• 한국가축번식학회지
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• 제26권4호
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• pp.395-399
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• 2002

Human chorionic gonadotropin (hCG) is a member of the glycoprotein hormone family which includes FSH. hCG TSH. These hormone family is characterized by a heterodimeric structure composed a common $\alpha$-subunit noncovalently linked to a hormone specific $\beta$-subunit. To determine u and $\beta$ -subunits can be synthesized as a single polypeptide chain (tethered-hCG) and also display biological activity, the tethered-hCC and -FSH molecule by fusing the carboxyl terminus of the hCG $\beta$-subunit to the amino terminus of the $\alpha$-subunit was constructed. To determine the importance of $\alpha$ COOH -terminal amino acid, we also deleted the $\alpha$ COOH-terminal amino acids. The expressing vectors were transfected into CHO-K 1 cells. The tethered-wthCG and -wtFSH was efficiently secreted. The $\alpha$ Δ83hCG and $\alpha$ Δ 83FSH mutants had no secretion. These results are the first conclusive evidence that COOH-terminal amino acids are very important for secretion in human glycoprotein hormone $\alpha$-subunit. These results demonstrated that the $\alpha$ Δ83hCG and $\alpha$ Δ 83FSH mutants could be play a pivotal role in the secretion of tethered-molecule.

• Journal of Applied Biological Chemistry
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• 제49권3호
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• pp.110-113
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• 2006

Prolyl endopeptidase (PEP, EC 3.4.21.26, also referred to as prolyl oligopeptidase) degrades proline containing, biologically active neuropeptides such as vasopressin, substance P and thyrotropin-releasing hormone by cleaving peptide bonds on carboxyl side of prolyl residue within neuropeptides of less than 30 amino acids. Evaluation of PEP levels in postmortem brains of Alzheimer's disease patients revealed significant increases in PEP activity. Therefore, a specific PEP inhibitor can be a good candidate of drug against memory loss. Upon our examination for PEP inhibitory activity from micronutrients, ascorbic acid (vitamin C) showed small but significant PEP inhibition (13% PEP inhibition at $8{\mu}g{\cdot}ml^{-1}$). Palmitic acid showed almost no PEP inhibition. However, 6-O-palmitoyl ascorbic acid ($\underline{1}$) showed 70% PEP inhibition at $8{\mu}g{\cdot}ml^{-1}$ indicating that hydrophobic portion of the compound $\underline{1}$ may facilitate the inhibitory effect. $IC_{50}$ value of compound $\underline{1}$ was $12.6{\pm}0.2{\mu}M$. The primary and secondary Lineweaver Burk and Dixon plots for compound $\underline{1}$ indicated that it is a non-competitive inhibitor with inhibition constant (Ki) value of $23.7{\mu}M$.

• Journal of Applied Biological Chemistry
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• 제48권4호
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• pp.207-212
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• 2005

Prolyl endopeptidase (PEP, EC 3.4.21.26), also referred to as prolyl oligopeptidase, has been suggested to participate in learning and memory processes by cleaving peptide bonds on carboxyl side of prolyl residue within neuropeptides of less than 30 amino acids, and is abundant in brains of amnestic patients. Therefore, compounds possessing PEP inhibitory activity can be good candidate of drug against memory loss. Upon examination for PEP inhibition from traditional medicinal plants having tonic, stimulating, and anti-amnestic effects, Corni Fructus (Cornus officinallis) showed significant PEP inhibition. Ursolic and oleanolic acids, components of Corni Fructus, inhibited PEP with $IC_{50}$ values of $17.2\;{\pm}\;0.5$ and $22.5\;{\pm}\;0.7\;{\mu}M$, respectively.

• Journal of Microbiology and Biotechnology
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• 제19권11호
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• pp.1306-1318
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• 2009

The filamentous ascomycete Sclerotinia sclerotiorum is well known for its ability to produce a large variety of hydrolytic enzymes. Two $\alpha$-amylases ScAmy54 and ScAmy43 predicted to play an important role in starch degradation were showed to produce specific oligosaccharides essentially maltotriose that have a considerable commercial interest. Primary structure of the two enzymes was established by N-terminal sequencing, MALDI-TOF masse spectrometry and cDNA cloning. The two proteins have the same N-terminal catalytic domain and ScAmy43 derived from ScAmy54 by truncation of 96 amino acids at the carboxyl-terminal region. Data of genomic analysis suggested that the two enzymes originated from the same $\alpha$-amylase gene and that truncation of ScAmy54 to ScAmy43 occurred probably during S. sclerotiorum cultivation. The structural gene of Scamy54 consisted of 9 exons and 8 introns, containing a single 1,500-bp open reading frame encoding 499 amino acids including a signal peptide of 21 residues. ScAmy54 exhibited high amino acid homology with other liquefying fungal $\alpha$-amylases essentially in the four conserved regions and in the putative catalytic triad. A 3D structure model of ScAmy54 and ScAmy43 was built using the 3-D structure of 2guy from A. niger as template. ScAmy54 is composed by three domains A, B, and C, including the well-known $(\beta/\alpha)_8$ barrel motif in domain A, have a typical structure of $\alpha$-amylase family, whereas ScAmy43 contained only tow domains A and B is the first fungal $\alpha$-amylase described until now with the smallest catalytic domain.

• 생명과학회지
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• 제6권3호
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• pp.185-192
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• 1996

박테리오파지 T7 gene 2.5 단백질은 single-stranded DNA 결합 단백질로 박태리오파지 T7의 DNA복제, 재조합, 및 수선에 필수적으로 요구된다. Gene 2.5 protein은 T7의 DNA 합성과 성장에 필수적인 단백질이다. Gene 2.5 Protein이 중요시 되는 이유는 이 단백질이 T7의 다른 복제 필수단백질인 T7의 다른 복제 필수단백질인 T7 DNA polymerase 와 gene 4 protein(helicase/primase)와 서로 상호작용할 것으로 제안되었기 때문이다. (Kim and Richardson, J. Biol. Chem., 1992;1994). 이 단백질의 단백질 상호작용을 가능하게 하는 domain은 carboxyl-terminal domain일 것으로 여러 실험에서 대두되었기에, 이 domain의 특성을 파악하기 위해 야생형과 변이체 gene 2.5 단백질들을 각각 GST에 융합한후 fusion 단백질을 정제하였다. 정제된 이 융합 단백질들의 carboxyl-terminal domain이 T7 복제 단백질들과 상호작용을 조사하는지를 조사하기 위해 affinity chromatography로 이용하였다. 실험 결과, 아생형 GST-gene 2.5 융합단잭질(GST-2.5 (WT))는 T7 DNA polymerase 와 상호작용을 하였지만. 변이형 융합단백질(GST-2.5$\Delta$21C)는 interaction을 하지 못했다. 이 결과는 carbohyl-terminal domain이 단백질-단백질 상호작용을 하는데 직접적으로 관여하는 것을 증명하였다. 또한,GST2.5(WT)는 gene 4 protein(helicase/primase)와 직접 상호작용을 하나. GST2.5$\Delta$21C는 상호작용을 하지 못하는 것으로 나타났다. 따라서 gene 4 proteins와의 상호작용에도 gene 2.5 protein의 carboxyl-terminal domain이 직접 관여 한다는 것이 증명되었다. 이상의 결과에서 gene 2.5 protein은 박테리오파지 T7 의 유전자 목제 시 단백질-단백질 상호작용에 관혀아며, 특히 gene 2.5 protein의 carboxyl-terminal domain이 이러한 상호작용에 직접적으로 관여하는 domain이라는 것을 알 수가 있었다.

• Archives of Pharmacal Research
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• 제29권5호
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• pp.384-393
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• 2006

Protein carboxylmethylation methylates the free carboxyl groups in various substrate proteins by protein carboxyl O-methyltransferase (PCMT) and is one of the post-translational modifications. There have been many studies on protein carboxylmethylation. However, the precise functional role in mammalian systems is unclear. In this study, immunoglobulin, a specific form of $\gamma-globulin$, which is a well-known substrate for PCMT, was chosen to investigate the regulatory roles of protein carboxylmethylation in the immune system. It was found that the anti-BSA antibody could be carboxylmethylated via spleen PCMT to a level similar to $\gamma-globulin$. This carboxylmethylation increased the hydrophobicity of the anti-BSA antibody up to 11.4%, and enhanced the antigen-binding activity of this antibody up to 24.6%. In particular, the Fc region showed a higher methyl accepting capacity with 80% of the whole structure level. According to the amino acid sequence alignment, indeed, 7 aspartic acids and 5 glutamic acids, as potential carboxylmethylation sites, were found to be conserved in the Fc portion in the human, mouse and rabbit. The carboxylmethylation of the anti-BSA antibody was reversibly demethylated under a higher pH and long incubation time. Therefore, these results suggest that protein carboxylmethylation may reversibly regulate the antibody-mediated immunological events via the Fc region.

• 농업과학연구
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• 제20권2호
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• pp.189-200
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• 1993

Ornithine Decarboxylase는 동물세포에 있어서 polyamines을 생합성하는 반응의 율속 단계에 있는 효소이다. 세포의 성장기간 동안에 이 효소는 mRNA와 단백질 수준에서 급격한 변화를 일으키며 조절된다. 이와 같은 polyamines농도의 급변화를 분자수준에서 탐구하기 위하여 ODC 특이 cDNA colne이 cDNA libray에서 분리되었다. cDNA의 clone이 이루어진 부분은 open reading frame과 3'-noncording region, 그리고 poly A의 tail이 있는 부분으로서 각각 456, 348, 14개의 nucleotides로서 구성되어 있었다. 인간, mouse, rat, hamster로부터 같은 지역내 추론된 C-말단으로부터의 아미노산의 비교는 단백질 상동성에 88%이상을 보였다. 이와같은 아미노산 염기순서에 있어서의 잘 보존된 특징은 ODC가 세포성정과 분화에 있어서 중요한 역할이 있음을 잘 나타내고 있다.

• Journal of Microbiology
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• 제38권4호
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• pp.275-280
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• 2000

Pseudomonas sp. S-47 is capable of catabolizing 4-chlorobenzoate (4CBA) as rarbon and energy sources under aerobic conditions via the mesa-cleavage pathway. 4CBA-dioxygenase and 4CBA-dihydrodiol dehydrogenase (4CBA-DD) catalyzed the degradation af 4CBA to produce 4-chlorocatechol in the pathway. In this study, the xylL gene encoding 4CBA-DD was cloned from the chromosomal DNA of Pseudomonas sp. S-47 and its nucleotide sequence was analyzed. The xylL gene was found to be composed of 777 nucleotide pairs and to encode a polypeptide of 28 kDa with 258 amino acid residues. The deduced amino acid sequence of the dehydrogenase (XylL) from strain S-47 exhibited 98% and 60% homologies with these of the corresponding enzymes, Pseudomonas putida mt-2 (XyIL) and Acinetobacter calcoaceticus (BenD), respectively. However, the amino arid sequences show 30% or less homology with those of Pseudomonas putida (BnzE), Pseudomonas putida Fl (TodD), Pseudomonas pseudoalcaligenes KF707 (BphB), and Pseudomonas sp. C18 (NahB). Therefore, the 4CBA-dihydrodiol dehdrogenase of strain S-47 belongs to the group I dehydrogenase involved in the degradation of mono-aryls with a carboxyl group.

• 한국양식학회지
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• 제12권2호
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• pp.91-100
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• 1999

The complete nucleotide sequence of olive flounder (Paralichthys olivaceus) hsp70-related rDNA was determined by RT- and RACE-PCR methods. A full-length of hsp70-related cDNA has an open reading frame of 1.95 kb encoding 650 amino acids with a calculated molecular weight of 71.1 kD. A corresponding hsp70-related protein contains a number of conserved elements including an ATP-binding domain, a nuclear localization signal and the carboxyl terminal motif, EEVD, which may have a role in chaperone function. Comparison of nucleotide and predicted amino acid sequence between olive flounder hsp70-related gene and hsp/hsc70 genes of other species revealed a high similarity with the cognate form of these genes. These results indicated that we recovered likely to be a olive flounder cognate hsc70 gene.

• Journal of Microbiology and Biotechnology
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• 제23권2호
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• pp.218-224
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• 2013

The aldo-keto reductases catalyze reduction reactions using various aliphatic and aromatic aldehydes/ketones. Most reductases require NADPH exclusively as their cofactors. However, NADPH is much more expensive and unstable than NADH. In this study, we attempted to change the five amino acid residues that interact with the 2'-phosphate group of the adenosine ribose of NADPH. These residues were selected based on a docking model of the YOL151W reductase and were substituted with other amino acids to develop NADH-utilizing enzymes. Ten mutants were constructed by site-directed mutagenesis and expressed in Escherichia coli. Among them, four mutants showed higher reductase activities than wild-type when using the NADH cofactor. Analysis of the kinetic parameters for the wild type and mutants indicated that the $k_{cat}/K_{m}$ value of the Asn9Glu mutant toward NADH increased 3-fold. A docking model was used to show that the carboxyl group of Glu 9 of the mutant formed an additional hydrogen bond with the 2'-hydroxyl group of adenosine ribose. The Asn9Glu mutant was able to produce (R)-ethyl-4-chloro-3-hydroxyl butanoate rapidly when using the NADH regeneration system.