• Title/Summary/Keyword: K-ATPase protein

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Isolation of Proteins that Specifically Interact with the ATPase Domain of Mammalian ER Chaperone, BiP

  • Chung, Kyung-Tae;Lee, Tae-Ho;Kang, Gyong-Suk
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.8 no.3
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    • pp.192-198
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    • 2003
  • BiP, immunoglobulin binding protein, is an ER homologue of Hsp70. However, unlit other Hsp70 proteins, regulatory protein(s) for BiP has not been identified. Here, we demo strafed the presence of potential regulatory proteins for BiP using a pull -down assay. Since BiP can bind any unfolded protein, only the ATPase domain of BiP was used for the pull -down assay in order to minimize nonspecific binding. The ATPase domain was cloned to produce recombinant protein, which was then conjugated to CNBr-activated agarose. The structural conformation and ATP hydrolysis activity of the recombinant ATPase domain were similar to those of the native protein, light proteins from metabolically labeled mouse plasmacytoma cells specifically bound to the recombinant ATPase protein. The binding of these proteins was inhibited by excess amounts of free ATPase protein, and was dependent on the presence of ATP. These proteins were eluted by ADP. Of these proteins, Grp170 and BiP where identified. while the other were not identified as known ER proteins, from Western blot analyses. The presence of the ATPase-binding proteins for BiP was first demonstrated in this study, and our data suggest similar regulatory machinery for BiP may exist in the ER, as found in prokaryotes and other cellular compartments.

Studies on the Myofibrilar Protein from Korean Duck Muscle (오리고기의 근원섬유 단백질에 관한 연구)

  • Chang, In-Yae;Nam, Hyun-Keun
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.9 no.1
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    • pp.45-50
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    • 1980
  • Myofibrilar Protein from Korean Duck Muscle was extracted and ATPase activities were studied. The results were as follows: 1. Mg-activated ATPase activity of Myofibril from Korean Duck, muscle exhibited a biphasic response, ATPase activity was high at a low ionic strength and low activity was showed at high ionic strength. 2. Effect of EDTA on the Myofibrillar protein ATPase activity was studied, it was investigated that the EDTA inhibition was showed at the concentration of $6.9{\mu}g$ and it above. 3. It showed that the effect of Ca++ on ATPase activity was decreased at the lower than $3{\mu}g$. Inhibition showed at the concentration of $6.9{\mu}g$ and it above. 4. It showed that the effect of Mg++ on ATPase activity was decreased at the lower than $3.6{\mu}g$. Inhibition showed at the concentration of $3.9{\mu}g$ and it above.

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Biochemical Properties of Myofibrillar Protein in Olive Flounder by the Sex (성별에 따른 넙치 근원섬유단백질의 생화학적 특성)

  • KIM Tae-Jin;YOON Ho-Dong;LEE Sang-Min;KIM Kyung-Gyl
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.30 no.3
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    • pp.349-354
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    • 1997
  • Myotibrillar ATPase activity, thermostability and composition of muscle protein were investigated to elucidate biochemical properties regard with rearing period and sex of olive flounder. Myofibrillar ATPase activity of male olive flounder reared for 6, 12 and 20 months was stronger than that of female one. $Mg^{2+}\;(+Ca^{2+})-ATPase$ activity of both female and male fish decreased with the elapse of rearing period, and the activity of male was higher than that of female far beyond the rearing periods. The high correlationship between the weight gain and myofibrillar ATPase activity was observed. The thor mostability of male myofibrillar protein was higher than that of female. Subunit composition of the myofibrillar and sarcoplasmic protein did not show difference between the both sex of the fish.

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Effect of Ginseng Saponin on the $Na^{+}$, $K^{+}$-ATPase of Dog Cardiac Sarcolemma

  • Lee, Shin-Woong;Lee, Jeung-Soo;Kim, Young-Hie;Jin, Kap-Duck
    • Archives of Pharmacal Research
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    • v.9 no.1
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    • pp.29-38
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    • 1986
  • The effects of ginseng saponins on the sarcolemmal $Na^{+}$, $K^{+}$-ATPase were compared to gypsophila saponin, sodium dodecylsulfate (SDS), and Triton X-100 to elucidate whether the effects are due to the membrane distruption, using a highly enriched preparation of cardiac sarcolemma prepared from dog ventricular myocardium. About 26% and 29% of vesicles in the preparation, enriched in ouabain-sensitive $Na^{+}$, $K^{+}$-ATP ase, $\beta$-adrenergic and muscarinic receptors are rightside-out and inside-out orientation, respectively. Ginseng saponins (triol>total> diol) inhibited $Na^{+}$, $K^{+}$-ATP ase activity, $Na^{+}$, $K^{+}$-ATPase activity and [$^{3}$H]ouabain binding of sarcolemmal vesicles. However, gypsophila saponin, SDS (0.4$\mu$g/$\mu$g protein) and Triton X-100 (0.6 $\mu$g/$\mu$g protein) caused about 1.35 and 1.40-fold increase in $Na^{+}$, $K^{+}$-ATPase activity and [$^{3}$H] oubain binding, respectively. Especially, the activating effect of gypsophila saponin on membrane Na+, K+ ATPase was detected at gypsophila saponin to sarcolemmal protein ratios as high as 100. Low dose of ginseng saponin (3$\mu$g/$\mu$g protein) decreased the phosphorylation sites and the concentration of ouabain binding sites (Bmax) without affecting the turnover number and affinity for ouabain binding, while gypsophila saponin, SDS(0.4 ug/ug protein), ahd Triton X-100 (0.6$\mu$g/$\mu$g protein) increased the Bmax. The results suggest that ginseng saponins cause a decrease in the number of active sites by interacting directly with $Na^{+}$, $K^{+}$-ATPase before disruption of membrane barriers of sarcolemmal vesicles.

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Vanadate-Induced Renal cAMP and Malondialdehyde Accumulation Suppresses Alpha 1 Sodium Potassium Adenosine Triphosphatase Protein Levels

  • Eiam-Ong, Somchit;Nakchui, Yuyen;Chaipipat, Mookda;Eiam-Ong, Somchai
    • Toxicological Research
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    • v.34 no.2
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    • pp.143-150
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    • 2018
  • It has been demonstrated that vanadate causes nephrotoxicity. Vanadate inhibits renal sodium potassium adenosine triphosphatase (Na, K-ATPase) activity and this is more pronounced in injured renal tissues. Cardiac cyclic adenosine monophosphate (cAMP) is enhanced by vanadate, while increased cAMP suppresses Na, K-ATPase action in renal tubular cells. There are no in vivo data collectively demonstrating the effect of vanadate on renal cAMP levels; on the abundance of the alpha 1 isoform (${\alpha}_1$) of the Na, K-ATPase protein or its cellular localization; or on renal tissue injury. In this study, rats received a normal saline solution or vanadate (5 mg/kg BW) by intraperitoneal injection for 10 days. Levels of vanadium, cAMP, and malondialdehyde (MDA), a marker of lipid peroxidation were measured in renal tissues. Protein abundance and the localization of renal ${\alpha}_1-Na$, K-ATPase was determined by Western blot and immunohistochemistry, respectively. Renal tissue injury was examined by histological evaluation and renal function was assessed by blood biochemical parameters. Rats treated with vanadate had markedly increased vanadium levels in their plasma, urine, and renal tissues. Vanadate significantly induced renal cAMP and MDA accumulation, whereas the protein level of ${\alpha}_1-Na$, K-ATPase was suppressed. Vanadate caused renal damage, azotemia, hypokalemia, and hypophosphatemia. Fractional excretions of all studied electrolytes were increased with vanadate administration. These in vivo findings demonstrate that vanadate might suppress renal ${\alpha}_1-Na$, K-ATPase protein functionally by enhancing cAMP and structurally by augmenting lipid peroxidation.

Molecular Cloning and Nucleotide Sequence of Amaranthus viridis Homologue of the H -Transporting ATPase Gene (비름에서 ATP 가수분해효소와 상동성을 가지는 유전자의 클로닝)

  • 한규웅
    • Journal of Life Science
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    • v.6 no.1
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    • pp.1-5
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    • 1996
  • Using differential hybridization, a cDNA clone was isolated fortuitously from Amaranthus viridis and sequenced. This nucleotide sequence exhibited 55.1% identity with vma6 which encodes the 36-kD subunit of the vacuolar proton transporting ATPase in Saccharmoyces cerevisiae. The predicted open reading frame encodes a protein of 221 amino acid sequence with a calculated molecular weight of 25,452 and reveals high levels of similarity with subunit D polypeptide of vacuolar H -ATP(e.g., 48.5, 52.1 and 49.3% identity to the vacuolar 36-kD chain of yeast, vacuolar 32-kD polypeptide IV of human and vacuolar 28-kD protein of bovine chromaffin granules, respectively). The hydropathy index computation revealed that this predicted protein is a peripheral protein. These results indicated that the predicted protein may play a sturctural role in the vaculor H -ATPase as does gamma subunit in V-type ATPase.

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PKC Isotype that Affects the Interaction of HRF with Na, K-ATPase (Na,K-ATPase와 IgE-Dependent Histamine Releasing Factor의 결합에 영향을 미치는 Protein Kinase C Isotype에 관한 연구)

  • Sohn Wern-Joo;Lee Kyunglim
    • 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.

Selective Interaction Between Chloroplast β-ATPase and TGB1L88 Retards Severe Symptoms Caused by Alternanthera mosaic virus Infection

  • Seo, Eun-Young;Nam, Jiryun;Kim, Hyun-Seung;Park, Young-Hwan;Hong, Seok Myeong;Lakshman, Dilip;Bae, Hanhong;Hammond, John;Lim, Hyoun-Sub
    • The Plant Pathology Journal
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    • v.30 no.1
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    • pp.58-67
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    • 2014
  • The multifunctional triple gene block protein 1 (TGB1) of the Potexvirus Alternanthera mosaic virus (AltMV) has been reported to have silencing suppressor, cell-to-cell movement, and helicase functions. Yeast two hybrid screening using an Arabidopsis thaliana cDNA library with TGB1 as bait, and co-purification with TGB1 inclusion bodies identified several host proteins which interact with AltMV TGB1. Host protein interactions with TGB1 were confirmed by biomolecular fluorescence complementation, which showed positive TGB1 interaction with mitochondrial ATP synthase delta' chain subunit (ATP synthase delta'), light harvesting chlorophyll-protein complex I subunit A4 (LHCA4), chlorophyll a/b binding protein 1 (LHB1B2), chloroplast-localized IscA-like protein (ATCPISCA), and chloroplast ${\beta}$-ATPase. However, chloroplast ${\beta}$-ATPase interacts only with $TGB1_{L88}$, and not with weak silencing suppressor $TGB1_{L88}$. This selective interaction indicates that chloroplast ${\beta}$-ATPase is not required for AltMV movement and replication; however, TRV silencing of chloroplast ${\beta}$-ATPase in Nicotiana benthamiana induced severe tissue necrosis when plants were infected by AltMV $TGB1_{L88}$ but not AltMV $TGB1_{L88}$, suggesting that ${\beta}$-ATPase selectively responded to $TGB1_{L88}$ to induce defense responses.

Na+/K+-ATPase Alpha Subunit in the Monogonont Rotifer, Brachionus koreanus: Molecular Cloning and Response to Different Salinity

  • Kim, Hokyun;Lim, Bora;Kim, Byung-Do;Lee, Young-Mi
    • Korean Journal of Environmental Biology
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    • v.34 no.2
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    • pp.97-106
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    • 2016
  • $Na^+/K^+$-ATPase is a membrane protein and plays a key role in osmotic regulation in living organisms. In the present study, a cDNA sequence encoding the $Na^+/K^+$-ATPase alpha subunit from the monogonont rotifer, Brachionus koreanus was cloned by rapid amplification of cDNA ends technique. To investigate the role of this enzyme in osmotic stress, enzymatic activities of $Na^+/K^+$-ATPase were measured after exposure to different salinities for 48 h. The full-length Bk $Na^+/K^+$-ATPase cDNA was 3069 bp-long, encoding a 1022-amino acid polypeptide. Bk $Na^+/K^+$-ATPase possesses eight membrane spanning regions and five conserved domains. Phylogenetic analysis showed that Bk $Na^+/K^+$-ATPase had high identity with those of other species, and was closely clustered with other Brachionus sp. These findings indicate that this protein was conserved both structurally and functionally. B. koreanus $Na^+/K^+$-ATPase activity was stimulated in both hyposaline (6 psu) and hypersaline (32 psu) conditions, suggesting that this protein may play a role in osmoregulation. This study would provide better understanding of the physiology of B. koreanus and this enzyme may be useful as a molecular marker for evaluation of osmotic stress in aquatic environment.

Phosphorylation, 14-3-3 protein and photoreceptor in blue light response of stomatal guard cells

  • Toshinori Kinoshita;Takashi Emi;Michio Doi;Shimazaki, Ken-ichiro
    • Journal of Photoscience
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    • v.9 no.2
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    • pp.335-337
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    • 2002
  • Blue light (BL) induces stomatal opening through activation of H$^{+}$ pump, which creates electrical gradient across the plasma membrane for $K^{+}$ uptake into guard cells. The pump is the plasma membrane H$^{+}$ -ATPase and is activated via phosphorylation of the C-terminus with concomitant binding of the 14-3-3 protein. The opening is initiated by the perception of BL through phototropin (phot), which are recently identified as BL receptors in stomatal guard cells. In this study, we provide the biochemical evidence for phots as BL receptors in stomatal guard cells. vfphot was phosphorylated reversibly by BL, and phosphorylation levels of vfphot increased earlier than those of the plasma membrane W-ATPase. BL-dependent phosphorylations of vfphot and H$^{+}$-ATPase showed similar fluence dependency. Staurosporin, an inhibitor of serine/threonine protein kinase, and diphenyleneiodonium chloride (DPI), an inhibitor of flavoprotein, inhibited BL-dependent phosphorylations of vfphot and H$^{+}$ -ATPase. These results indicate that vfphot acts as a BL-receptor mediating stomatal opening.l opening.

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