• Preventive Nutrition and Food Science
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• 제7권4호
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• pp.432-436
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• 2002

A proteomic map of Hanwoo loin was obtained using 2-D SDS-PAGE and mass spectrometric analysis: 27 bovine proteins plus 2 proteins having similarities to other mammal proteins out of 52 proteins analyzed. The identified proteins consisted of 50 % basic house keeping proteins involved in metabolism, 30% muscle proteins, and other miscellaneous proteins. Many proteins on the 2-D gel with different molecular weights and isoelectric points were identified as same proteins due to posttranslational modification. As many of the identified house keeping proteins showed the high sequence similarities to other mammal equivalent proteins, searching the mammal databases could confirm the annotation. The preliminary identification of the proteome in bovine loin tissue could reveal the functions of proteins at over 50 % of chance with high fidelities. Using the established loin proteome map, proteomic difference between 1 yr and 2 yr Hanwoo loin tissues were compared on 2D gel. Regardless of the difficulty normalizing protein concentrations and sample-to-sample variations, three unidentified proteins and myoglobin were selected as up-regulated proteins during the fat deposition period. This study contributes to a move thorough and holistic understanding of beef meat, helping to build the basis for future identification of new markers for good quality meat.

• 생명과학회지
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• 제8권6호
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• pp.695-701
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• 1998

한국산 꽃뱀(Natrix tigrina Lateralis)의 간 마이크로좀에 존재하는 mixed function oxidase system 구성 성분들의 함량과 P45O 의존성 monooxygenase의 활성도를 조사하고 이들을 흰쥐(Sp. D)의 것과 상호 비교하였다. 꽃뱀에서의 P45O, b5 함량 및 NADPH-cytochrome c reductase 활성도는 흰쥐에서 보다 낮았으며, 7-ethoxycoumarin O-deethylase와 benzphetamine N-demethylase 활성도 역시 흰쥐에서 보다 상당히 낮았다. 그러나 aryl hydrocarbon hydroxylase와 testosterone hydroxylase 활성도는 흰쥐와 비교할 때 거의 비슷하거나 오히려 높았다. Testosterone의 수산화 반응에 대한 선택특이성을 조사한 결과, 꽃 뱀은 7$\alpha$ 위치에서, 흰쥐는 6$\beta$ 위치에서 가장 높은 수산화 반응물을 생성했다. 그러나 testosterone의 C2와 C3 위치에서의 수산화 반응에 대한 선택특이성은 꽃뱀과 흰쥐에서 비슷하였다. Radioimmunoassay (RlA)를 실시하여 5종 (CYP2B, CYP1A1, CYP1A2, CYP3A 및 CYP2El)의 P45O 동위효소의 구성비를 비교한 결과, 꽃뱀에서는 CYP1A1/1A2가, 흰쥐에서는 CYP2El이 각각 비교적 많이 존재하였다. 부분정제한 P45O을 SDS-PAGE와 RIA로 분석한 결과, 꽃뱀의 간 마이크로좀에 존재하는 P45O중에는 흰쥐와는 다른 종류의 P45O 동위효소가 존재함을 시사하였다.

• Journal of Microbiology and Biotechnology
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• 제22권4호
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• pp.535-540
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• 2012

${\beta}$-D-Xylosidase (E.C. 3.2.1.37) from Bifidobacterium breve K-110, which hydrolyzes ginsenoside Ra1 to ginsenoside Rb2, was cloned and expressed in Escherichia coli. The ($His_6$)-tagged recombinant enzyme, designated as XlyBK-110, was efficiently purified using $Ni^{2+}$-affinity chromatography (109.9-fold, 84% yield). The molecular mass of XylBK-100 was found to be 55.7 kDa by SDS-PAGE. Its sequence revealed a 1,347 bp open reading frame (ORF) encoding a protein containing 448 amino acids, which showed 82% identity (DNA) to the previously reported glycosyl hydrolase family 30 of Bifidobacterium adolescentis ATCC 15703. The $K_m$ and $V_{max}$ values toward p-nitrophenyl-${\beta}$-D-xylopyranoside (pNPX) were 1.45mM and 10.75 ${\mu}mol/min/mg$, respectively. This enzyme had pH and temperature optima at 6.0 and $45^{\circ}C$, respectively. XylBK-110 acted to the greatest extent on xyloglucosyl kakkalide, followed by pNPX and ginsenoside Ra1, but did not act on p-nitrophenyl-${\alpha}$-L-arabinofuranoside, p-nitrophenyl-${\beta}$-D-glucopyranoside, or p-nitrophenyl-${\beta}$-D-fucopyranoside. In conclusion, this is the first report on the cloning and expression of ${\beta}$-D-xylosidase-hydrolyzing ginsenoside Ra1 and kakkalide from human intestinal microflora.

• 자연과학논문집
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• 제8권2호
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• pp.57-61
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• 1996

Cellulomonas fimi에서 유래한 $\beta$-glucosidase 유전자를 갖고 있는 대장균으로부터 $\beta$-glucosidase 효소를 정제하였다. 전기 영동과 크로마토그라피 실험을 수행함으로써 정제된 효소의 분자량은 56,000 달톤이며 단일 폴리펩티드로 구성되어 있음을 알 수 있었다. 정제된 $\beta$-glucosidase 효소는 당이 $\beta$-결합을 하고 있는 cellobiose, PNPG, PNPC 등의 기질에 대하여 작용하여 분해시킬 수 있었으나, $\alpha$-결합을 갖고있는 maltose는 분해할 수 없었으므로, $\beta$-결합에 대한 기질 특이성을 갖고 있음을 알았다. 철, 수은, 구리 등의 중금속 이온들에 의해 효소 활성이 저해되었고 DTT에 의해 효소의 활성이 활성화됨을 보임으로써 $\beta$-glucosidase 효소의 활성화 부위는 -SH 기가 중요하게 작용하고 있음을 시사하였다.

• 한국식품과학회지
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• 제31권3호
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• pp.586-592
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• 1999

그루, 은파 및 탑동 등의 한국산 소맥분의 단백질 함량은 수입 소맥과 비교하여 상당히 많은 양 함유되어 있으나 탑동을 제외한 한국밀의 반죽 물성은 매우 약하여 단백질의 함량이 많음에도 불구하고 연질맥과 같은 반죽물성을 나타내었고, 특히 그루는 가장 많은 양의 단백질을 함유하지만 전형적인 sticky dough의 문제점을 나타내었다. 수입산과 국내산의 분류에 관계없이 단백질 함량이 많은 그루, 은파, 및 탑동 등의 한국 밀들은 DNS와 같이 acid insoluble protein의 함량이 상대적으로 많으며 water soluble과 salt soluble 단백질의 함량은 상대적으로 적었다. 특히 수입 DNS 밀은 전체 단백질의 함량에 대한 40% 이상이 acid insoluble protein이며 SDS insoluble protein 함량 또한 가장 많은 양 함유되어 있었다. 반면 한국산 소맥은 대부분 연질맥인 WW와 같이 SDS insoluble protein의 함량이 상대적으로 적었다. 또한 품종이나 제품성에 관계없이 밀가루의 단백질 함량이 높을 수록 단백질의 S-S와 SH 함량이 증가하였으며, 이러한 결과는 외국문헌에서 알려진 밀가루의 특성과 특별한 차이가 없었다. 그러나 탑동밀을 제외한 4종의 한국 밀에서 gluten의 특징적인 116 kD 이상의 high molecular weight protein subunit이 관찰되었으며, 이러한 subunit은 high molecular weight glutenin subunit에 해당되는 것으로 외국문헌의 경우 subunit 2.2로 분류하기도 한다. 또한 116 kD영역에 나타나는 triplet band는 탑동을 제외한 4 종의 우리밀은 doublet로 나타나므로 단백질의 high molecular weight subunit region에서 경질맥인 탑동밀의 구조가 가장 수입밀과 일치하는 것을 관찰할 수 있었다.

• BMB Reports
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• 제38권5호
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• pp.526-532
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• 2005

A lectin with in-vitro anticancer activity against established human cancer cell lines has been purified by affinity chromatography on asialofetuin-linked amino activated silica beads from the tubers of Arisaema tortuosum, popularly known as Himalayan Cobra lily, a monocot plant from the family Araceae. The bound Arisaema tortuosum lectin (ATL) was eluted with glycine-HCl buffer, pH 2.5. ATL was effectively inhibited by asialofetuin, a complex desialylated serum glycoprotein as well as by N-acetyl-D-lactosamine, a disaccharide. It gave a single band corresponding to a subunit molecular weight of 13.5 kDa in SDS-PAGE, pH 8.8 both under reducing and non reducing conditions. When subjected to gel-filtration on Biogel P-200, it was found to have a molecular weight of 54 kDa, suggesting a homotetramer structure, in which individual polypeptides are not bound to each other with disulfide bonds. ATL is a glycoprotein with 0.9% carbohydrate content, stable up to $55^{\circ}C$ and at pH 2 to 10. The lectin had no requirement for divalent metal ions i.e. $Ca^{2+}$ and $Mn^{2+}$ for its activity. However, as reported for other monocot lectins, ATL gave multiple bands in isoelectric focusing and Native PAGE, pH 8.3. The lectin was found to inhibit in vitro proliferation of human cancer cell lines HT29, SiHa and OVCAR-5.

• Journal of Microbiology and Biotechnology
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• 제10권3호
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• pp.307-311
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• 2000

Abstract Extracellular chitinase was purified from the culture liquid of the marine bacterium, Bacillus sp. LJ-25 , and its enzymatic properties were examined. The purified chitinase exhibited a single band on SDS-PAGE and the molecular weight was estimated to be approximately 50 kDa. The optimum pH and temperature for the enzymatic activity were 7.0 and $35^{\circ}C$, respectively. The activity of the chitinase was strongly inhibited by $Zn^{2+}$ and slightly inhibited by $Ba^{2+},{\;}Co^{2+},{\;}Mn^{2+},{\;}and{\;}Cu^{2+}$. The purified chitinase did not hydrolyze $p-nitrophenolN-acetyl-{\bata}-D-glucosaminide{\;}(GlcNAc)_2$ and Micrococcus lysodeikticus cells, which are known to be the substrates for exo-type chitinase. Among the hydrolyzates of colloidal chitin, $(GlcNAc)_2$ was in the highest concentration with small amounts of GlcNAc and $(GlcNAc)_3$..

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.53-53
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• 2003

Calumenin was previously identified as a high affinity Ca$\^$2+/ binding protein in mouse cardiac sarcoplasmic reticulum (SR). For the present study, a 48 kDa skeletal homologue of calumenin was identified by sucrose-density gradient of rabbit skeletal SR membranes, concanavalin A treatment, 2D-gel electrophoresis, $\^$45/Ca$\^$2+/ overlay, Stains-all staining, and MALDI-TOF analysis. We attempted to clone the skeletal calumenin by RT-PCR based on mouse cardiac and human calumenin sequences. The deduced amino acid sequence (315 residues) of the skeletal calumenin showed high identity to mouse cardiac calumenin (90％). As seen in the cardiac calumenin, the deduced sequence contains a 19 amino acid N-terminal signal sequence and a HDEF C-terminal sequence, a putative retrieval signal to ER. Also, the skeletal calumenin contains one N-glycosylation site, three PKC phosphorylation sites, eight casein kinase 2 phosphorylation sites, and 6 EF-hand domains. GST-calumenin showed a conformational change and increased mobility in the presence of Ca$\^$2+/ in SDS-PAGE. Three calumenin interacting proteins (ryanodine receptor 1, glycogen phosphorylase, and phosphofructo kinase) were identified by pull-down assay with GST-calumenin and solubilized SR. All the interactions were Ca$\^$2+/dependent. The present results suggest that calumenin plays an important role in Ca$\^$2+/ homeostasis of muscle cells.

• 한국자기공명학회논문지
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• 제16권1호
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• pp.34-45
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• 2012

Melanocortin-4 receptor (MC4R) subtype is associated with obese humans. Especially, in a patient with severe early-onset obesity, novel heterozygous mutation in the MC4R gene was detected, resulting in an exchange of aspartic acid to asparagine in $90^{th}$ amino acid residue located in the predicted second trans-membrane domain (TM2). Mutations in the melanocortin-4 receptor (MC4R) gene are the most frequent monogenic causes of severe obesity which have been described as heterozygous with loss of function. In order to compare structure difference between MC4R wild type (MC4R-TM2-wt) and mutant (MC4R-TM2-D90N), we designed both MC4R-TM2-wt and MC4R-TM2-D90N construct in pET 21b vector. In this study, we optimized high-yield purification procedure for recombinant TM2-D90N. Eluted recombinant protein was resolubilized under urea condition for thrombin cleavage reaction and we conducted the high-performance liquid chromatography (HPLC) with reverse phase column under 1% acetonitrile, 0.01% TFA buffer solution. The molecular size of purified target peptide was confirmed by Tricine-SDS page analysis. To characterize MC4R-TM2-D90N, we have performed $^{15}N$-isotope labeling of peptide using M9 media and purified labeled target peptide for hetero-nuclear NMR spectroscopy.

• 한국패류학회지
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• 제12권2호
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• pp.109-121
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• 1996

Histochemical experiment was carry out respectively to confirm the properties of the salis (Achatina fulica and Incilaria fruhstorferi). SDS-PAGE was carried out to compare and invertigate the distribution aspects of protein patterns between the two species. Five types(A, B, F, H and I)of gland cells with four neutral mucopolysaccharide cells and one acid mucopolysaccharide cells and one acid mucopolysaccharide cell were observed in acinous of Achatina fulica, while six types were observed in acinous of Incilaria fruhstorferi: ond acid mucopolysaccharide cell(type-A) and four neutral mucopolysaccharide cells(type-B, C, D and F) and one cell that acid mucopolysaccharide is only mimbrane that surrounded granule(type-E). The results are follows:The thpe-A fland cell is commonly observed between the two species. The type-A gland cell in Achatina fulica possesses a nucleus with a developed heterdchromatin, and the cytoplasm was filled with round granules. The granules were surrounded with an uncertain boundary mimbrane and confirmed with neutral mucopolysaccharides, but is confirmed acid mucopolysaccharide in Incilaria fruhstorferi.The type-B gland cell is obwerved in the two species, too. The type-B gland cell in Achatina fulica was round shaped, and included an evenly alrge nucleus. The uncleoplasm included granules that were confirmed in the neutral mucopolysaccharides of the two species. The type-C and D gland cells exist only in Incilaria fruhstorferi, nucleoplasm was well developed heterochromatins. The type-E gland cell appears in the acinous surrounded the salivary gland of Incilaria fruhstorferi. Thdse granules appear irregular irregular shape and size and the cytoplasm is formed in alveolar. The type-F gland cells are commonly observed in the salivary glands of the two species. They are similar with the type-B gland cell, but the granular shape is comparatively small and irregular, and possess the neutral mucos granules. The type-H gland cells are mainly seen in only Achatina, and in nucleus is a well developed heterochromatin. The cytoplasm is filled with round small granules with acid mucopolysaccharide for alcianophilia observed. The type-I cell was small cell with an irregular shape and only observed in the gland cells of Achatina fulica. The heterochromatins were developed in the nucleus and the granules are not observed in cytoplasm.Secretory ducts of saliva are composed of the interlobular duct and interlobar secretory duct. In Achatina fulica the interlobular duct consists of a simple cuboidal epithelium, while the endothelium of intralobar secretory duct of Incilaria fruhstorferi consists of a simple squamous epithelium and in the cytoplasm is filled with granules(type-G secretory cell). A SDS-PAGE was carried out to confirm that the protein band pattern consist of salivary gland. In conclusions, five more bands in Achatina fulica and three bands in Incilaria fruhstorferi were confirmed in MW<29 kDa. one main band coincides comparatively with both and is between 29-45 kDa. There are four main bands in Achatina fulica and two main bands in Incilaria fruhstorferi between 45-66.5 kDa respectively. The bands in Achatina fulica seem more complex than in incilaria fruhstorferi.