• Bulletin of the Korean Chemical Society
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• v.3 no.3
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• pp.122-129
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• 1982

In order to extend our understanding on the multiple inhibition enzyme kinetics, a general equation of an enzyme reaction in the presence of two different reversible inhibitors was derived by what we call "match-box mechanism" under the combined assumption of steady-state and quasi-equilibrium for inhibitor binding. Graphical methods were proposed to analyze the multiple inhibition of an enzyme by any given sets of different inhibitors, i.e., competitive, noncompetitive, and uncompetitive inhibitors. This method not only gives an interaction factor $({\alpha})$ between two inhibitors, but also discerns ${\alpha}_1$ and ${\alpha}_2$ with and without substrate binding, respectively. The factors involved in the dissociation constants of inhibitors can also be evaluated by the present plot. It is also shown that the present kinetic approach can be extended to other forms of activators or hydrogen ions with some modification.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.04a
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• pp.261-261
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• 1994

Bovine milk xanthine oxidase (E.C.1.1.3.22, XO) purchased from Sigma Chemical Co. had the three protein fragments below 150 kDa on 7.5％ SDS-PAGE, which did not show enzyme activity. To remove these fragments, the enzyme preparation was further purified through Sephadex G-200 column chromatography. Two peaks exhibiting enzymatic activity were separated very closely to the void volume, which were revealed as two different enzyme forms, dimeric and monomeric, confirmed by activity staining on native PAGE. Anti sera-against each of the two enzyme forms were raised by subcutaneous injection at multiple sites on the back of rabbits during 4 weeks. On the immunodiffusion test, it was found that both of the antisera of the two forms could react with each other, which implied that their epitopes were identical In the Western blot analysis of mouse liver cytosol fraction, it was found that rabbit anti-XO antibody bound well with the protein band of monomeric mouse liver XO of about 150kDa. Based on this result, mouse liver cDNA 1 ibrary was screened by in situ hybridizat ion wi th rabbi t anti -XO antibody as probe. Through the immunological screening, recombinant phages giving positive signal by the production of XO were selected and further purified. To validate these clones, purified phages were lysogenized in E. coli Y1089 and their lysates were analysed for enzyme activity and immunoreactivity, It was verified that lysates of the purified recombinant phage lysogens exhibited the enzymatic activity as well as bound wi th XO antibody, when induced by IPTG. The above results assert that selected recombinant phage carries mouse liver XO gene.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.5 no.1
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• pp.18-22
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• 2005

Pompe disease is a genetic disorder caused by a deficiency of acid ${\alpha}$-glucosidase (GAA). This enzyme defect results in lysosomal glycogen accumulation in multiple tissues and cell types, with cardiac, skeletal, and smooth muscle cells the most seriously affected. Infantile-onset Pompe disease is uniformly lethal. Affected infants present in the first few months of life with hypotonia, generalized muscle weakness, and a hypertrophic cardiomyopathy, followed by death from cardiorespiratory failure or respiratory infection, usually by 1 year of age. Late-onset forms is characterized by a lack of severe cardiac involvement and a less severe short-term prognosis. Enzyme replacement therapy for Pompe disease is intended to address directly the underlying metabolic defect via intravenous infusions of recombinant human GAA to provide the missing enzyme. We experienced one case of Pompe disease in 3-years old boy that has improved his exercise ability and cardiac function after GAA enzyme replacement therapy.

• Journal of Plant Biology
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• v.13 no.2
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• pp.11-14
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• 1970

Identification and observations of leucine aminopeptidase (LAP) and multiple molecular forms of the enzyme, isozymes, were made with a technique of starch-gel electrophoresis for various legume pollen. Plants tested other than Leguminosae demonstrated either no indication of the presence or at least tract of enzymes and the isozymes, although all legume pollen tested showed strong LAP patterns. The electrophoretic patterns of LAP failed to be shown if the extracts were heated or otherwise denatured. Extent of zymogrammatic appearance of LAP and the isozymes were characteristic of a species.

• BMB Reports
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• v.38 no.6
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• pp.633-638
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• 2005

Biosynthesis of prostanoids is regulated by three sequential enzymatic steps, namely phospholipase $A_2$ enzymes, cyclooxygenase (COX) enzymes, and various lineage-specific terminal prostanoid synthases. Prostaglandin E synthase (PGES), which isomerizes COX-derived $PGH_2$ specifically to $PGE_2$, occurs in multiple forms with distinct enzymatic properties, expressions, localizations and functions. Two of them are membrane-bound enzymes and have been designated as mPGES-1 and mPGES-2. mPGES-1 is a perinuclear protein that is markedly induced by proinflammatory stimuli, is down-regulated by anti inflammatory glucocorticoids, and is functionally coupled with COX-2 in marked preference to COX-1. Recent gene targeting studies of mPGES-1 have revealed that this enzyme represents a novel target for anti-inflammatory and anti-cancer drugs. mPGES-2 is synthesized as a Golgi membrane-associated protein, and the proteolytic removal of the N-terminal hydrophobic domain leads to the formation of a mature cytosolic enzyme. This enzyme is rather constitutively expressed in various cells and tissues and is functionally coupled with both COX-1 and COX-2. Cytosolic PGES (cPGES) is constitutively expressed in a wide variety of cells and is functionally linked to COX-1 to promote immediate $PGE_2$ production. This review highlights the latest understanding of the expression, regulation and functions of these three PGES enzymes.

• YAKHAK HOEJI
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• v.41 no.2
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• pp.212-219
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• 1997

Multiple forms of extracellular phospholipase $A_2$ have been detected in human amniotic fluid (HAF). When HAF was subjected to heparin-Sepharose column chromatography, phospholipase $A_2$ activity was detected in both heparin-non binding and binding fraction. The activity of heparin-non binding fraction was further purified by sequential uses of column chromatographies on butyl-Toy-opearl 650M and DEAE-Sephacel. DEAE-Sephacel fraction contained three different phospholipase $A_2$ activities (Peak I, II, III). The molecular weight of DEAE-Sephacel fraction phospholipase $A_2$ determined by SDS-PAGE were about 52KDa (Peak I). Peak II, III required micromolar $Ca^{2+}$ ion for its maximum activity, but Peak I enzyme showed calcium independent phospholipase $A_2$ activity and showed broad range of pH (6.0~10.0) optimum. All these enzymes were not recognized by a monoclonal antibody raised against phospholipase $A_2$ from human synovial fluid. These results suggest that HAF might contain multiple forms of extracellular phospholipase $A_2$, which may neither belong to the 14KDa group II phospholipase $A_2$ family nor cytosolic phospholipase $A_2$.

• The Korean Journal of Pharmacology
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• v.16 no.2 s.27
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• pp.15-24
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• 1980

[${\alpha}$]-Amylase catalyses the hydrolysis of starch, glycogen, and related poly- and oligosac-charide by random cleavage of ${\alpha}$-D-(l-4) glucan linkage. In man large amounts of amylase are secreted into the digestive tract by the salivary and exocrine pancreatic gland, minimal amount being produced also in other tissues. It has been known that ${\alpha}$-amylase exists in multiple molecular forms, isoenzyme which can be separated from each other because of difference in their physicochemical properties. By using various methods, several groups of investigator have separated the many isoenzyme in serum, saliva and pancreatic juice. Furthermore, changes of the normal serum isoenzyme pattern is diagnostically useful even when the total serum enzyme activity is noninformative, such as the clinical use of isoenzyme of serum lactate dehydrogenase. Procarboxypeptidase-A which is one of the pancreatic enzymes is also present as isoenzymes. Four forms of procarboxypeptidase-A haye been found in the bovine enzyme and three forms of the porcine enzyme. In human pancreatic juice four forms of procarboxypeptidase-A isoenzyme were found by isoelectric focusing method. Recently, the so-called isoamylase analysis was developed for the diagnostic use of amylase in pancreatic diseases. In alcohotic patients, the serum concentration of pancreatic isoamylase is subnormal and this lowered activity provides strong evidence for pancreatic exocrine insufficiency. The purpose of this study was to elucidate the variations of the isoenzyme of amylase and procarboxypeptidase-A in serum, saliva and pancreatic juice of the experimental animals. The results are as follow. 1) Three main forms of isoenzyme of amylase by isoelectric focusing were found in pancreatic juice of normal rabbit. However, many new bands were appeared in the pancreatic juice of cholic acid administered animal intravenously while the infusion of cholic acid or elastase into pancreatic duct produced the decrease of number of the fractions on the isoelectric focusing. In the case of serum isoenzyme from normal animal, two major and a few minor isoamylases were observed. By injecting alcohol intravenousely the fractions of serum isoamylase were significantly decreased and in contrary to the pattern in the pancreatic juice the infusion of cholic acid or elastase into pancreatic duct exhitited a significant decrease of the isoenzyme of amylase fractions. In saliva from normal animal three main isoamylase were produced of the administration of alcohol. 2) In the case of procarboxypeptidase-A isoenzyme, two major fractions which have isoelectric point at 6.2 and 6.4 and other two minor bands were observed in the pancreatic juice of normal rabbit. By the treatment of the juice with trypsin, only one band was produced on the isoelectric focusing. No procarboxypeptidase was appeared on the electrofocusing by the infusion of cholic acid or phospholipase A into the pancreatic duct of rabbit. However, a single major fraction of procarboxypeptidase-A was appeared at 3 hr after simple ligation of the pancreatic duct. No significant changes were observed in the juice of the alcohol or cholic acid administered group.

• Korean Journal of Food Science and Technology
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• v.27 no.5
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• pp.673-679
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• 1995

Pectinesterase(PE) has been definitively established as the causative agent for clarification of citrus juice and gelation of frozen concentrates. This enzyme is present in multiple forms in citrus fruit. Although representing a minor fraction of the total PE activity, thermostable pectinesterase(TSPE) accounts for the severe time/temperature processing treatment required to inactivate PE. This study was undertaken to separate and purify thermolabile pectinesterase(TLPE) and TSPE from the crude PE extracts of Valencia orange rag-pulp powder. The approach taken was to carry out the three kind of chromatographies of CM-Sephadex C-50, Phenyl Sepharose CL-4B and CM-Biogel A to the unheated crude PE or the heated crude PE. All of them used could increase the purity of PEs and, specially, Phenyl Sepharose CL-4B chromatography could separate crude PE as the mixture of PEs into two forms of TLPE and TSPE. The purified TLPE had specific activity of 1,005 units/mg, yield of 13.6% and purification of 35 fold, while the TSPE separated from the unheated crude PE showed specific activity of 3,115 units/mg, yield of 1.5% and purification of 100.5 fold, and another TSPE from the heated crude PE was found to be specific activity of 1,803 units/mg, yield of 15.4% and purification of 140 fold.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.832-838
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• 1999

Two different types of lipases (lipase I and lipase II) secreted into culture medium by Rhizopus sp. L-I were purified using a hydrophobic chromatography and were partially characterized. Both enzymes were monomeric as revealed by SDS-PAGE and gel filtration. The molecular masses of the enzymes were identified as 45 kDa (lipase I) and 69 kDa (lipase II). The isoelectric points were estimated to be 3.6 and 5.2 for lipase I and lipase II, respectively. pH and temperature activity optima for lipase I were as 7.5 and $50^{\circ}C$, respectively, whereas the corresponding parameters for lipase II were 6.0 and $45^{\circ}C$. The amino terminal sequences of lipase I and lipase II, determined by Edman degradation, were found to be Leu-Val-Met-Ile-Gln-Arg and Leu-Val-Met-Lys-Gln-Arg, respectively. By western blotting analysis, the two lipases were found to have a common antigenic determinant. Immuno-electron cytochemistry conducted with polyclonal anti-lipase I antibody indicated the enzyme located in both the periplasm and the adjacent vesicles of fungal hyphae. Fortunately, the sites on the cell envelope where lipase was exported into the culture medium was also identified.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.71-71
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• 1993

Carboxylesterase is widely distributed in the tissues of vertebrates, insects, plants and mycobacteria. Among various tissues of animals and humans, the highest esterase activity with various substrates is found in the liver. Kidney has moderate carboxylesterase activity in the proximal tubules. Considerable esterase activity is also found in the small intestine epithet elial cells and serum of mammals. Besides these tissues, carboxylesterase has been found in the lung, testis, adipose tissue, nasal mucosa and even in the central nervous system. Hepatic microsomal carboxylesterase catalyzes the hydrolysis of a wide variety of endogenous and exogenous compounds such as carboxylester, thioester and aromatic amide. Since carboxylesterases are important for metabolic activation of prodrugs and detoxification of xenobiotics, differences in substrate specificity and immunological properties of this enzyme are important in connection with choosing a suitable laboratory animal for the evaluation of biotransformation and toxicity of drugs. On the other hand, liver, kidney, intestine and serum were found to contain multiple forms of carboxylesterases in animal species and humans. In fact, we have purified more than fifteen isoforms of carboxylesterases from microsomes of liver, kidney and intestinal mucosa of nine animal species and humans. and characteristics of these isoforms were compared each other in terms of their physical and immunochemical properties. On the other hand, we have reported that hepatic microsomal carboxylesterases are induced by many exogenous compounds such as phenobarbital, polycyclic aromatic hydrocarbons, Aroclor 1254, aminopyrine and clofibrate. Later, we showed that some isoforms of hepatic carboxylesterase were induced by glucocorticoids such as dexamethasone and 16 ${\alpha}$-carbonitrile, but other isoforms were rather inhibited by these compounds. These findings indicate that involvement of carboxylesterases in the metabolism and toxicity of drugs should be explained by the isoforms involved. Since 1991, we have carried out detailed research investigating the types of carboxylesterases involved in the metabolic activation of CPT-11, a derivative of camptothecin, to the active metabolite, SN-38. The results obtained strongly suggest that some isoforms of carboxylesterase of liver microsomes and intestinal mucosal membrane are exclusively involved in CPT-11 metabolism. In this symposium, the properties of carboxylesterase isoforms purified from liver, kidney and intestine of animal species and humans are outlined. In addition, metabolism of CPT-11, a novel antitumor agent, by carboxylesterases in relation to the effectiveness will also be discussed.