Molecular Pharmacological Interaction of Phenylbutazone to Human Neutrophil Elastase

Kooil Kang 대한생리학회-대한약리학회 1998 The Korean Journal of Physiology & Pharmacology Vol.2 No.3

<P> Human neutrophil elastase (HNElastase, EC 3.4.21.37), a causative factor of inflammatory diseases, was purified by Ultrogel AcA54 gel filtration and CM-Sephadex ion exchange chromatography. HNElastase was inhibited by phenylbutazone in a concentration dependent manner up to 0.4 mM, but as the concentration increased, the inhibitory effect gradually diminished. Binding of phenylbutazone to the human neutrophil elastase caused strong Raman shifts at 200, 440, and 1194 cm<SUP>⁣1</SUP>. The peak at 1194 cm<SUP>⁣1</SUP> might be evidence of the presence of -N=N-ヶ radical. The core area of the elastase, according to the visual molecular model of human neutrophil elastase, was structurally stable. A deeply situated active center was at the core area surrounded by hydrophobic amino acids. Directly neighboring the active site was one positively charged atom and two atoms carrying a negative charge, which enabled the enzyme and the drug to form a strong interaction. Phenylbutazone may form a binding, similar to a key & lock system to the atoms carrying opposite charges near the active site of the enzyme molecule. Furthermore, the hydrophobicity of the surrounding amino acid near the active site seemed to enhance the binding strength of phenylbutazone. Binding of phenylbutazone near the active site may cause masking of the active site, preventing the substrate from approaching the active site and inhibiting elastase activity.

THE ROLE OF NEUTRAL PROTEINASES IN CELLULAR INVASION

Kang, Kooil 고신대학교 의학부 1986 高神大學校 醫學部 論文集 Vol.2 No.1

Simultaneous Separation of Pure Neutrophils and Lymphocytes from Normal Human Blood by Step Gradient

Kang, Kooil,Jeong, Hye Young,Ghim, Sa-Youl 大韓免疫學會 1987 大韓免疫學會誌 Vol.9 No.1

Neutrophil protease의 생화학적·약리학적 작용을 연구하기 위한 선행단계로서, Hypaque-Ficoll step gradient에 의하여 혈액속의 neutrophil과 lymphocyte의 분리를 시도하였다. 지금까지는 neutrophil이나 lymphocyte 중 한가지만 순수하게 분리하는 방법이 개발되어 있으나, 이 방법을 수행함으로써 고순도의 neutrophil과 lymphocyte를 동시에 얻을 수 있는데, 3-step gradient(비중 1.080~l.133g/ml)에 동일한 양의 신선한 혈액을 사용하여, 실내온도에서 300g로 25분간 원심분리 하는 것이 가장 적절하였다. 위와 같이 최적조건으로 분리하였을시, 비중 1.133g/m3와 1.122g/m1용액 사치에 선명하게 형성되는 3번째 band는 순도 96%이상의 neutrophil을 함유하였으며, 80% 이상의 세포가 회수되었고, 99% 이상의 세포가 살아 있음을 확인하였다. 또한, 그 위에 나타나는 2번째 band인 lymphocyte는 93.7%의 순도를 보여주었다.

Tetracycline계 항균제에 의한 사람 호중구 Elastase의 효소 활성도 억제 및 그 작용 기전

김우미,강구일 고신대학교 의학부 1993 高神大學校 醫學部 論文集 Vol.9 No.1

Human neutrophil elastase, which is known to be a possible mediator in the pathophysiology of connective tissue breakdown, was effectively inhibited by tetracycline, oxytetracycline and demeclocycline. Among them, oxytetracycline showed the most potent inhibitory effect on elastase activity. Tetracycline inhibited human neutrophil elastase non-competitively, however oxytetracycline inhibited human neutrophil elastase competitively. Ki values of tetracycline and oxytetracycline were 4.9mM and 0.39mM, respectively. De-dimethylaminotetracycline, which showed no antibiotic activity since the active dimethylamino radical was removed from the position #4 of the tetracycline, inhibited the activity of human neutrophil elastase by tetracyclines not depend on the dimethylamino radical which is a critical active site for antibiotic effect, rather it depend on the hydoxyl radical of tetracyclines. The property of inhibiting elastase may be an additional molecular biochemical mechanism of action of these drugs as an inhibitor of tissue destruction at the inflammatory sites.

INHIBITION OF HUMAN NEUTROPHIL ELASTASE BY DIFFERENT CLASSES OF ANTIBIOTICS

Ghim, Sa-Youl,Kang, Kooil 고신대학교 의학부 1988 高神大學校 醫學部 論文集 Vol.4 No.1

사람의 혈액에 있는 호중구 엘라스테이즈에 관한 연구의 진척은, 약물로 사용가능한 생체내 혹은 인공합성의 저해제 개발을 동반해 오고 있다. 정제된 호중구 엘라스테이즈에 12종의 항생제를 처리하였더니 20mM 미만의 lC_(50)을 보인 것은 oxytetracycline, cefamandole, gentamicin, amikacin, cloxacillin, methicillin, chloramphenicol 등이었고, 10mM 농도에서 50% 이상의 저해율을 나타낸 것은 methicillin, cefamandole, oxytetracycline 등이었다. 그 결과는 세포벽 합성이나 단백질 합성을 저해하는 일반적 항생제의 약리학적 기전과는 다른 차원에서 항생제가 숙주의 조직을 보호할 수 있는 가능성을 보여준 모델이 존재한다는 것을 in vitro 실험에서 확인할 수 있었다.

사람중성구 Cathepsin G의 분리와 Anti-Human Cathepsin G Antibody의 in vivo 합성 및 분리

배성준,김사열,강구일 고신대학교 의학부 1989 高神大學校 醫學部 論文集 Vol.5 No.1

Human neurtrophil cathepsin G was purified by two step procedure involving ultrogel AcA54 gel filtration and CM-Sephadex ion exchange chromatography. Highly purified human neutrophil cathepsin G was injected to rabbit for in vivo synthesis of anti-human cathepsin G antibodies. For purificaion of Ab, precipitation by ammonium sulfate, DEAD-cellulose ion exchange chromatography and CNBr-activated sepharose 4B affinity chromatography was applied Purified Antibodies detected by immumoprecipitation showed specificity against human neutrophil cathepsin G but not human neutrophil elastsae.

RAPID PURIFICATION AND MOLECULAR CHARACTERIZATION OF HUMAN NEUTROPHIL ELASTASE

Ghim, Sa-Youl,Bae, Sung Jun,Kang, Kooil 고신대학교 의학부 1989 高神大學校 醫學部 論文集 Vol.5 No.1

Elastase에 의해 야기되는 질병의 예방 및 치료 응용을 위하여는, 먼저 이 효소가 고순도로 분리되고 그 근본적인 성질의 해명이 이루어져야 한다. 고순도의 elastase를 얻기위해, 사람의 전혈로부터 Hypaque-Ficoll step gradient에 의해 순수한 neutrophil을 얻은 다음 이것을 extract로 만들어 액체크로마토그래피를 실시하였다. Ultrogel AcA54 gel filtration을 거친 후 CM-sephadex 이온교환 크로마토그래피를 적용하였는데, 이때 유출 완충액 속에 0.1% Brij 35를 첨가하여 elastase의 순도를 더욱 높일 수 있었다. 이 정제된 elastase는 SDS-PAGE를 통해 MW 28,000, 29,000, 30,000의 서로 다른 3개 의 밴드로 확인되었으며, 그간 수많은 연구자들간에 elastase의 분자량에 대해 심한 견해 차이를 보여온 것은 그 분자적 성질이나 분자량이 유사한 cathepsin G가 혼재하거나 elastase분자내 혹은 분자간 S-S결합의 차이에 의함을 알 수 있었다.

비스테로이드성 항염증제에 의한 사람 백혈구 Cathepsin G의 효소 활성도 억제

배성준,김우미,강구일 고신대학교 의학부 1990 高神大學校 醫學部 論文集 Vol.6 No.2

Human leukocyte cathepsin-Gs are active participant in the active phase of inflammations like rheumatoid arthntis, emphysema and glomerular injury Inhibition effects of human leukocyte cathpsin-Gs by non-steroidal anti-inflammatory drugs were vanous Among them, especially, sulindac, salicylate, phenylbutazone, oxyphenbutazone, and salicyluric acid inhibited human leukocyte cathepsin-Gs effectively IC50s of each drug were 8.3mM, 14.3mM, 6.5mM, 11mM, and 15mM respectively. The drugs which have same chemical structure and same degree ofinhibition effect on cyclooxygenase showed different degree or no effect on inhibition of cathepsin G These different inhibition effects may be, beside of hinhibition of cyclooxygenase in the prostaglandin synthesis pathway, another posible mechanism of antitinflammatory effect of NSAIDs and the effect could be a direct protection of tissue destruction in inflammatory diseases.

비스테로이드성 항염증제가 n-Formyl-Methionyl-Leucyl-Phenylalanine에 의한 사람 중성구의 이동에 미치는 영향

김우미,박승언,강구일 고신대학교 의학부 1993 高神大學校 醫學部 論文集 Vol.9 No.2

Migration of neutrophil to the inflammatory site is an important phenomenon in inflammatory diseases. To investigate whether non-steroidal anti-inflammatory drugs(NSAIDs), which inhibit cyclooxygenase in prostaglandin synthesis pathway, have a specific property interfering migration of neutrophil or not. we studied the effects of seven NSAIDs on n-formyl-peptide-induced migration of neutrophil using a 48-well micro chemotaxis assembly. Oxyphenbutazone, phenylbutazone, sulindac. zomepirac and ibuprofen at effective plasma concentration supressed the migration of neutrophil up to 66.4, 33.7, 33.3, 36.2, and 23.5 % , respectively. Salicylate showed no effect on the migration of neutrophil, however, indomethacin showed stimulation effect. Even though same class of indol derivatives, sulindac inhibits and indomethacin stimuletes the chemotaxis of neutrophil. From these results, we suggested that there might be another mode of pharmacological action of NSAIDs for anti-inflammatory effect, in addition to cyclooxygenase inhibition, direct acting on the chemotaxis of neutrophil.

고려 홍삼 및 가토의 안구 수정체로부터 사람 중성구 Elastase 의 활성도 억제성분 분리

강구일,김우미,이대희,김신동,양호성,최강주 ( Kooil Kang,Woo Mi Kim,Dae Heui Lee,Sin Dong Kim,Ho Sung Yang,Kang Ju Choi ) 생화학분자생물학회 1994 BMB Reports Vol.27 No.2

Human neutrophil elastase(HNElastase, EC 3, 4, 21, 11) is a curative factor to elucidate inflammatory diseases. Korean ginseng which has been known as an effective agent on inflammatory diseases, and mammalian eye lens was an organ which has never been involved any inflammatory diseases, were expected to contain some specific chemical compounds which could inhibit HNElastase. Four different compounds were separated from ginseng extract by HPLC with methanol and C^(18) reverse phase column chromatography. The fraction which was eluted at 5∼6 min, showed inhibition effect on HNE. 0.28 ㎎/㎖ of This compound inhibited the activity of HNE up to 97% of control activity and IC_(50) was 0.076 ㎎/㎖. Rabbit eye lens proteins including nucleus and cortical portion were analyzed separately by size exclusion chromatography. Two different proteins were separated from nucleus lens protein. The protein which contained in the first peak, showed inhibitory effect of HNElastase. Four different proteins also separated from cortical protein, these proteins showed anti-elastase activities but the anti-elastase activities were less than those of nucleus protein. From these results, we strongly suggest that Korean ginseng and rabbit eye lens protein contain specific compounds which could inhibit human neutrophil elastase. Therefore, these compounds might be unique materials for developing some anti-inflammatory agents.