• BMB Reports
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• v.34 no.6
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• pp.537-543
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• 2001

In the present study, an in vitro ELISA system to assess the interaction between Src homology (SH)2 domains and phosphotyrosine that contain peptides was established using purified GST-conjugated SH2 proteins and synthetic biotinylated phosphotyrosine that contain oligopeptides. The SH2 domains bound the relevant phosphopeptides that were immobilized in the streptavidin-coated microtiter plate in a highly specific and dose-dependent manner. The epidermal growth factor receptor (EGFR)-, T antigen (T Ag)-, and platelet-derived growth factor receptor (PDGFR)-derived phosphopeptides interacted with the growth factor receptor binding protein (Grb)2/SH2, Lck/SH2, and phosphatidyl inositol 3-kinase (PI3K) p85/SH2, respectively. No cross-reactions were observed. Competitive inhibition experiments showed that a short phosphopeptide of only four amino acids was long enough to determine the binding specificity. Optimal concentrations of the GST-SH2 fusion protein and phosphopeptide in this new ELISA system for screening the binding blockers were chosen at 2nM and 500nM, respectively. When two candidate compounds were tested in our ELISA system, they specifically inhibited the Lck/SH2 and/or p85/SH2 binding to the relevant phosphopeptides. Our results indicate that this ELISA system could be used as an easy screening method for the discovery of specific binding blockers of protein-protein interactions via SH2 domains.

• Korean Journal of Food Science and Technology
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• v.21 no.2
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• pp.242-251
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• 1989

The salted-dried mullet(Mugil japonicus) roe is a kind of traditional food particulary in the area of Young-am gun, Chunnam province. This study was conducted to conform the scientific processing conditions and to evaluate the nutritional quality and changes of major components during storage times. The manufacturing method was that the fresh roe was salted for about 20 hours for the preparation of salted-dried roe, washed by clean waters, drained, shaped a flat piece with 1.2cm thickness by pressing, and spreaded sesame oils on the surface of the salted roe periodically during wind drying for 20 days. The dried roe was blanched in heated water$(80^{\circ}C/3min)$ and packaged the dried product for storages. The fractional compositions of free lipid of wind dried roe were 40% of neutral lipids, 12% of glycolipids and 9% of phospholipids and those of bound lipids were 13% of neutral lipids. 10% of glycolipids and 13% of phospholipids respectively. The major fatty acids of the roe were $C_{16:0}$, $C_{18:0}$, $C_{18:1}$, $C_{18:2}$ and $C_{20:0}$ which was consisted of free and bound lipids in wind drying method during processing and storages. Total amino acids were 99.87g/100g and major amino acids were Glu, Pro, Leu, Lys and CySH and the protein score was average 155% and the chemical score was average 109%. Free amino acids was 1,376mg% that had 50.61% of Pro and the major kinds of those were Tyr and CySH.

• Journal of Microbiology and Biotechnology
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• v.30 no.5
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• pp.633-641
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• 2020

Microbial rhodopsins are a superfamily of photoactive membrane proteins with the covalently bound retinal cofactor. Isomerization of the retinal chromophore upon absorption of a photon triggers conformational changes of the protein to function as ion pumps or sensors. After the discovery of proteorhodopsin in an uncultivated γ-proteobacterium, light-activated proton pumps have been widely detected among marine bacteria and, together with chlorophyll-based photosynthesis, are considered as an important axis responsible for primary production in the biosphere. Rhodopsins and related proteins show a high level of phylogenetic diversity; we focus on a specific class of bacterial rhodopsins containing the '3 omega motif.' This motif forms a stack of three non-consecutive aromatic amino acids that correlates with the B-C loop orientation and is shared among the phylogenetically close ion pumps such as the NDQ motif-containing sodium-pumping rhodopsin, the NTQ motif-containing chloride-pumping rhodopsin, and some proton-pumping rhodopsins including xanthorhodopsin. Here, we reviewed the recent research progress on these 'omega rhodopsins,' and speculated on their evolutionary origin of functional diversity.

• The Korean Journal of Mycology
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• v.10 no.4
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• pp.147-154
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• 1982

To find antitumor components with low toxicity from natural resources, antitumor test of the water extract of the carpophores of Trametes sanguinea (L. ex Fr.) Lloyd. was undertaken. The carpophores of this fungus were collected in Gyeong Gi Province and extracted with hot water. The extract was purified by dialyzing through Visking tube and a protein-bound polysaccharide fraction was obtained. The fraction was tested for antitumor activity against sarcoma 180 implanted in mice. The tumor inhibition ratio of the fraction against the tumor was 72.4% at the dose of 10mg/kg/day for the period of ten days. The tumor in one of the eight mice was completely regressed. The antitumor components were found to be a polysaccharide and a protein. The hydrolysis of the polysaccharide moiety yielded three monosaccharides, and from the hydrolysate of the protein moiety 13 amino acids were identified.

• The Korean Journal of Mycology
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• v.10 no.4
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• pp.165-171
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• 1982

To investigate antitumor components of Korean higher fungi, the carpophores of Ramaria formosa $(Fr.)Qu\acute{e}l$. were collected in Gang Won Province and extracted with hot water. The extract was concentrated and precipitated by four volumes of ethanol. The precipitate was centrifugated and purified by dialyzing through Visking tube and a protein-bound polysaccharide fraction was obtained. The fraction was tested for antitumor activity against sarcoma 180 implanted in mice. The tumor inhibition ratio of the fraction was 66% in the dose of 50mg/kg/day for the period of ten days. The tumor in two of the eight mice was completely regressed. The components of the fraction were found to be a polysaccharide and a protein. The chemical analysis of the fraction showed that the polysaccharide moiety consisted of four monosaccharides and that the protein moiety contained fourteen amino acids.

• Proceedings of the Ginseng society Conference
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• 1988.08a
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• pp.1-10
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• 1988

Previously. we reported that the intraperitoneal administration of ginseng crude saponin increased: (I) nuclear RNA polymerase activity. (2) nuclear RNA synthesis. (3) cytoplasmic RNA synthesis. (4) cytoplasmic heavy polyrioosome content. (5) amino acid incorporation in vitro of microsome and polysome isolated rat liver. and (6) the incorporation rate of labeled amino acids into serum protein. In addition, a spectacular increase in the rough endoplasmic reticulum of hepatocyte administered crude saponin for four weeks orally was shown through electron microscopy. An increase in polysomal content in membrane-hound ribosome was shown through ultracentrifugation. Recently, successive intraperitoneal. administration .of $ginsenosid-Rb_2$ was given to streptozotocin (STZ) diaoetic rats of hypoproteinemia. The blood urea nitrogen and hepatic urea concentration were decreased significantly. The total protein and alhumin levels in the serum were increased in comparison to control values. In contrast. the $ginsenoside-Rb_2$ treated group of STZ diahetic rats showed a significant increase in liver RNA. total ribosome and membrane-bound ribosomal contents. The administration of $ginsenoside-Rb_2$ increased the incorporation rate of labeled - precursor into total serum protein. Additionally $ginsenoside-Rb_2$ improved the nitrogen balance of diabetic rats. On the bases of these experimental results, ginseng saponin has a metabolic stimulatory or anabolic action on RNA and protein synthesis.

• The Korean Journal of Mycology
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• v.13 no.1
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• pp.11-21
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• 1985

Antitumor components were obtained from the cultured mycelia of Pleurotus pulmonarius by ethanol precipitation. The protein-bound polysaccharide was subjected to DEAE-­Sephadex column chromatography and Sephadex G-200 gel filtration. The antitumor fraction $C_1$ was isolated. The inhibition ratio of fraction $C_1$ was 81.8 % in the doses of 10 mg/kg/day for 10 days. The antitumor fraction $C_1$ consisted of a polysaccharide and a protein. The protein-moiety was composed of 14 amino acids. From the peritoneal cell populations in the mice given antitumor fraction $C_1$, the injection of the fraction caused the influx of peritoneal macrophages at two days when compared with those of soluble starch. This was named pulmonaran after its species name.

• Journal of Microbiology and Biotechnology
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• v.13 no.6
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• pp.897-905
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• 2003

Phospholipase D (PLD) and ADP-ribosylation factor (ARF) were partially purified on a series of column chromatography, and their biochemical properties were characterized to understand the regulatory mechanism of PLD activation by ARF protein in the antigen-induced immune responses in guinea pigs. Heparin Sepharose and high-Q Sepharose column chromatographies were used for the purification of PLD, and Sephadex G-25, DEAE Sephacel, Source 15 PHE (HIC), Superdex-75, and Uno-Q column chromatographies were used for the purification of ARF. The purified PLD and ARF proteins were identified with anti-rabbit PLD- or ARF-specific antibodies, showing about 64 or 85 kDa for the molecular mass of PLD and 29 or 35 kDa for the sizes of ARF. Partial cDNA of ARF3 was cloned by RT-PCR in guinea pig lung tissue and its nucleotides and amino acids were sequenced. Guinea pig ARF3 showed 92% of nucleotides sequence identity and 100% of amino acid sequence homology with human ARF3. The ARF-regulated PLD activity was measured in the oleate or ARFs-containing mixed lipid vesicles. The purified and recombinant ARF (rARF) activities were assessed with the $GTP{\gamma}S$ binding assay. The PLD activity was induced by oleate in a dose-dependent manner. The purified ARF and recombinant ARF3 increased PLD activity in guinea pig lung tissues. These data show that the activity of membrane-bound PLD can be regulated by the cytosolic ARF proteins, suggesting that ARF proteins in guinea pig lung can act as a regulatory factor in controlling the PLD activity in allergic reaction.

• Molecules and Cells
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• v.41 no.9
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• pp.842-852
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• 2018

Notch signaling is an evolutionarily conserved pathway and involves in the regulation of various cellular and developmental processes. Ligand binding releases the intracellular domain of Notch receptor (NICD), which interacts with DNA-bound CSL [CBF1/Su(H)/Lag-1] to activate transcription of target genes. In the absence of NICD binding, CSL down-regulates target gene expression through the recruitment of various corepressor proteins including SMRT/NCoR (silencing mediator of retinoid and thyroid receptors/nuclear receptor corepressor), SHARP (SMRT/HDAC1-associated repressor protein), and KyoT2. Structural and functional studies revealed the molecular basis of these interactions, in which NICD coactivator and corepressor proteins competitively bind to ${\beta}-trefoil$ domain (BTD) of CSL using a conserved ${\varphi}W{\varphi}P$ motif (${\varphi}$ denotes any hydrophobic residues). To date, there are conflicting ideas regarding the molecular mechanism of SMRT-mediated repression of CSL as to whether CSL-SMRT interaction is direct or indirect (via the bridge factor SHARP). To solve this issue, we mapped the CSL-binding region of SMRT and employed a 'one- plus two-hybrid system' to obtain CSL interaction-defective mutants for this region. We identified the CSL-interaction module of SMRT (CIMS; amino acid 1816-1846) as the molecular determinant of its direct interaction with CSL. Notably, CIMS contains a canonical ${\varphi}W{\varphi}P$ sequence (APIWRP, amino acids 1832-1837) and directly interacts with CSL-BTD in a mode similar to other BTD-binding corepressors. Finally, we showed that CSL-interaction motif, rather than SHARP-interaction motif, of SMRT is involved in transcriptional repression of NICD in a cell-based assay. These results strongly suggest that SMRT participates in CSL-mediated repression via direct binding to CSL.

• IMMUNE NETWORK
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• v.2 no.2
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• pp.102-108
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• 2002

Background: This study was aimed to differentiate two forms of CTLA-4 (CD152) in activated peripheral blood lymphocyte and clarify the mechanism how cytoplasmic form of this molecule is targeted to cell surface. Methods: For this purpose we generated 2 different anti-human CD152 peptide antibodies and 5 different N'-terminal deletion mutant CTLA4Ig fusion proteins and carried out a series of Western blot and ELISA analyses. Antipeptide antibodies made in this study were anti-CTLA4pB and anti-CTLA4pN. The former recognized a region on extracellular single V-like domain and the latter recognized N'-terminal sequence of leader domain of human CD152. Results: In Western blot, the former antibody recognized recombinant human CTLA4Ig fusion protein as an antigen. And this recognition was completely blocked by preincubating antipeptide antibody with the peptide used for the antibody generation at the peptide concentration of 200 ug/ml. These antibodies were recognized human CD152 as a cytoplasmic sequestered- and a membrane bound- forms in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocyte (PBL). These two forms of CD152 were further differentiated by using anti-CTLA4pN and anti-CTLA4pB antibodies such that former recognized cytosolic form only while latter recognized both cytoplasmic- and membraneforms of this molecule. Furthermore, in a transfection expression study of 5 different N'-terminal deletion mutant CTLA4Ig, mutated proteins were secreted out from transfected cell surface only when more than 6 amino acids from N'-terminal were deleted. Conclusion: Our results implies that cytosolic form of CTLA-4 has leader sequence while membrane form of this molecule does not. And also suggested is that at least N'-terminal 6 amino acid residues of human CTLA-4 are required for regulation of targeting this molecule from cytosolic- to membrane- area of activated human peripheral blood T lymphocyte.