• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.4
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• pp.344-350
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• 2006

Thermoplasma acidophilum is a thermoacidophilic archaeon that grows optimally at $59^{\circ}C$ and pH 2. Along with another thermoacidophilic archaeon, Sulfolobus solfataricus, it is known to metabolize glucose by the non-phosphorylated Entner-Doudoroff (nED) pathway. In the course of these studies, the specific activities of glyceraldehyde dehydrogenase and glycerate kinase, two enzymes that are involved in the downstream part of the nED pathway, were found to be much higher in T. acidophilum than in S. solfataricus. To characterize glycerate kinase, the enzyme was purified to homogeneity from T. acidophilum cell extracts. The N-terminal sequence of the purified enzyme was in exact agreement with that of Ta0453m in the genome database, with the removal of the initiator methionine. Furthermore, the enzyme was a monomer with a molecular weight of 49kDa and followed Michaelis-Menten kinetics with $K_m$ values of 0.56 and 0.32mM for DL-glycerate and ATP, respectively. The enzyme also exhibited excellent thermal stability at $70^{\circ}C$. Of the seven sugars and four phosphate donors tested, only DL-glycerate and ATP were utilized by glycerate kinase as substrates. In addition, a coupled enzyme assay indicated that 2-phosphoglycerate was produced as a product. When divalent metal ions, such as $Mn^{2+},\;CO^{2+},\;Ni^{2+},\;Zn^{2+},\;Ca^{2+},\;and\;Sr^{2+}$, were substituted for $Mg^{2+}$ the enzyme activities were less than 10% of that obtained in the presence of $Mg^{2+}$. The amino acid sequence of T. acidophilum glycerate kinase showed no similarity with E. coli glycerate kinases, which belong to the first glycerate kinase family. This is the first report on the biochemical characterization of an enzyme which belongs to a member of the second glycerate kinase family.

• Journal of Life Science
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• v.23 no.2
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• pp.190-196
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• 2013

The sea squirt, Halocynthia roretzi, has experienced mass mortality due to softness syndrome. The identification of disease-induced genes can provide insights into the development of this syndrome. To identify the genes, we performed differentially expressed gene (DEG) analysis. The expression of the phosphoglycerate kinase (HrPGK) gene was significantly decreased in diseased sea squirts compared to normal ones. We confirmed the result of the DEG analysis through RT-PCR and real-time PCR. In addition, we detected single nucleotide polymorphisms at position -106 (A/T) and -254 (G/T) in the HrPGK gene promoter by genotyping analysis. At the -106 site of the HrPGK gene, the frequency of the AA allele in disease-resistant sea squirts was about two-fold higher than that of sensitive ones, and the frequency of the TT allele in the disease-resistant sea squirts was about six-fold lower. At the -254 site of the HrPGK gene, the frequency of the GT and the GG allele was approximately two-fold higher and two-fold lower, respectively, in the disease-resistant sea squirts compared to the disease-sensitive ones. Analysis of the relationship between the genotypic variation at the -106/-254 promoter and the expression of HrPGK mRNA showed that HrPGK mRNA expression was higher in the -106/-254 AA/GT genotype samples than in the -106/254 TT/GG genotype ones. These results show that sea squirts harboring the AA/GT genotype may have more resistance to mortality than the sea squirts with other genotypes.

• Applied Biological Chemistry
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• v.41 no.5
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• pp.355-362
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• 1998

The minor form of phosphofructokinase (EC 2.7.1.11; PFK), which was suggested to be of plastid origin from the host fraction of chickpea nodules, was isolated as a small protein with apparent molecular mass near 220 kDa and purified to a high degree. SDS-PAGE and western blot indicated that the enzyme was made up of a homotetrameric structure (55 kDa). The enzyme had sharp pH profiles with maximal activities at pH 8 and displayed Michaelis-Menten kinetics with respect to Fru-6-P and nucleoside triphosphate substrate at the pH optimum (pH 8) and at pH 7. MgATP was the most effective phosphoryl donor. Phosphoenolpyruvate was a potent inhibitor of minor PFK activity, and the enzyme was also strongly inhibited by 3-phosphoglycerate, 2-phosphoglycerate, and to a lesser extent, PPi. Minor PFK was weakly activated by KCl, NaCl and Pi, and was inhibitory at high concentration of KCl and Pi.

• Microbiology and Biotechnology Letters
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• v.35 no.3
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• pp.184-190
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• 2007

Approximately 2.0 kb of the promoter region of the Pichia pastoris phosphoglycerate kinase gene (PGK1) was reduced to a 266 bp fragment and this minimized portion was used for construction of a new episomal constitutive expression vector in P. pastoris. As an approach to developing a constitutive expression vector in P. pastoris, the GAP promoter region of the Pichia expression vector pGAPZB was replaced with sequentially deleted PGK1 promoter fragments fused to a beta-galactosidase gene. When a lacZ gene was used as a reporter gene, PGK1 promoter strength was lower than that of the constitutive GAP promoter but it was higher than TEF1. We report here the development of the pPGKZ-E vector as a new episomal expression vector for heterologous gene expression by removing non-essential regions of the PGK1 promoter. This broadens the choice of episomal expression vectors for controlled constitutive expression in P. pastoris.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.219-222
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• 2000

Physiological changes of Escherichia coli during the fed-batch fermentation process were characterized in this study. Overall cellular protein samples prepared at the different stage of fermentation were separated by 2-dimensional gel electrophoresis (2-DE), and differently expressed 15 proteins, Phosphotransferase enzyme I, GroEL, Trigger factor, ${\beta}$ subunit of ATP synthase, Transcriptional regulator KDGR, Phosphoglycerate mutase 1, Inorganic pyrophosphatase, Serine Hydroxymethyl-transferase, ${\alpha}$ subunit of RNA polymerase, Elongation factor Tu, Elongation factor Ts, Tyrosine-tRNA ligase, DnaK suppressor protein, Transcriptional elongation factor, 30S ribosomal protein S6 were identified using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). When bacterial cells grow to high cell density, and IPTG-inducible heterologous protein is produced, expression level of overall cellular proteins was decreased. According to their functions in the cell, identified proteins were classified into three groups, proteins involved in transport process, small-molecule metabolism, and synthesis and modification of macromolecules.

• Journal of Animal Science and Technology
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• v.53 no.1
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• pp.15-22
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• 2011

The purpose of this study was to analyse of association between growth traits and single nucleotide polymorphisms (SNPs) polymorphism of phosphoglycerate kinase 2 (PGK 2) gene in pigs. The birth weight of piglet influences on weaning weight and survival rate that are import economic traits in pig industry. Also, these growth traits are representative factor to decrease a period getting to marketing weight as well as growth rate in pig. The PGK 2 is an isozyme that catalyzes the first ATP-generating step in the glycolytic pathwayand important enzyme related with energy metabolism. Twenty of SNPs were discoveredby genome structure analysis that compares the sequence on promoter and transcription region of PGK 2 gene in porcine chromosome 7. An association between PGK 2 SNPs and growth traits was analyzed in $F_2$ reciprocal-crossbred population between korean native pig (KNP) and Landrace. Association analysis indicated that polymorphism of the PGK 2 gene promoter region has significant effects on weight at birth (p<0.01) and weight at 3 weeks of age (p<0.0001). These results suggest that PGK 2 gene polymorphism was associated with energy metabolism and physiological function of growth in pig.

• Journal of Life Science
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• v.21 no.1
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• pp.146-154
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• 2011

In this study, physiological changes in a thermotolerant yeast Saccharomyces cerevisiae KNU5377 cell exposed to 48-hour alcohol fermentation at $40^{\circ}C$ were investigated. After 12 hours of alcohol fermentation at $40^{\circ}C$, the $C_{16:1}$ unsaturated acid of plasma membrane increased to 1.5 times more than the $C_{16:0}$ saturated fatty acid, and to about 2 times more for the $C_{18:1}$ unsaturated fatty acid. Fermentation at both $30^{\circ}C$ and $37^{\circ}C$ fermentation showed the same pattern as that done at $40^{\circ}C$. The pH of the alcohol-fermentation medium was reduced to pH 4.1 from a starting pH of 6.0 through the 12-hr fermentation and then maintained this level during the continuing fermentation. With the process of fermentation, the remaining glucose was reduced, but its amount remaining during the $40^{\circ}C$-fermentation was less reduced than those fermented at $30^{\circ}C$ and $37^{\circ}C$. In the study investigating the changing pattern of cellular proteins in the alcohol-fermenting cells, the SDS-PAGE and 2-D data indicated the most expressed dot was phosphoglycerate kinase, which is one enzyme involved in glycolysis. Why this enzyme was most expressed in the cells exposed to unfavorable conditions such as high temperature, increasing concentration of produced alcohol and long time exposure to other stress factors remains unsolved.

• Journal of Microbiology and Biotechnology
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• v.28 no.8
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• pp.1339-1345
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• 2018

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, which catalyzes aldol cleavage and condensation reactions, has two distinct substrate-binding sites. The substrate-binding mode at the catalytic site and Schiff-base formation have been well studied. However, structural information on the phosphate-binding loop (P-loop) is limited. Zymomonas mobilis KDPG aldolase is one of the aldolases with a wide substrate spectrum. Its structure in complex with the substrate-mimicking 3-phosphoglycerate (3PG) shows that the phosphate moiety of 3PG interacts with the P-loop and a nearby conserved serine residue. 3PG-binding to the P-loop replaces water molecules aligned from the P-loop to the catalytic site, as observed in the apostructure. The extra electron density near the P-loop and comparison with other aldolases suggest the diversity and flexibility of the serine-containing loop among KDPG aldolases. These structural data may help to understand the substrate-binding mode and the broad substrate specificity of the Zymomonas KDPG aldolase.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1994.11a
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• pp.147-172
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• 1994

Aziridinylbenzoquinones such as 3, 6-diaziridinyl-1, 4-benzoquinone (DZQ) and its 2, 5-methyl analog (MeDZQ) require bioreductive activation in order to elicit their anticancer activities. To determine the involvement of DTD in the activation of these drugs, we have used a ligation-mediated polymerase chain reaction to map the intracellular alkylation sites in a sing1e copy gene at the nucleotide level. We have performed this analysis in two human colon carcinoma cells, one proficient (HT-29) and one deficient (BE) in DT-diaphorase (DTD) activity. In the DTD proficient HT-29 cell line, DZQ and MeDZQ were found to alkylate both 5'-(A/T)G(C)-3' and 5'-(A/T)A-3' sequences. This is consistent with the nucleotide preferences observed when DZQ and MeDZQ are activated by purified DTD to reactive metabolites capable of alkylating DNA in vitro [Lee, C. -S., Hartley, J. A., Berardini, M. D., Butler, J., Siegel., D., Ross, D., & Gibson, N. W. (1992) Biochemistry, 31: 3019-3025]. Surprisingly in the DTD-deficient BE cell line a pattern of alkylation induced by DZQ and MeDZQ similar to that observed in the DTD-proficient HT-29 cells was observed. This suggests that reductive enzymes other than DTD can be involved in activating DZQ and MeDZQ to DNA reactive species in vivo.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.8
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• pp.1083-1088
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• 2012

The breeding value of marbling score in skeletal muscle is an important factor for evaluating beef quality. In the present study, we investigated proteins associated with the breeding value of the marbling score for bovine sirloin to select potential biomarkers to improve meat quality through comparative proteomic analysis. Proteins isolated from muscle were separated by two-dimensional gel electrophoresis. After analyzing images of the stained gel, seven protein spots for the high marbling score group were identified corresponding to changes in expression that were at least two-fold compared to the low marbling score group. Four spots with increased intensities in the high marbling score group were identified as phosphoglycerate kinase 1, triosephophate isomerase, acidic ribosomal phosphoprotein PO, and capping protein (actin filament) Z-line alpha 2. Spots with decreased intensities in the high marbling score group compared to the low score group were identified as 14-3-3 epsilon, carbonic anhydrase II, and myosin light chain 1. Expression of myosin light chain 1 and carbonic anhydrase 2 was confirmed by Western blotting. Taken together, these data could help improve the economic performance of cattle and provide useful information about the underlying the function of bovine skeletal muscle.