Functional study on an extracellular metalloprotese from Vibrio vulnificus sp. strain MO6 24/0

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교차 연결된 피브린을 분해하는 기능과 프로트롬빈을 트롬빈으로 활성화시키는 기능을 동시에 갖는 단백질 분해효소 vEP-MO6를 Vibrio vulnificus sp. strain MO6 24/0 균주로부터 분리, 정제하여 그 특성을 규명하였다. 이 효소를 정제하기 위하여 첫 번째로, 배양액에 분비된 단백질을 포화농도 70%의 ammonium sulfate로 농축시켰으며, HiPrep-Q anion exchange 컬럼 크로마토그래피와 Superdex 75 size-exclusion 컬럼 크로마토그래피를 순차적으로 수행하였다. vEP-MO6를 SDS-polyacrylamide gel에서 전기영동한 결과 36 kDa 크기의 단일 폴리펩티드로 이루어졌음을 확인하였으며, azocasein과 blood coagulation 과정에 관여하는 단백질들을 가수분해함을 확인하였다. vEP-MO6 단백질 분해효소는 효소활성 분석에 사용한 여러 가지 합성기질 중에서 전형적인 plasminogen activator(uPA) 기질인 S-2444에서 가장 높은 활성을 보였다. vEP-MO6는 금속이온 chelator인 EDTA에 의해 활성이 억제된 반면, 세린 단백질 분해효소 저해제들인 DFP 와 PMSF 그리고 시스테인 단백질 분해효소 저해제인 TLCK 에는 활성이 억제되지 않은 것으로 보아, vEP-MO6는 메탈로 단백질 분해효소 계통의 효소임을 알 수 있었다. vEP-MO6의 최적온도와 pH는 각각 40°C와 8.0이었다. vEP-MO6는 여러 가지 2가 금속이온들 중에서 Ni^(2+)과 Cu^(2+)에 의해 강하게 억제되었고, Ca^(2+)이온에는 영향을 받지 않았다. Fibrinogen-agarose plate assay를 통한 turbid ring 형성 실험과 트롬빈 활성화 기질인 Boc-VPR-pNA를 이용한 트롬빈 활성실험을 통해 vEP-MO6가 프로트롬빈으로부터 트롬빈 계열의 효소를 생성함을 확인하였다. Fibrin-agarose plate assay를 통한 clear zone 형성 실험과 플라스민 활성화 기질인 S-2251을 이용한 fibrin polymer turbidity 변화 실험을 통해 vEP-MO6의 피브린 분해능을 확인하였다.
이상의 결과로부터, Vibrio vulnificus sp. strain MO6 24/0 균주로부터 분리한 vEP-MO6는 교차 연결된 피브린을 분해하는 기능과 프로트롬빈을 활성화시키는 기능을 동시에 갖는 Ca^(2+) 비 의존성 금속함유 단백질 분해효소임을 알 수 있다.|A novel cross-linked fibrin-degrading and prothrombin-activating enzyme (vEP-MO6) was purified and characterized from Vibrio vulnificus sp. strain MO6 24/0. The extracellular proteins secreted in the culture supernatant were precipitated with ammonium sulfate at 70% saturation concentration followed by further purification steps involving HiPrep-Q column anion exchange chromatography and Superdex 75 size-exclusion chromatography. The vEP-MO6 protease appeared to compose of a single polypeptide with apparent molecular weight of about 36 kDa, as judged by SDS-PAGE. The purified enzyme digested azocasein and a few other proteins that are associated with the blood coagulation pathway. Among the chromogenic substrates tested, S-2444 (Glu-Gly-Arg-pNA) that is a typical substrate for plasminogen activator, could be the most suitable substrate for the amidolytic activity of vEP-MO6 protease. The enzymatic activity of vEP-MO6 protease was clearly inhibited by a metal chelating agent (EDTA), but not by inhibitors of serine protease (DFP and PMSF) and cysteine protease (TLCK), suggesting that it is a member of the metalloprotease. The optimal temperature and pH for the proteolytic activity of vEP-MO6 were 40°C and 8.0, respectively. Among the divalent caions, Ni^(2+) and Cu^(2+) were the strongest inhibitors for vEP-MO6 protease activity. Other divalent cations such as Ca^(2+) was not required for vEP-MO6 protease activity. The inhibitory effect of EDTA could be reversed by the addition of excess Mg^(2+) and Mn^(2+), suggesting that these cations can be activators for vEP-MO6. The turbid ring formation assay on fibrinogen-agarose plate and the thrombin activity assay with thrombin-specific chromogenic substrate, Boc-VPR-pNA showed that vEP-MO6 actually cleaves prothrombin to a thrombin-like enzyme. The fibrinolytic activity of vEP-MO6 could be also confirmed by the clear zone formation assay on fibrin-agarose plate and the change in turbidity of fibrin polymer as measured by spectrophotometric method.
All these results presented in the present study strongly suggest that vEP-MO6 is a Ca^(2+)-independent prothrombin-activating and cross-linked fibrin-degrading enzyme belonging to the family of metalloprotease.
Alternative Title
비브리오 균주의 분비성 금속함유 단백질 분해효소의 기능에 관한 연구
Alternative Author(s)
Kwon, Ju Young
朝鮮大學校 大學院
일반대학원 생물신소재학과
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Table Of Contents
Ⅱ-1. Materials = 8
Ⅱ-2. Cultivation of bacterial strain = 8
Ⅱ-3. Protease activity assay = 9
Ⅱ-4. Determination of protein concentration = 9
Ⅱ-5. Enzyme Purificiation = 10
Ⅱ-5-1. Ammonium sulfate precipitation = 10
Ⅱ-5-2. Anion exchange chromatography on HiPrep 16/10 Q FF column = 10
Ⅱ-5-3. Size-exclusion chromatography on Superdex 75 10/300 GL column = 11
Ⅱ-6. Polyacylamide gel electrophoresis = 12
Ⅱ-7. Characterization of vEP-MO6 protease = 12
Ⅱ-7-1. Determination of molecular weight by SDS-PAGE = 12
Ⅱ-7-2. Fibrinolytic activity assay = 12
Ⅱ-7-3. Determination of substrate specificity = 13
Ⅱ-7-4. Fibrin-agarose plate assay = 13
Ⅱ-7-5. Analysis of prothrombin activation = 14
Ⅱ-7-6. Effects of protease inhibitors on vEP-MO6 protease = 15
Ⅱ-7-7. Effects of temperature and pH on vEP-MO6 protease = 15
Ⅱ-7-8. Effects of various metal ions on vEP-MO6 protease = 15
Ⅱ-7-9. N-terminal amino acid sequencing = 16
Ⅲ-1. Purification of extracellular protease = 17
Ⅲ-2. N-terminal amino acid sequencing = 23
Ⅲ-3. Substrate specificity of vEP-MO6 protease = 23
Ⅲ-4. Plasmin activity assay of vEP-MO6-cleaved plasminogen = 29
Ⅲ-5. Effects of various protease Inhibitors on vEP-MO6 protease = 32
Ⅲ-6. Effects of metal ions and chelating agent on vEP-MO6 protease = 32
Ⅲ-7. Effects of pH and temperature on vEP-MO6 protease = 36
Ⅲ-8. Effect of Ca^(2+) ion on vEP-MO6 protease = 36
Ⅲ-9. Prothrombin activation by vEP-MO6 protease = 40
Ⅲ-10. Fibrinolytic activity of vEP-MO6 protease = 43
Ⅳ. 적요 = 48
Ⅴ. References = 50
朝鮮大學校 大學院
권주영. (2004). Functional study on an extracellular metalloprotese from Vibrio vulnificus sp. strain MO6 24/0.
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