively and the zinc binding properties, kinetic parameters, and pH dependence of each mutant are determined in order to examine the role of tyrosine residue in matrilysin catalysis. Both mutants contain two zinc atoms per mol of enzyme, indicating that either tyrosime does not affect the zinc binding property of the enzyme. Y216F and Y219F mutants are highly active and the kcat/Km values are only decreased 1.1-1.5-fold compared to the wild-type enzyme. The decrease in the activity of the mutants is essentially due to the increase in Km value. The pH dependencies of the kcat/Km values for both mutants are similar to the corresponding dependencies obtained with the wild type enzyme. The pKa values at the alkaline side of both mutants are not changed. These kinetic and pH dependence results indicate that the ionization of active site tyrosine residue of matrilysin is not reflected in the kinetics of peptide hydrolysin as catalyzed by astacin.

• Journal of Nutrition and Health
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• v.5 no.2
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• pp.91-103
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• 1972

Zinc is one of the essential trace elements in the living organism for growth and health. The first identified metalloenzyme, carbonic anhydrase, is a zinc compound and several others have been described since. Among zinc deficiency syndromes in animals porcine parakeratosis has been successfully treated with zinc supplements, and in man a syndrome of anemia, hypogonadism, hepatosplenomegaly, and dwarfism, prevalent in parts of Iran and Egypt, has been ascribed to lack of zinc in the diet. Dietary zinc excess in the rat is manifested by a hypochromic, microcytic anemia, poor growth, reduction in liver catalase and cytochrome oxidase. The present study is an attempt to delineate the changes of tissue contents of trace elements, especially of iron, copper and zinc in liver and kidneys of the rats. Weanling albino rats, weighing 60 to 80gm. were used in this experiments. The rats were housed in cages with aluminum floors and received feed and distilled water ad libitum. Animals were divided into three groups, control, low zinc diet and high zinc diet groups. The high zinc diet group was subdivided into 0.5% Zn and 0.7%Zn groups. The supplementary copper or iron was added to the high dietary zinc groups. The animals were sacrificed and the tissues were washed several times with deionized water. The wet digested samples were analyzed by Hitachi Model 207 atomic absorption spectro-photometer for the determination of iron, copper and zinc in the liver and kidney. Hemoglobin level in the blood was measured by cyanmethemoglobin method. The results of this study are as follows: 1) All rats fed high zinc diets and low zinc diets gained less weight than control. Weight gain was not improved by the supplementary copper or iron and both. 2) Hemoglobin concentration was decreased significantly in the rats fed high zinc diets and less in the low zinc diet. Supplementary copper and iron to the higher zinc diet appeared to give some improvement of anemia. 3) The iron contents of the liver and kidneys were significantly decreased in the high zinc groups and the reduction was more significantly in the rats receiving higher zinc diet (0.7%). The supplementary copper caused a further depression of liver iron. On the other hand, the iron, added to the high zinc diet lessoned the severity of the decrease in liver iron and caused kidney iron to be maintained almost at the level found in the rats fed by zinc and supplementary copper diet. 4) High zinc diets did not change copper content of the liver and kidney. Supplementary copper elevated the concentration in the liver and kidney and added iron had no effect on the accumulation of copper in the liver and kidneys. 5) The high zinc diets caused marked increases of zinc content in the liver and kidney. Supplementary iron to the high zinc diet caused increases of zinc contents of liver and kidneys.

• Molecules and Cells
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• v.43 no.9
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• pp.831-840
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• 2020

The β-class of carbonic anhydrases (β-CAs) are zinc metalloenzymes widely distributed in the fungal kingdom that play essential roles in growth, survival, differentiation, and virulence by catalyzing the reversible interconversion of carbon dioxide (CO₂) and bicarbonate (HCO₃^-). Herein, we report the biochemical and crystallographic characterization of the β-CA CafA from the fungal pathogen Aspergillus fumigatus, the main causative agent of invasive aspergillosis. CafA exhibited apparent in vitro CO₂ hydration activity in neutral to weak alkaline conditions, but little activity at acidic pH. The high-resolution crystal structure of CafA revealed a tetramer comprising a dimer of dimers, in which the catalytic zinc ion is tetrahedrally coordinated by three conserved residues (C119, H175, C178) and an acetate anion presumably acquired from the crystallization solution, indicating a freely accessible "open" conformation. Furthermore, knowledge of the structure of CafA in complex with the potent inhibitor acetazolamide, together with its functional intolerance of nitrate (NO₃^-) ions, could be exploited to develop new antifungal agents for the treatment of invasive aspergillosis.

• Journal of Life Science
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• v.23 no.12
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• pp.1557-1561
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• 2013

Carbonic anhydrase isozymes are a widespread, zinc-containing metalloenzyme family. The enzyme catalyzes the reversible inter-conversion of $CO_2$ and $HCO_3$. This reaction is the main role played by CA enzymes in physiological conditions. This enzyme has been found in virtually all organisms, and at least 16 isozymes have been isolated in mammals. Unlike mammals, there is little information available regarding CA isozymes in the tissues of non-mammalian groups, such as fish. Carbonic anhydrase is very important in the osmotic and acid-base regulation in fish. It is well-known that the gills of fish play the most important role in acid-base relevant ion transfer, the transfer of $H^+$ and/or $HCO_3^-$, for the maintenance of systemic pH. Rainbow trout, Oncorhynchus mykiss, is the most important freshwater fish species in the aquaculture industry of Korea, with annual production increasing each year. In addition, environmental toxicology research has shown that rainbow trout is known to be the species that is most susceptible to environmental toxins. Consequently, carbonic anhydrase was detected in rainbow trout, Oncorhynchus mykiss. The isolated protein showed the specific band with a molecular weight of 30 kDa and pI of 7.0, and it was identified as being carbonic anhydrase. The immunohistochemical result demonstrated that the carbonic anhydrase was located in the epithelial cells of the gills.

• Journal of Life Science
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• v.10 no.2
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• pp.210-217
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• 2000

A kinetic profile of the catalytic domain of stromelysin-1 (SCD) using the fluorescent peptide substrate has been determined by the stopped-flow technique. The pH profile has a pH optimum of about 5.5 with an extended shoulder above pH 7. Three pKa values, 5.0, 5.7, and 9.8 are found for the free enzyme state and two pH independent Kcat/Km values of 4.1$\times$104 M-1 s-1 and 1.4$\times$104 M-1 s-1 at low and high pH, respectively. The profile is quite different in shape with other MMP family which has been reported, having broad pH optimum with two pKa values. The substrate specificity of SCD towards fluorescent heptapeptide substrates has been also examined by thin layer chromatography. The cleavage sites of the substrates have been identified using reverse-phase HPLC method.SCD cleaves Dns-PLA↓L↓WAR and Dns-PLA↓L↓FAR at two positions. However, the Dns-PLA↓LRAR, Dns-PLE↓LFAR, adn Dns-PLSar↓LFAR are cleaved exclusively at one bond. The double cleavages of Dns-PLALWAR and Dns-PLALFAR by SCD are in marked contrast to the close structurally related matrilysin. A notable feature of SCD catalysis agrees with the structural data that the S1' pocket of SCD is deeper than that of matriysin. The differences observed between SCD and matrilysin may form the basis of understanding the structural relationships and substrate specificities of the MMP family in vivo.

• Journal of Life Science
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• v.10 no.2
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• pp.218-227
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• 2000

Bone morphogenetic protein 1 (BMP-1) is part of a complex capable of inducing ectopic bone formation in mammals. Studies on TGF-β1 processing and Drosophila dorsal-ventral patterning have focused attention on BMP-1 as important in mediating the biological activity of this bone inducing complex. Herein, the bacterial expression, refolding, purification, and initial characterization of the BMP-1 proteolytic domain (BPD) are described. A semi-quantitative fluorescence-based thin layer chromatography assay was developed to assist in rapidly screening for optimal renaturation conditions. According to a preliminary screen for optimal conditions for the refolding of BPD , a detectable proteolytic activity against a high turnover substrate for astacin, a homologous protease from crayfish was observed. The conditions identified have allowed the expression of sufficient amounts of BPD for the characterization of the protein. Its proteolytic activity exhibits the same cleavage specificity as astacin against seven substrates that were previously synthesized for studying astacin. Furthermore, this activity is inhibited by the metal chelator 1,10-phenanthroline but not by its analogue 1,7-phenanthroline. The collagenase inhibitor Pro-Leu-Gly hydroxamate was found to inhibit both astacin and BPD activity. The results presented in this paper argue that BMP-1 does in fact possess an intrinsic proteolytic activity.

• Journal of Life Science
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• v.24 no.2
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• pp.186-190
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• 2014

Carbonic anhydrase isozymes (CAs) are widespread zinc-containing metalloenzyme family. The enzyme catalyzes the reversible interconversion of $CO_2$ and $HCO_3$. This reaction is the main role of CA enzymes in physiological conditions. CA III, one of the CA isozymes, has been identified in many tissues. It is distinguished from the other isozymes by several characteristics, particularly by a lower specific activity and by its resistance to acetazolamide. However, the physiological function of CA III in fish is unknown. In this study, we examined the detection of CAs in the Shaggy sea raven Hemitripterus villosus, using SDS-PAGE, isoelectric focusing (IEF), and western blot analysis. We detected a significant protein band with molecular weight about 30 kDa from the tissues of H. villosus by SDS-PAGE and western blotting. A specific band of CA III with pI 7.0 was detected by IEF and western blotting in gill and muscle. The immunoreaction of anti-CA III expressed in the gill of H. villosus was much stronger than other tissues. One explanation for this result is that the fish gill is the only organ that is exposed to the external environment and that plays an important role in acid-base relevant ion transfer, the transfer of $H^+$ and/or $HCO{_3}^-$, for the maintenance of systemic pH. This is the first report on the identification of a carbonic anhydrase III-like protein from H. villosus.