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인체 Melanoma Plasminogen Activator 의 정제 및 특성
정광회,김경호,김유삼 ( Kwang Hoe Chung,Kyong Ho Kim,Yu Sam Kim ) 생화학분자생물학회 1985 BMB Reports Vol.18 No.3
Plasminogen activators are produced by many transformed cells and by a few normal cell lines and tissues. Human melanoma cell line G-361 was cultured and plasminogen activator was purified from the culture medium by the column chromatography on zinc chelate-Sepharose 6B, ConA-agarose, and Sephadex G-150 superfine. The molecular weight of the purified plasminogen activator was determined to be about 71,000. The maximum activity of the activator showed at about pH 7.5. The fibrinolytic activity of the plasminogen activator was inhibited by diisopropyl flurophosphate. The melanoma plasminogen activator was appeared to be immunologically identical with the tissue plasminogen activator, but unrelated to urokinase. The melanoma plasminogen activator bound significantly to fibrin clots in vitro, while urokinase did not.
Purification and Characterization of Plasminogen Activator Secreted by Human Melanoma Cell G-361
정광회,김경호,김유삼,Chung, Kwang-Hoe,Kim, Kyong-Ho,Kim, Yu-Sam 생화학분자생물학회 1985 한국생화학회지 Vol.18 No.3
인체 melanoma 세포 G-361 을 세포배양하였을 때 분비된 plasminogen activator를 zinc chelate-Sepharose 6B 흡착 크로마토그라피법, Concanavalin-A agarose 흡착 크로마토그라피법, Sephadex G-150 젤 여과법등을 사용하여 정제하였고 그 특성을 조사하였다. 정제된 plasminogen activator는 SDS-polyacrylaminde gel에서 전기이동한 결과 하나의 major band을 나타냈고, 그 분자량은 약 71,000 dalton이었다. 이 plasminogen activator 가 가장 큰 활성을 나타내는 최적 pH는 8.0이었고, 열에 대하여는 매우 안정하였다. 또한 diisopropyl fluorophosphate 처리에 의하여 활성이 억제되는 것으로 보아 일종의 serine protease라는 사실이 확인되었으며, 인위적으로 만든 fibrin clot에 대한 친화성이 urokinase와 비교하였을 때 약 9배 높았다. Tissue plasminogen activator를 항원으로 하여 토끼로 부터 항체를 얻었고 이 항체를 가지고 melanoma 세포 배양액에서 추출한 plasminogen activator와 Ouchterlony double immunodiffusion한 결과 하나의 precipitation band를 얻었다. 그러고 이 항혈청은 melanoma plasminogen activator의 활성을 90% 이상 억제하였다. 그러나, tPA에 대한 항혈청은 urokinase와는 전혀 면역학적으로 반응하지 않았다. Plasminogen activators are produced by many transformed cells and by a few normal cell lines and tissues. Human melanoma cell line G-361 was cultured and plasminogen activator was purified from the culture medium by the column chromatography on zinc chelate-Sepharose 6B, ConA-agarose, and Sephadex G-150 superfine. The molecular weight of the purified plasminogen activator was determined to be about 71,000. The maximum activity of the activator showed at about pH 7.5. The fibrinolytic activity of the plasminogen activator was inhibited by diisopropyl flurophosphate. The melanoma plasminogen activator was appeared to be immunologically identical with the tissue plasminogen activator, but unrelated to urokinase. The melanoma plasminogen activator bound significantly to fibrin clots in vitro, while urokinase did not.
Purification of Human Interferon-${\beta}$ from Recombinant E. coli
이진규,이석재,최문기,정광회,신광순,백승복,Lee, Jin-Kyu,Lee, Seok-Jae,Choe, Moon-Kee,Chung, Kwang-Hoe,Shin, Kwang-Soon,Paik, Sung-Bok 생화학분자생물학회 1990 한국생화학회지 Vol.23 No.2
유전자 재조합 인간 Interferon-${\beta}$를 초음파 처리, 8 M Guanidine-HCl 추출, inclusion body의 용해, 희석, Blue Sepharose CL-6B column chromatography, HPLC gel filtration 방법 등을 이용하여 대장균으로부터 정제하였다. 정제된 IFN-${\beta}$의 specific activity는 $3.1{\times}10^8$ IU/mg이었고 정제도는 1,902이었다. 정제된 IFN-${\beta}$는 환원된 상태와 환원되지 않은 상태에서 SDS polyacrylamide gel electrophoresis 한 결과 모두 단일 띠로 나타났으며 분자량은 18,000 dalton이었다. N-말단 아미노산을 조사한 결과 재조합 인간 IFN-${\beta}$는 천연형 인간IFN-${\beta}$와 같이 N-말단이 methionine임이 밝혀졌다. 전자 현미경을 이용하여 inclusion body 형성을 조사한 결과 IFN-${\beta}$ 유전자를 가지고 있는 대장균에서는 inclusion body의 형성을 확인할 수 있었으나 숙주(wild type)에서는 확인되지 않았다. 최종적으로 정제된 IFN-${\beta}$의 정제도는 HPLC gel filtration 한 결과 99% 이상으로 나타났다. Recombinant human interferon-${\beta}$ was purified to homogeneity from E. coli by methods of sonication, extraction with 8 M Guanidine HCl, solubilization of inclusion body, dilution, Blue Sepharose CL-6B column chromatography and HPLC gel filtration. Specific activity of purified IFN-${\beta}$ was $3.1{\times}10^8$ IU/mg protein and the purification was 1,902 fold. The purified IFN-${\beta}$ was a single band on SDS polyacrylamide gel electrophoresis under reducing condition and non-reducing condition and its molecular weight was estimated to 18,000 dalton. The results of N-terminal analysis showed that recombinant human IFN-${\beta}$ has N-terminal methionine same as natural human IFN-${\beta}$. The inclusion bodies were observed in the E. coli cells harboring IFN-${\beta}$ gene but not observed in the host cells (MM 294). The purity of finally purified IFN-${\beta}$ was more than 99% by HPLC gel filtration.
HELP ( 헤파린 매개성 체외 저밀도지단백 피브리노겐 침전법 ) 치료로 완치된 당뇨병성 족부 괴저증
윤영섭(Young Sup Yoon),정광회(Kwang Hoe Chung),이동일(Dong Il Lee),이성란(Sung Rahn Lee),최승혁(Seung Hyuk Choi),심원흠(Won Heum Shim),장양수(Yang Soo Jang) 대한내과학회 1998 대한내과학회지 Vol.55 No.5
HELP(Heparin-mediated extracorporeal low-density lipoprotein fibrinogen precipitation) is an LDL apheresis system which has been utilized as a last therapeutic option for drug resistant hypercholesterolemia. Recently the scope of the treatment has been expanded to coronary artery obstructive disease(CAOD) or peripheral arterial obstructive disease(PAOD) with dyslipidemia. We applied six sessions of HELP therapy with a week interval to a patient with diabetic gangrene of both feet and PAOD who had elevated LDL-cholesterol and fibrinogen despite maximal drug therapy. Lipids, Lp(a) and fibrinogen were measured on plasma samples before and after treatment. Changes of symptoms and physical findings were reported before, immediately after and 3 month after treatment. In every session, LDL cholesterol level was reduced more than 40%, Mean LDL cholesterols were reduced from 133.5 mg/dL to 55.0 mg/dL(59%). Total cholesterol(104.5 mg/dL;51% decrease), triglycerides(142.0 mg/dL;47% decrease) and Lp(a)(24.3 mg/dL; 58% decrease) levels were also reduced. Mean HDL cholesterol was reduced to 6.3 mg/dL(25%) with prompt recovery to pretreatment level within one week. Mean fibrinogen decreased from 571.0 mg/dL to 253.3 mg/dL(58%) without bleeding complications. Two episodes of dizziness with spontaneous resolution were observed during or over three days after treatment. Two sessions of HELP made diabetic gangrene on toes improved. After six sessions, the gangrenes showed near-complete healing. So we report a case of a patient who had persistent diabetic gangrene of both feet despite proper revascularization, which was completely healed by HELP without significant side effects.