• Applied Microscopy
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• v.30 no.4
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• pp.413-421
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• 2000

We have isolated a large cylindrical protein complex from hyperthermophile archaeon Thermococcus profundus. Structural analysis by image processing of electron micrographs suggests that the complex is composed of two stacked rings of eight subunits each; the ring enclose a central channel. The purified protein was shown to be a homomultimer of 60 kDa subunit (P60 complex). It exhibits an extremely thermostable ATPase activity with a temperature optimum of $80^{\circ}C$. This protein complex may play an important role in the adaptation of thermophile archaeon to life at high temperature.

• Journal of Microbiology and Biotechnology
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• v.23 no.6
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• pp.818-825
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• 2013

We isolated and functionally characterized the ${\alpha}$- and ${\beta}$-subunits (ApCpnA and ApCpnB) of a chaperonin from Aeropyrum pernix K1. The constructed vectors pET3d-ApCpnA and pET21a-ApCpnB were transformed into E. coli Rosetta (DE3), BL21 (DE3), or CodonPlus (DE3) cells. The expression of ApCpnA (60.7 kDa) and ApCpnB (61.2 kDa) was confirmed by SDS-PAGE analysis. Recombinant ApCpnA and ApCpnB were purified by heat-shock treatment and anion-exchange chromatography. ApCpnA and ApCpnB were able to hydrolyze not only ATP, but also CTP, GTP, and UTP, albeit with different efficacies. Purified ApCpnA and ApCpnB showed the highest ATPase, CTPase, UTPase, and GTPase activities at $80^{\circ}C$. Furthermore, the addition of ApCpnA and ApCpnB effectively protected citrate synthase (CS) and alcohol dehydrogenase (ADH) from thermal aggregation and inactivation at $43^{\circ}C$ and $50^{\circ}C$, respectively. In particular, the addition of ATP or CTP to ApCpnA and ApCpnB resulted in the most effective prevention of thermal aggregation and inactivation of CS and ADH. The ATPase activity of the two chaperonin subunits was dependent on the salt concentration. Among the ions we examined, potassium ions were the most effective at enhancing the ATP hydrolysis activity of ApCpnA and ApCpnB.

• The Korean Journal of Physiology
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• v.19 no.1
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• pp.15-23
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• 1985

Previous biochemical studies indicate that $(Na^++K^+)-ATPase$ is composed of two subunits, ${\alpha}$ and ${\beta}$, in a form of ${\alpha}_2{\beta}_2$ with a molecular weight of approximately 300,000 daltons. There is also suggestive evidence that the $Na^+$, $K^+$ pump in human erythrocytes occurs in a complex with some glycolytic enzymes. We assessed here in situ assembly size of the $(Na^++K^+)-ATPase$ of human erythrocytes by applying classical target theory to radiation inactivation data of the ouabain-sensitive sodium flux and ATP hydrolysis of intact cells and ghosts. Cells(in the presence of cryoprotective agent) and ghosts were irradiated at $-45^{\circ}C$ to $-50^{\circ}C$ with an increasing dose of a 1.5 MeV electron beam, and after thawing, the pump and/or enzyme activities were assayed. Each activity measured was decreased as a simple exponential function of radiation dose, from which a radiation sensitive volume (target size) was calculated. When intact cells were used, the target size of both $(Na^++K^+)-ATPase$ and $Na^+$, $K^+$ pump was found to be approximately 600,000 daltons. This target size of the ATPase was reduced to approximately 325,000 daltons if the cells were pretreated with strophanthidin. When ghosts were used, the target size of the ATPase was again approximately 325,000 daltons. Our target size measurement suggests that, in intact cells, the $(Na^++K^+)-ATPase/Na^+,K^+$ pump exists either as a dimer of $(\alpha\beta)_2$ which is a functional unit or as a monomer of $(\alpha\beta)_2$ but in tight complex with other enzyme or enzymes. The results also suggest that this dimeric or heterocomplex association is dissociated during ghost preparation and strophanthidin treatment.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.31 no.1
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• pp.24-30
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• 2005

The flow of saliva is controlled entirely by nervous stimuli. The present study was aimed to explore the role of sympathetic and parasympathetic nerves in the regulation of sodium transporters and water channels in the salivary gland. Rats were denervated of their sympathetic and parasympathetic nerves to the submandibular gland, and the expression of sodium transporters and water channels was determined. The expression of either ${\alpha}-1$ or ${\beta}-1$ subunit of Na, K-ATPase was not significantly affected by the sympathetic denervation. On the contrary, the expression of both subunits was decreased by the parasympathetic denervation. The expression of ${\alpha}-,\;{\beta}-$, and ${\gamma}$-subunits of ENaC was not significantly affected by the sympathetic denervation, but was increased by the parasympathetic denervation. On the contrary, the expression of NHE3 was markedly decreased by both the sympathetic and the parasympathetic denervation. The sympathetic denervation significantly increased the expression of AQP1, while the parasympathetic denervation was without effect. The sympathetic and parasympathetic denervation significantly increased the expression of AQP4. The sympathetic denervation did not affect the expression of AQP5, but the parasympathetic denervation significantly decreased it. These results suggest that sympathetic and parasympathetic nerves have tonic effects on the regulation of sodium transporters and AQP water channels in the salivary gland. The sympathetic and parasympathetic denervation may then result in alterations of secretory rate and electrolyte composition of the saliva.

• Proceedings of the Korea Crystallographic Association Conference
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• 2002.11a
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• pp.25-25
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• 2002

CodWX, encoded by the cod operon in Bacillus subtilis, is a member of the ATP-dependent protease complex family, and is homologous to the eukaryotic 26S proteasome. It consists of two multimeric complexes: two hexameric ATPase caps of CodX and a protease chamber consisting of CodW dodecamer. Prior structural studies have shown that the N-terminal threonine residue is solely functional as a proteolytic nucleophile in ATP-dependent proteases such as HslV and certain β-type subunits of 20S proteasome, which have a primary sequence similarity of -50% and -20% with CodW respectively. Here we present a 3.0 Å resolution crystal structure of CodW, which is the first N-terminal serine protease among the known proteolytic enzymes. In spite of the same fold and the conserved contacts between subunits with HslV in E. coli and H. influenza, this structure shows the five additional residues extending from conserved Thr1 among the other ATP-dependent pretense and extraordinary basic proteolytic chamber.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.32 no.1
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• pp.1-7
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• 2006

The present study was aimed to explore the role of sympathetic and parasympathetic nerves in the regulation of sodium transporters and water channels in the salivary gland. Rats were denervated of their sympathetic and parasympathetic nerves to the submandibular gland, and the glandular expression of sodium transporters and water channels was determined by Western blot analysis. The expression of either ${\alpha}1$ or ${\beta}1$ subunit of Na, K-ATPase was not significantly affected either by the sympathetic or by the parasympathetic denervation. The expression of subunits of epithelial sodium channels was significantly increased both in the denervated and contralateral glands either by the sympathetic or by the parasympathetic denervation. Neither the sympathetic nor the parasympathetic denervation significantly altered the expression of aquaporin-1 (AQP1). Nor was the expression of AQP4 affected significantly by the parasympathetic or the sympathetic denervation. On the contrary, the expression of AQP5 was significantly increased not only by the parasympathetic but also by the sympathetic denervation. These results suggest that sympathetic and parasympathetic nerves have tonic regulatory effects on the regulation of certain sodium transporters and AQP water channels in the salivary gland.

• Journal of Photoscience
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• v.11 no.3
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• pp.121-127
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• 2004

The${\beta}$-glucosidases occur widely in all living organisms and has in general a tendency to form oligomers of varying numbers of subunits or aggregates, although the functional implications of such diverse oligomerization schemes remain unclear. In particular, the assembly mode of the oat ${\beta}$-glucosidase is very unique in that it multimerizes by linear stacking of a hexameric building block to form long fibrillar multimers. Some structural proteins such as actin and tubulin assemble into long fibrils in a helical fashion and several enzymes such as GroEL and Pyrodictium ATPase functional complexes, 20S proteasome of the archaebacterium Thermoplasma acidophilum, and lutamine synthetase fromblue-green algae, assemble into discrete oligomers upto 4 stacked rings to maintain their enzymatic activities. In particular, oat ${\beta}$-glucosidase exists in vivo as a discrete long fibrillar multimer assembly that is a novel structure for enzyme protein. It is assembled by linear stacking of hollow trimeric units. The fibril has a long central tunnel connecting to the outer medium via regularly distributed side fenestrations. The enzyme active sites are located within the central tunnel and multimerization increases enzyme affinity to the substrates and catalytic efficiency of the enzyme. Although it is suggested that oligomerization may contribute to the enzyme stability and catalytic efficiency of ${\beta}$-glycosidases, the functional implications of such diverse oligomerization schemes remain unclear so far.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.17 no.4
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• pp.280-290
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• 1984

An extensive study has been made on the relationship between the freshness and the compositions of the muscle protein of prawn, Pandalus japonica during the storage under partially frozen condition. The variations of the subunit distribution for sarcoplasmic protein and myofibrillar protein extracted from the samples by changes of freshness were discussed by sodium dodecylsulfate-poly-acrylamide gel (SDS-PAG) electrophoresis. On the other hand, the denaturation constant ($K_D$) of the myofibrillar protein extracted from the prawn stored at $-3^{\circ}C\;and\;-20^{\circ}C$ were successively compared. The prawn muscle contained about $18\%$ of protein with the composition of $32\%$ in sarcoplasmic protein, $56\%$ in myofibrillar protein, $10\%$ in residual intracellular protein and $2\%$ in stroma. The indices for estimating freshness of the muscle were approached to the early stage of putrefaction on the 26th day of the storage with $25.29mg\%$ of total volatile basic nitrogen, $31.36\%$ of K-value and 8.83 of pH. The content of the myofibrillar protein was remarkably decreased with the time during the storage while that of residual intracellular protein was increased. The $K_D$ values of the myofibrillar protein were $9.03{\times}10^{-6}sec^{-1}\;at\;-3^{\circ}C\;and\;4.42{\times}10^{-6}sec^{-1}\;at\;-20^{\circ}C$. The results of the analysis of SDS-PAG electrophoretograms indicated that the sarcoplasmic protein and the myofibrillar protein were composed of 12 subunits and 17 subunits in the muscle of instantaneously killed prawn ana were changed into 8 subunits and 22 subunits in the muscle stored for 26 days, respectively. It is noticeable that 30,000, 41,000, 107,000, 136,000, 170,000 173,000, 185,000, and 198,000 daltons of the newly appeared 8 subunits were found in the myofibrillar protein from the prawn muscle stored for 26 days. The amino acid composition of the muscle protein showed that the most of amino acids were slightly decreased with the days of the storage. With respect to the free amino acid composition of the muscle of instantaneously killed prawn, glycine, proline, arginine, alanine and taurine comprised $93\%$ of the total free amino acids. Taurine, valine, leucine, phenylalanine, serine, lysine, methionine, isoleucine and histidine were increased during the storage period but exceptionally proline was decreased.

• Proceedings of the Korea Environmental Mutagen Society Conference
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• 2002.11a
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• pp.80-88
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• 2002

Recent studies demonstrate that collagen IV selectively pro-motes the repair of physiological processes in sublethally injured renal proximal tubular ceils (RPTC). We sought to further define the mechanisms of cell repair by measuring the effects of toxicant injury and stimulation of repair by L-ascorbic acid-2-phosphate (AscP), exogenous collagen IV, or function-stimulating integrin antibodies on the expression and subcellular localization of collagen-binding integrins (CBI) in RPTC. Expression of CBI subunits ${\alpha}_1$, ${\alpha}_2$, and ${\beta}_1$ in RPTC was not altered on day 1 after sublethal injury by S-(1,2-dichlorovinyl)-L-cysteine (DCVC). On day 6, expression of ${\alpha}_1$ and ${\beta}_1$ subunits remained unchanged, whereas a 2.2-fold increase in ${\alpha}_2$ expression was evident in injured RPTC. CBI localization in control RPTC was limited exclusively to the basal membrane. On day 1 after injury, RPTC exhibited a marked inhibition of active $Na^+$ transport and a loss of cell polarity characterized by a decrease in basal CBI localization and the appearance of CBI on the apical membrane. On day 6 after injury, RPTC still exhibited marked inhibition of active $Na^+$ transport and localization of CBI to the apical membrane. However, DCVC-injured RPTC cultured in pharmacological concentrations of AscP (500 ${\mu}$M)or exogenous collagen IV (50 ${\mu}$g/ml) exhibited an increase inactive $Na^+$ transport, relocalization of CBI to the basal membrane, and the disappearance of CBI from the apical membrane on day 6. Function-stimulating antibodies to CBI ${\beta}_1$ did not promote basal relocalization of CBI despite stimulating the repair of $Na^+$/$K^+$-ATPase activity on day 6 after injury. These data demonstrate that DCVC disrupts integrin localization and that physiological repair stimulated by AscP or collagen IV is associated with the basal relocalization of CBI in DCVC-injured RPTC. These data also suggest that CBI-mediated repair of physiological functions may occur independently of integrin relocalization.

• Journal of Life Science
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• v.24 no.8
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• pp.915-926
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• 2014

The ribosome is a protein synthesizing machinery and a ribonucleoprotein complex that consists of three ribosomal RNAs (23S, 16S and 5S) and 54 ribosomal proteins in bacteria. In the course of ribosome assembly, ribosomal proteins (r-protein) and rRNAs are modified, the r-proteins bind to rRNAs to form ribonucleoprotein complexes which are folded into mature ribosomal subunits. In this process, a number of non-ribosomal trans-acting factors organize the assembly process of the components. Those factors include GTP- and ATP-binding proteins, rRNA and r-protein modification enzymes, chaperones, and RNA helicases. During ribosome biogenesis, they participate in the modifications of ribosomal proteins and RNAs, and the assemblies of ribosomal proteins with rRNAs. Ribosomes can be assembled from a discrete set of components in vitro, and it is notable that in vivo ribosome assembly is much faster than in vitro ribosome assembly. This suggests that non-ribosomal ribosome assembly factors help to overcome several kinetic traps in ribosome biogenesis process. In spite of accumulation of genetic, structural, and biochemical data, not only the entire procedure of bacterial ribosome synthesis but also most of roles of ribosome assembly factors remain elusive. Here, we review ribosome assembly factors involved in the ribosome maturation of Escherichia coli, and summarize the contributions of several ribosome assembly factors which associate with 50S and 30S ribosomal subunits, respectively.