• 미생물학회지
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• 제29권4호
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• pp.230-237
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• 1991

Citrate synthase 1 (mitochondrial) and citrate synthase 2 (nonmitochondrial) were purified from Saccharomyces cerevisiae. The physical and enzymatic characteristics of citrate synthase 2 were ananlyzed in comparison with citrate synthase 1. Both isoenzymes were shown to be dimeric proteins of identical subunits, and the molecular weights of the subunits were estimated to be 48.3kDa for citrate synthase 1 and 47.0kDa for citrate synthase 2, respectively. The optimal pH value for enzyme activity was pH 7.5 for both isoenzymes. However, the optimal temperature for the activity was strikingly different; while the activity of citrate synthase 1 reached its peak at 65.deg.C, that of citrate synthase 2 was maximal at 40.deg.C. Citrate synthase 2 showed much lower thermal and pH stability than citrate synthase 1. In addition, citrate synthase 2 was affected much more by the metal ions such as $Zn^{2+}$ , $Mn^{2+ , and $Co^{2+} than citrate synthase 1. Among the several possible regulatory metabolites tested, ATP showed the strongest inhibitory effect on both enzymes. ADP and NADH were found to have greater effect on citrate synthase 2 than on citrate synthase 1. Kinetic analysis revealed that citrate synthase 2 has approximately 7- and 3.5-fold lower affinity to acetyl CoA and to oxaloacetate, respectively, than citrate synthase 1.

• BMB Reports
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• 제42권12호
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• pp.812-816
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• 2009

To screen chaperone proteins from Schizosaccharomyce pombe (S. pombe), we prepared recombinant citrate synthase of the fission yeast as a substrate of anti-aggregation assay. Purified recombinant citrate synthase showed citrate synthase activity and was suitable for the substrate of chaperone assay. Several heat stable proteins including aspartyl aminopeptidase (AAP) for candidates of chaperone were screened from the supernatant fraction of heat-treated crude extract of S. pombe. The purified AAP migrated as a single band of 47 kDa on SDS-polyacrylamide gel electrophoresis. The native size of AAP was estimated as 200 kDa by a HPLC gel permeation chromatography. This enzyme can remove the aspartyl residue at N-terminus of angiotensin I. In addition, AAP showed the heat stability and protected the aggregation of citrate synthase caused by thermal denaturation. This study showed that S. pombe AAP is a moonlight protein that has aspartyl aminopeptidase and chaperone activities.

• 미생물과산업
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• 제12권2호
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• pp.30-35
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• 1986

Almost all of the aerobic organisms contain citric acid cycle (or, tricarboxylic acid cycle). This cycle is involved both in energy metabolism and biosynthetic reactions; generation of NADH which derives the synthesis of chemical energy, ATP, and provision of intermediates needed for the biosynthesis. Because of its importance in the cellular metabolism, the regulation of the TCA cycle and its component enzymes has been extensively studied by many biologists (7,28). Citrate synthase is resposible for the initial step of the cycle and has been recognized as the rate limiting step (14,121,26). Understanding of the mechanism of the expression of citrate synthase should be a key step for the elucidation of the regulation of the TCA cycle in the cell metabolism.

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.425-431
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• 1996

The enzymatic characteristics of Alcaligenes latus were investigated by measuring the variations of various enzyme activities related to biosynthesis and degradation of poly-${\beta}$-hydroxybutyrate (PHB) during cultivation. All PHB biosynthetic enzymes, ${\beta}$-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase, were activated gradually at the PHB accumulation stage, and the PHB synthase showed the highest value among three enzymes. This indicates that the rate of PHB biosynthesis is mainly controlled by either ${\beta}$-ketothiolase or acetoacetyl-CoA reductase rather than PHB synthase. The enzymatic activities related to the degradation of PHB were also measured, and the degradation of PHB was controlled by the activity of PHB depolymerase. The effect of supplements of metabolic regulators, citrate and tyrosine, was also investigated, and the activity of glucose-6-phosphate dehydrogenase was increased by metabolic regulators, especially by tyrosine. The activities of ${\beta}$-ketothiolase and acetoacetyl-CoA reductase were also activated by citrate and tyrosine, while the activity of PHB depolymerase was depressed. The increased rate and yield of PHB biosynthesis by metabolic regulators may be due to the increment of acetyl-CoA concentration either by the repression of the TCA cycle by citrate through product inhibition or by the activation of sucrose metabolism by the supplemented tyrosine.

• Mycobiology
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• 제43권3호
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• pp.272-279
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• 2015

To screen molecular chaperones similar to small heat shock proteins (sHsps), but without ${\alpha}$-crystalline domain, heat-stable proteins from Schizosaccharomyces pombe were analyzed by 2-dimensional electrophoresis and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Sixteen proteins were identified, and four recombinant proteins, including cofilin, NTF2, pyridoxin biosynthesis protein (Snz1) and Wos2 that has an ${\alpha}$-crystalline domain, were purified. Among these proteins, only Snz1 showed the anti-aggregation activity against thermal denaturation of citrate synthase. However, pre-heating of NTF2 and Wos2 at $70^{\circ}C$ for 30 min, efficiently prevented thermal aggregation of citrate synthase. These results indicate that Snz1 and NTF2 possess molecular chaperone activity similar to sHsps, even though there is no ${\alpha}$-crystalline domain in their sequences.

• 미생물학회지
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• 제25권3호
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• pp.189-197
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• 1987

배지성분으로 첨가된 glutamate의 농도가 균의성장고 tylosin 생합성에 미치는 영향을 조사하였다. 그 결과 oxaloacetate를 공동기질로 사용하는 효소의 활성에 의하여 균의 성장과 tylosin의 생합성이 조절됨을 알았다. 즉, citrate synthase와 aspartate aminotransferase의 활성은 균의 성장에 아주 긴요하며 methylmalonyl-CoA carboxytransferase의 활성은 tylosin 생합성에 아주 중요한 효소임을 알았다. Glutamate의 농도는 우의 효소의 활성에 직접적으로 영향을 주고 있음을 알았다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제11권6호
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• pp.516-521
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• 2006

The use of gltA gene, as a new biomarker for environmental stress biomonitoring, was investigated because of its key position as the first enzyme of the tricarboxylic acid (TCA) cycle. A recombinant bioluminescent Escherichia coli strain, EBJM2, was constructed using a plasmid carrying the citrate synthase (gltA) promoter transcribing the Photorhabdus luminescens IuxCDABE genes (gltA::luxCDABE). The responses from this strain were studied with five different classes of toxicants: DNA damage chemicals, phenolics, oxidative-stress chemicals, PAHs, and organic solvents. EBJM2 responded strongly to DNA damage chemicals, such as mitomycin C (MMC) and methyl-nitro-nitrosoguanidine (MNNG) and nalidixic acid with the strongest responses. In contrast, tests with several compounds from the other four classes of toxicants gave no significant response. Therefore, EBJM2 was found to be sensitive to DNA damage chemicals.

• 한국미생물·생명공학회지
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• 제13권3호
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• pp.179-184
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• 1985

성적 화합성 heterothallic 2부체와 1부체인 Saccharomycopsis lipolytica에서 시트르산 생산성을 비교하였다. 교잡과 세포융합에 의한 2부체는 random spore 분석과 단상화에 의하여 확인되었다. ATCC 44601과 IFO 1209 균주는 IFO 1550와 IFO 1551 균주보다 시트르산과 이소시트르산을 많이 생산하였다. 2부체에 의한 시트르산 생산양식은 양친 1부체 균주의 중간정도이였다. IFO 1550와 IFO 1551 균주의 citrate synthetase와 isocitrate lyase의 비활성은 ATCC 44601과 IFO 1209 균주에서 보다 높았고 이들 효소의 비활성과 시트르산 생산간에는 상관관계가 없었다.

• BMB Reports
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• 제33권5호
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• pp.407-411
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• 2000

The effects of heat shock, or all-trans retinoic acid, on the expression of the C-reactive protein mRNA in the HT-29 human colon carcinoma cells, as well as the functional role of the C-reactive protein as a molecular chaperone, were studied. The expression level of the C-reactive protein mRNA in the HT-29 cells was increased time-dependently when exposed to heat-shock, and dose-dependently when treated with all-trans retinoic acid. The activities of transglutaminase C and K in the HT-29 cells were significantly increased when treated with all-trans retinoic acid. The C-reactive protein prevented thermal aggregation of the citrate synthase and stabilized the target enzyme, citrate synthase. The C-reactive protein promoted functional refolding of the urea-denatured citrate synthase up to 40-70%. These results suggest that the C-reactive protein, which is induced in human colon carcinoma cells, when heated or treated with all-trans retinoic acid has in a part functional activity of the molecular chaperone.

• Journal of Microbiology and Biotechnology
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• 제19권12호
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• pp.1628-1634
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• 2009

Small heat shock proteins (sHSPs) function as molecular chaperones that protect cells against environmental stresses. In the present study, the genes of hsp17.6 and hsp17.7, cytosolic class I sHSPs, were cloned from a tropical plant, Ageratina adenophorum. Their C-terminal domains were highly conserved with those of sHSPs from other plants, indicating the importance of the C-terminal domains for the structure and activity of sHSPs. The recombinant HSP17.6 and HSP17.7 were applied to determine their chaperone function. In vitro, HSP17.6 and HSP17.7 actively participated in the refolding of the model substrate citrate synthase (CS) and effectively prevented the thermal aggregation of CS at $45^{\circ}C$ and the irreversible inactivation of CS at $38^{\circ}C$ at stoichiometric levels. The prior presence of HSP17.7 was assumed to suppress the thermal aggregation of the model substrate CS. Therefore, this report confirms the chaperone activity of HSP17.6 and HSP17.7 and their potential as a protectant for active proteins.