• 대한치과교정학회지
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• 제9권1호
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• pp.9-14
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• 1979

The purpose of this study was to pursue the biosynthesis of proteins of human and bovine periodontal ligaments in vitro system. The excised periodontal ligaments from human and bovine were incubated in Krebs-glucose medium containing $^3H$-proline. After incubation the incubated periodontal ligaments were homogenized and the proteins were treated with 0.1%sodium dodecyl sulfate and $\beta$-mercaptoethanol. Separation of the protein fractions was performed with agarose gel column chromatography and SDS acrylamide gel electrophoresis. The results indicated as follow: 1. Only a small percentage of $^3H$-proline incorporated into proteins was hydroxylated to $^3H$-hydroxyproline. 2. The labeled proteins in periodontal ligaments showed a wide distribution of molecular weight. But only small amounts of labeled protein were found that were characteristics of the molecular weight of collagen. 3. In all of the combined fractions of gel filtration, the degree of hydroxylation was small.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권1호
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• pp.61-64
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• 2000

A series of 5-oxaproline peptide derivatives was synthesized and evaluated for its ability to inhibit the prolyl 4-hydroxylase in vitro. Structure-activity studies show that the 5-oxaproline sequences, prepared by the 1,3-dipolar cycloaddition of the C-methoxycarbonyl-N-mannosyl nitrone in the presence of the ethylene, are more active than the corresponding proline derivatives. Prolyl 4-hydroxylase belongs to a family of $Fe^{2+}-dependent$ dioxygenase, which catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in -Gly-Xaa-Pro-Gly- of procollagen chains. In this paper we discover the more selective N-Cbz-Gly-Phe-Pro-Gly-OEt $(K_m\;=\;520\;{\mu}M)$ sequences which are showed stronger binding than others in vitro. Therefore, we set out to investigate constrained tetrapeptide that was designed to mimic the proline structure of pep tides for the development of prolyl 4-hydroxylase inhibitor. From this result, we found that the most potent inhibitor is N-Dansyl-Gly-Phe-5-oxaPro-Gly-OEt $(K_i\;=\;1.6\;{\mu}M)$. This has prompted attempts to develop drugs which inhibit collagen synthesis. Prolyl 4-hydroxylase would seem a particularly suitable target for antifibrotic therapy.

• 한국식품영양과학회지
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• 제21권3호
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• pp.241-246
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• 1992

L-Ascorbic acid (AsA) 생합성이 불가능한 guinea pig을 실험 동물로 하여 collagne 함량이 높은 조직인 폐 및 피부중의 AsA함량과 동일조직중 collagne 함량이 높은 조직인 폐 및 피부중의 AsA 함량과 동일조직중 collagen의 proline잔기의 수산화율을 조사하여 collagne 생합성에 대한 조직중 AsA의 영향에 대해서 알아보았다. Guinea pig(체중 약 250g)를 AsA 무투여군(A), 투여군(B), 300mg/day 투여군(C)으로 나눠 14일간 사육한 후, 마취하에서 개복하여 복부 대동맥으로부터 채혈함과 동시에 간장과 폐를 적출하였으며, 등부위의 피부를 채취하여 분석용 시료로 하였다. 이들 시료로부터 혈청중 alkaline phosphatase (ALP)활성과 각조직중의 AsA 함량, proline 함량 및 그 수산화율, (1-$^{14}$ C) proline 의 incorporation 양을 측정하였다. 그 결과, AsA 토여군인 B, C군의 경우 순조로운 체중증가와 함께 혈청중 ALP활성도 정상값을 나타냈으며 현저한 ALP활성 저하가 관찰되었다. 한편, AsA 함량이 높을수록 (1-14C) proline 의 incorporation 양이 많고 collagne 중의 hydroxyproline 함량도 증가하는 것으로 나타나, 조직중의 collagen합성량과 AsA함량과의 사이에는 높은 상관관계가 존재함이 확인되었다.

• 한국독성학회:학술대회논문집
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• 한국독성학회 2002년도 Molecular and Cellular Response to Toxic Substances
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• pp.161-161
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• 2002

The abnormal accumulation of collagen is progressive and often results in impairment of liver function, i.e. liver cirrhosis. Collagen synthesis requires several posttranslational events. Prolyl 4-hydroxylase is the key enzyme in collagen synthesis that catalyzes the hydroxylation of peptide-bound proline residues to 4-hydroxyroline.(omitted)

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.1
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• pp.154.1-154.1
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• 2003

Hydroxyproline (HYP) is a post-translational product of proline hydroxylation catalyzed by an enzyme prolyl 4-hydroxylase which plays a crucial role in the synthesis of all collagens, because the 4-hydroxyproline residues are essential for the folding of the newly synthesized collagen polypeptide chains into triple-heical molecules. Considering the role of collagen and its significance in many clinically important diseases such as liver cirrhosis, a great deal of attention has been directed toward the development of an assay at cell-based system. (omitted)

• 한국식품영양과학회지
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• 제28권6호
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• pp.1332-1338
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• 1999

Intermolecular collagen cross links stabilize collagen fibrils and are necessary for normal tensile strength in collagen fibrils. Once the fibrils are aligned, hydroxyllysine, hydroxylysine derived aldehyde modified enzymatically, reacts with hydroxylysine to form the dehydrodihydroxylysinonorleucine (DHLNL), an immature crosslink. Pyridinoline, one of matured cross links is presumably formed nonenzymatically through condensation of DHLNL and hydroxylysine residue. It is widely distributed in hard connective tissues such as cartilage, bone and tendon. L ascorbic acid(AsA) is well known to be required for the enzymatic hydroxylation of proline and lysine in collagen fibrils. The purpose of this study is to clarify the role of AsA on the biosynthesis of DHLNL in vitro. We examined the effect of AsA on the formation of hydroxylysine and DHLNL in collagen. Pyridinoline and DHLNL were measured as a function of time. The contents of DHLNL was increased, reached maximum within 2 hr and was held until 24 hr, then it decreased slowly. On the contrary, pyridinoline increased gradually after 24 hr and continued to increase for 2 weeks. Moreover, the contents of DHLNL remarkably decreased at 60 min after incubation, the contents of DHLNL was decreased by addition of AsA or dehydroascorbic acid(DHA). These results suggest that the supplementation of AsA causes decrease in DHLNL formation and pyridinoline formed by nonenzymatic reaction of DHLNL.

• 한국결정학회:학술대회논문집
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• 한국결정학회 2003년도 춘계학술연구발표회
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• pp.15-15
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• 2003

To discover new drugs more quickly and more efficiently, pharmaceutical companies and biotechnology firms are increasingly turning to the genomics and the structural proteomics technologies. Structural-proteomics can provide a foundation for this through the determination and analysis for protein structure on a genomics scale. Among many structures determined by CGI, we will present with the representative examples drawn from our work on novel structures or complex structures of the disease-related proteins. The alpha subunit of Hypoxia-inducible factor (HIF) is targeted for degradation under normoxic conditions by an ubiquitin-ligase complex that recognizes a hydroxylated proline residue in HIF. Hydroxylation is catalysed by HIF prolyl 4-hydroxylases (HIFPH) which are fe(II) and 2-oxoglutarate (2-OG) dependent oxygenases. Here, we discuss the first crystal structure of the catalytic domain of HIFPH in complexes, with the Fe(II)/2-OG at 1.8Å. These structures suggest that the Ll region (residues 236-253), which is also conserved in mammals, form a 'lid' that closes over the active site. The structural and mutagenesis analyses allow us to provide a focus for understanding cellular responses to hypoxia and a target for the therapeutic manipulation.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.28-28
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• 2003

To discover new drugs more quickly and more efficiently, pharmaceutical companies and biotechnology firms are increasingly turning to the genomics and the structural proteomics technologies. Structural-proteomics can provide a foundation for this through the determination and analysis for protein structure on a genomics scale. Among many structures determined by CGI, we will present with the representative examples drawn from our work on novel structures or complex structures of the disease-related proteins. The alpha subunit of Hypoxia-inducible factor (HIF) is targeted for degradation under normoxic conditions by an ubiquitin-ligase complex that recognizes a hydroxylated proline residue in HIF, Hydroxylation is catalysed by HIF prolyl 4-hydroxylases (HIFPH) which are Fe(II) and 2-oxoglutarate (2-OG) dependent oxygenases. Here, we discuss the first crystal structure of the catalytic domain of HIFPH in complexes, with the Fe(II)/2-OG at 1.8 ${\AA}$. These structures suggest that the L1 region (residues 236-253), which is also conserved in mammals, form a ‘lid’ that closes over the active site. The structural and mutagenesis analyses allow us to provide a focus for understanding cellular responses to hypoxia and a target for the therapeutic manipulation.

• Applied Biological Chemistry
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• 제39권2호
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• pp.134-139
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• 1996

연근해산 수산물중 부산물의 양이 많은 붕장어껍질, 말쥐치껍질 및 화살오징어껍질을 대상으로 수산 연제품의 품질개선제로 사용하기 적절한 수산물껍질 젤라틴의 원료를 검색하였다. 콜라겐함량은 붕장어껍질(24.69%)이 가장 많았고, 다음으로 말쥐치껍질(20.03%)이었으며 화살오징어껍질은 12.62%에 불과하였다. 콜라겐의 조성은 어류껍질의 경우 가용성획분$(67.4%{\sim}72.3%)$이 불용성획분보다 높았으나, 화살오징어껍질의 경우 불용성획분(69.6%)이 가용성획분보다 높았고, 아미노산조성은 가용성획분 및 불용성획분 간에 차이가 없었다. 연근해산 수산물껍질의 콜라겐은 ${\alpha}$ chain과 ${\beta}$ chain으로 구성되어 있었고, ${\alpha}$ chain은 동일종이 아닌 hetero형 이었다. Imino acid 조성비, proline의 수산화정도 및 열변성온도는 붕장어껍질 콜라겐이 가장 높았고, 다음으로 말쥐치껍질 콜라겐 및 화살오징어껍질 콜라겐의 순이었고 또한 이들 껍질로부터 추출한 젤라틴의 물리적 특성도 열변성온도의 경향과 같았다.

• Biomolecules & Therapeutics
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• 제15권4호
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• pp.261-265
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• 2007

Hydroxyproline (HYP) is a post-translational product of proline hydroxylation catalyzed by an enzyme prolyl 4-hydroxylase (P4H) which plays a crucial role in the synthesis of all collagens. Considering the role of collagen and its significance in many clinically important diseases such as liver fibrosis, a great deal of attention has been directed toward the development of an assay at cell-based system. The reason is that cell-based assay system is more efficient than enzyme-based in vitro system and takes much less time than in vivo system. Several assay procedures developed for P4H are laborious, time-consuming and not feasible for the massive-screening. Here, we report the cell-based assay method of prolyl 4-hydroxylase in immortalized rat hepatic stellate HSC-T6 cells. To optimize the cell culture condition to assay for HYP content, various concentrations of reagents were treated for different times in HSC-T6 cells. Our data showed that the treatment with ascorbate in a hypoxic condition for 24 h resulted in the maximal increase of HYP by 1.8 fold. Alternatively, cobalt chloride ($5\;{\mu}M$) and ascorbate ($50\;{\mu}M$) in normoxic states exhibited similar effect on the production of HYP as in a hypoxic condition. Therefore, cobalt chloride can be substituted for a hypoxic condition when an anaerobic chamber is not available. Rosiglitazone and HOE077, known as inhibitors of collagen, synthesis decreased P4H enzyme activity by 32.3% and 15%, respectively, which coincided with previous reports from liver tissues. The level of the smooth muscle ${\alpha}$-actin, a marker of activated stellate cells, was significantly increased under hypoxia, suggesting that our experimental condition could work for screening the anti-fibrotic compounds. The assay procedure took only 3 days after treatment with agents, while assays from the primary stellate cells or liver tissues have taken several weeks. Considering the time and expenses, this assay method could be useful to screen the compounds for the inhibitor of prolyl 4-hydroxylase.