• The Korean Journal of Pharmacology
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• v.26 no.1
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• pp.35-49
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• 1990

A highly purified $(Na^+,\;K^+)-ATPase$ from the rectal gland of Squalus acanthias and from the electric organ of Electrophorus electricus has been used to raise antibodies in rabbits. The 97,000 dalton catalytic subunit and glycoprotein derived from the rectal gland of spiny shark were also used as antigens. The two $(Na^+,\;K^+)-ATPase$ holoenzymes and the two shark subunits were antigenic. In Ouchterlony double diffusion experiments, these antibodies formed precipitation bands with their antigens. Antibodies prepared against the two subunits of shark holoenzyme also formed precipitation bands with their antigens and shark holoenzyme, but not with eel holoenzyme. These observations are in good agreement with inhibitory effect of these antibodies on the catalytic activity of $(Na^+,\;K^+)-ATPase$ both from the shark and the eel, since there is very little cross-reaction between the shark anticatalytic subunit antibodies and the eel holoenzyme. The maximum antibodies titer of the anticatalytic subunit antibodies is found to be 6 weeks after the initial single exposure to this antigen. Multiple injections of the antigen increased the antibody titer. However, the time required to produce the maximum antibody titer was approximately the same. These antibodies also inhibit catalytic activity of $(Na^+,\;K^+)-ATPase$ vesicles reconstituted by a slow dialysis of cholate after solubilization of the enzyme in a presonicated mixture of cholate and phospholipid. In these reconstituted $(Na^+,\;K^+)-ATPase$ vesicles, effects of these antibodies on the fluxes of $Na^+$, $Rb^+$, and $K^+$ were investigated. Control or preimmune serum had no effect on the influx of $^{22}Na^+$ or the efflux of $^{86}Rb^+$. Immunized sera against the shark $(Na^+,\;K^+)-ATPase$ holoenzyme, its glycoprotein or catalytic subunit did inhibit the influx of $^{22}Na^+$ and the efflux of $^{86}Rb^+$. It was also demonstrated that these antibodies inhibit the coupled counter-transport of $Na^+$ and $K^+$ as studied by means of dual labeling experiments. However, this inhibitory effect of the antibodies on transport of ions in the $(Na^+,\;K^+)-ATPase$ vesicles is manifested only on the portion of energy and temperature dependent alkali metal fluxes, not on the portion of ATP and ouabain insensitive ion movement. Simultaneous determination of effects of the antibodies on ion fluxes and vesicular catalytic activity indicates that an inhibition of active ion transport in reconstituted $(Na^+,\;K^+)-ATPase$ vesicles appears to be due to the inhibitory action of the antibodies on the enzymatic activity of $(Na^+,\;K^+)-ATPase$ molecules incorporated in the vesicles. These findings that the inhibitory effects of the antibodies specific to $(Na^+,\;K^+)-ATPase$ or to its subunits on ATP and temperature sensitive monovalent cation transport in parallel with the inhibitory effect of vesicular catalytic activity by these antibodies provide direct evidence that $(Na^+,\;K^+)-ATPase$ is the molecular machinery of active cation transport in this reconstituted $(Na^+,\;K^+)-ATPase$ vesicular system.

• Animal cells and systems
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• v.14 no.1
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• pp.1-10
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• 2010

Previously we showed that CHIP, a co-chaperone of Hsp70 and E3 ubiquitin ligase, can promote the degradation of mutant SOD1 linked to familial amyotrophic lateral sclerosis (fALS) via a mechanism not involving SOD1 ubiquitylation. Here we present evidence that CHIP functions in the interaction of mutant SOD1 with 26S proteasomes. Bag-1, a coupling factor between molecular chaperones and the proteasomes, formed a complex with SOD1 in an hsp70-dependent manner but had no direct effect on the degradation of mutant SOD1. Instead, Bag-1 stimulated interaction between CHIP and the proteasome-associated protein VCP (p97), which do not associate normally. Over-expressed CHIP interfered with the association between mutant SOD1 and VCP. Conversely, the binding of CHIP to mutant SOD1 was inhibited by VCP, implying that the chaperone complex and proteolytic machinery are competing for the common substrates. Finally we observed that mutant SOD1 strongly associated with the 19S complex of proteasomes and CHIP over-expression specifically reduced the interaction between S6/S6' ATPase subunits and mutant SOD1. These results suggest that CHIP, together with ubiquitin-binding proteins such as Bag-1 and VCP, promotes the degradation of mutant SOD1 by facilitating its translocation from ATPase subunits of 19S complex to the 20S core particle.

• International Journal of Industrial Entomology and Biomaterials
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• v.15 no.2
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• pp.115-122
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• 2007

As the genome of B.mori is available in GenBank and the EST database of B.mori is expanding, identification of novel genes of B.mori is conceivable by data-mining techniques. We used the in silico cloning method to get the vacuolar-type $H^+-ATP$ synthetase (V-ATPase) c subunit (16 kDa proteolipid subunit) gene of B.mori and analysed with bioinformatics tools. The result was confirmed by RT-PCR and sequencing. The V-ATPase c subunit cDNA contains a 468 bp ORF. The ORF encoded a 155-residue protein that showed extensive homology with V-ATPase c subunits from other 15 species and contained four membrane-spanning helices. Tissue expression pattern analysis revealed that V-ATPase c expressed strongly in Malpighian tubules, not in fat body. This gene has been registered in GenBank under the accession number EU082222.

• The Korean Journal of Zoology
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• v.37 no.4
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• pp.524-530
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• 1994

An Arpase complex has been purified to apparent homogeniety from the extract of chick skeletal muscle using conventional column chromatographies and glycerol density gradient centrifugation. This eWe has a sedimentation coefficient of 15S as determined by the gradient centrifugation and therefore is referred to as the 15S ATPase. It behaves as a 600-kOa molecule upon gel filtration analysis using a Superose-6 column. However, the ATPase runs as a 95-kDa polvpeptide when analyzed by polvacrvlamide gel electrophoresis in the presence of sodium dodecyl sulfate. Thus, the Arpase is likely to consist of six identical subunits of 95 KDa. It has a Km value of 0.6 mM for ATP and is maximally active at pH 9.

• Biomedical Science Letters
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• v.6 no.3
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• pp.165-170
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• 2000

Vacuolar (H$^{+}$)-ATPase (V-ATPase) is an intracellular protein which consists of multiple subunits. It carries out acidification by pumping protons in the cell. This enzyme has also been found in the synaptic vesicles and may play an important role in the neurotransmission. We cloned cDNA fragments encoding the 16 kDa subunit of V-ATPase from the rat brain by RT-PCR and PCR using total RNA or recombinant phage DNA as templates. They contained the full coding sequences (468 bp) and one nucleotide at 3' region turned out to be different (A to C) when compared to the liver counterpart. However, this polymorphic difference did not cause any significant change in the primary structure of the protein because both GCA and GCC code for alanine. Our study would contribute to the understanding of the function of 16 M)a V-ATPase in the brain and of the mechanisms of neurotransmission.

• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.260-266
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• 2005

IgE-dependent histamine releasing factor (HRF), previously known as P23/P21 or translationally controlled tumor protein (TCTP), induces the degranulation of histamine in mast cell and basophil. Yeast two hybrid results showed that HRF interacts with the alpha subunit of Na, K-ATPase, suggesting that HRF is a regulator for governing the activity of Na, K-ATPase. In this study, we examined the interaction of HRF and Wa,K-ATPase after treatments of various PKC isotype inhibitors. Membrane fractionation, pull-down assay and immunoprecipitation results showed that PKC $\alpha,\;PKC\;\beta,\;\delta$ subunits are involved in the phosphorylation of HRF. However, these results did not correlate with the results of histamine release assay since histamine release assay results suggested that some PKC isotype inhibitors induced the histamine release in RBL-2H3 cell.

• The Korean Journal of Zoology
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• v.37 no.4
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• pp.531-537
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• 1994

Partial internal amino acid sequences of the 15S ATPase from chick skeletal muscle were determined and found to be identical to the corresponding regions of the Mg2+_ATPase from Xenopus laevis oocytes, that is a close homolog of N-ethvlmaleimide-sensitive factor (called NSF) in hamster and Sec18p in yeast, both of which are believed to plaN an essential role in vesicle fusion in secretory process. Thus, the 15S Arpase in chick skeletal muscle maw also belong to a protein family of the "vesicle fusion proteins". Unlike the Mg2'-Afpase with an isoelectric point (pl) of 5.5, however, the 15S Arpase was separated into four spots with pls of 4.9,6.4 and 6.9 upon analysis by twoiimensional gel electrophoresis. In addition, the anti-15S ATPase IgG was found to be capable of interacting with the 265 protease complex upon analysis by immunoprecipitation. Moreover, immunoblot analysis revealed that the anti-155 Arpase IgG recognizes three subunits, ko of which show the same mobilities as the 510-kDa subunit 4 and 48-kDa subunit 7 of the 26S protease complex that are known to contain a highly consented ATP-binding motif. These results surest that a common antigenic site, likely the consensus nucleotide-binding site, exists in the 15S ATPase and the 26S pretense complex and hence both the enzymes maw also be related ATPases.d ATPases.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.181-181
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• 1996

The (Na, K) ATPase is a membrane ion transporting ATPase composed of an ${\alpha}$ catalytic subunit and a ${\beta}$ glycoprotein subunit. The topology of the rat ${\alpha}$1 and ${\beta}$1 subunits has been studied by insertion of epitope(s) : at the NH2-terminus and COOH-terminus and between Glu117 and Glul18, Lys828 and Arg829, Gln900 and Trp901, and Va1939 and Phe940 of the ${\alpha}$ subunit; and at the NH2-terminus and COOH-terminus and between Glu228 and Tyr229 of the ${\beta}$ subunit. The epitope-tagged ${\alpha}$l, constructs were expressed in HeLa cells to select for stable cell lines expressing a functional (Na, K)ATPase. All constructs, except for the one tagged between Gln900 and Trp901, resulted in ouabain-resistant colonies indicating that modified proteins retained functional integrity. The epitope-tagged ${\beta}$ constructs were transiently expressed in Cos-7 cells. The orientation of the epitopes with respect to the cell membrane was revealed by indirect immunofluorescence performed on permeabilized and non-permeabilized cells expressing the (Na, K)ATPase chains. The results indicate that the ${\alpha}$ subunit has 4 transmembrane segments in the COOH terminal membrane bound domain between residues 760 and 938, and that both the NH2-terminus and the COOH-terminus are in the cytosol; it was not determined whether there are more transmembrane segments between residue 938 and the COOH-terminus. The ${\beta}$ subunit has only one transmembrane spanning region with the NH2-terminus in the cytosol and the COOH-terminus on the extracytoplasmic surface of the plasma membrane.

• Korean Journal of Environmental Agriculture
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• v.17 no.4
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• pp.296-300
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• 1998

The analysis of biochemical changes in tobacco plant as increase of NaCl concentraion was conducted. Total protein content and soluble protein content were decreased as NaCl concentration was increased, in that steady decreased until 120mM NaCl and largely decreased at 150mM NaCl. The expression of 74Kd subunit was increased until 60mM NaCl. However, the amount of 74Kd protein was decreased from 90mM NaCl. There was no difference for expression of other protein subunits. Chlorophyll a content was significantly decrease as NaCl concentration was increased, but chlorophyll b content was not much decreased. The slow increase up to 120mM NaCl and large increase at 150mM NaCl for ATPase and peroxidase activities indicated that 120mM NaCl could be a limiting concentration for salt injury.

• Herbal Formula Science
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• v.10 no.1
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• pp.113-129
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• 2002

The present study designed to investigate whether hwangryunhaedok-tang show an anti-hypertensive effect and elucidate its possible mechanism in spontaneously hypertensive rats. The systolic blood pressures (SBP) were significantly decreased as an oral administration of hwangryunhaedok-tang compared with their control group. The urine volume was significantly increased by administration of hwangryunhaedok-tang but urinary sodium (UNaV), potassium (UKV), chloride excretion (UCIV) were not remarkably affected. The urinary creatinine excretion rate (UcrV) was significantly increased in rats administered with hwangryunhaedok-tang in association with increase of creatinine clearance (Ccr). The urine osmolality (Uosmol) was significantly decreased in SHR administered with hwangryunhaedok-tang without being changed in solute-free water reabsorption ( TcH20). The expressions of Aquaporin 2 (AQP-2). 3 and ${\alpha}\;1$, ${\beta}\;1$ subunits of Na.K-ATPase were determined by Western blot analysis to assess the role of these proteins in association with changes of renal functions in SHR administered with hwangryunhaedok-tang. The expression of AQP-2 and 3 protein was significantly down-regulated in the kidney of SHR administered with hwangryunhaedok-tang compared with those in control rats without being altered expression of ${\alpha}\;1$, ${\beta}\;1$ subunits of Na,K-ATPase. In the in vitro assay, Angiotensin converting enzyme (ACE) was inhibited by hwangryunhaedok-tang in a dose-dependent manner. Berberine and/or palmatine, which are well known as a main components of hwangryunhaedok-tang, also have an ACE inhibitory effects in a dose-dependent manner. Taken together, these results suggest that hwangryunhaedok-tang lowered blood pressure through the increase of diuresis caused by down-regulation of water channels and the inhibition of Angiotensin converting enzyme.