• 제목, 요약, 키워드: zymography

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자이모그라피 기술의 문제점과 해결 (Problems and Solutions of Zymography Techniques)

  • 강대욱;최낙식
    • 생명과학회지
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    • v.29 no.12
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    • pp.1408-1414
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    • 2019
  • 효소는 세제, 식품, 사료, 의약품 및 의료용 분야 등 산업 전반적인 응용 분야에서 널리 사용되고 있으며, 산업 제품 및 공정에서 주요 요인이다. 효소를 선별, 확인, 및 특성 분석을 위해 zymography 기술이 일상적으로 사용됩니다. Zymography 기술은 SDS-전기영동을 통해 단백질을 분리한 후 포함된 기질을 겔 상에서 분해하는 기능성 효소를 검출하는 데 널리 사용되는 단순하고 민감하며 정량화가 가능한 기술이다. 이 방법은 비 환원 조건하에서 SDS-전기영동 겔에서 전기영동에 의한 단백질의 분리와 겔 상에서 효소 활성을 검출하는 다목적 2 단계의 기술이다. 이는 SDS-전기영동 겔에 기질을 중합시키고 전기영동 분리 후 효소 반응 완충용액에서 복원된 가수분해 효소에 의해 분해되는 것을 기반으로 하는 기술이다. 미생물 배양액, 식물 추출물, 혈액, 조직 배양액, 식품 속 효소 및 메타 프로테옴을 포함한 어떤 종류의 생물학적 시료들을 zymography에 적용하고 분석이 가능하다. Zymography의 장점은 전처리 없이 혼합된 시료를 적용하여 SDS-전기영동 겔 상에서 활성을 지닌 단백질을 직접 육안으로 검출이 가능할 뿐만 아니라 나노그람(nanogram) 수준에서 활성을 확인이 가능하다. 그래서 이 zymography 기술은 다양한 분야에 응용이 가능하다. 하지만, 이러한 장점이 오히려 단점으로 작용하여 실험적 오류를 범할 수 있는 경우가 많다. 본 총설에서 zymography 기술의 장점, 단점, 및 문제점 해결에 관해서 서술하였다.

Identification of Recombinant Subtilisins

  • CHOI , NACK-SHICK;YOO, KI-HYUN;YOON, KAB-SEOG;CHANG, KYU-TAE;MAENG, PIL-JAE;KIM, SEUNG-HO
    • Journal of Microbiology and Biotechnology
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    • v.15 no.1
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    • pp.35-39
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    • 2005
  • To identify the activity of recombinant subtilisins (subtilisin BPN' and subtilisin Carlsberg), three different zymography methods, SDS-fibrin zymography (SDS-FZ), reverse fibrin zymography (RFZ), and isoelectric focusingfibrin zymography (IEF-FZ), were used. The recombinant subtilisins BPN' and Carlsberg did not migrate into the electrophoretic field based on a Laemmli buffer system, instead forming a "binding mode" at the top part of the separating gels with the SDS-FZ and RFZ techniques. Yet, this problem was resolved when using IEF-FZ with a pH range from 3 to 10. In addition, all these methods enabled the activity of a recombinant pro-subtilisin DJ-4 to be detected without a refolding pathway.

HT-1080 세포에서 만형자 용매 추출물의 암전이 억제효과 (Anti-invasive Effect of the Solvent-partitioned Fractions from Viticis Fructus in PMA-induced HT-1080 Cells)

  • 손재민;김호준;공창숙;서영완
    • 생명과학회지
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    • v.28 no.3
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    • pp.293-299
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    • 2018
  • 본 연구는 PMA (Phorbol 12-myristate 13-acetate)에 의해 MMP 활성이 증가된 섬유육종세포에서 MMP-2와 -9의 mRNA 발현 및 단백질 활성에 대한 만형자의 억제 효과를 zymography와 RT-PCR 방법에 의해서 측정하였다. 만형자 시료는 dichloromethane에 의해서 두 번 추출되었으며 동일한 과정을 methanol를 사용하여 반복하였다. 각각의 용매에 의해서 얻어진 추출물을 합한 후에 zymography를 이용하여 이 추출물의 MMP-2와 -9에 대한 억제효과를 측정한 결과 유의적인 억제효과를 나타내었다. 억제활성 성분을 추적하기 위하여 극성에 따른 추출물의 용매분획을 실시하여 n-hexane, 85% aq. MeOH, n-butanol, 및 water 분획층을 얻었다. 이 4가지 용매분획에 대한 MMP 억제활성을 측정하였으며 측정한 결과 85% aq. MeOH 분획층이 zymography와 RT-PCR 실험에서 MMP-2와 -9에 대해 가장 강한 억제효과를 나타내었다. 이상의 결과는 만형자 추출물이 암전이 억제제 개발을 위한 좋은 원천이 될 수 있는 가능성이 있음을 제시한다.

유근피(楡根皮)에 존재하는 matrix metalloproteinase-9 억제 물질의 분리 및 정제 (Separation and purification of substance having matrix metalloproteinase-9 inhibition effect in Ulmus davidiana Plancn. var. japonica Nakai)

  • 한기정;이광수;공광훈;조성희
    • 분석과학
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    • v.16 no.3
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    • pp.179-184
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    • 2003
  • 극성이 다른 여러 가지 용매를 사용하여 느릅나무의 뿌리 껍질(이하 유근피)을 추출하였다. MeOH 추출물이 SK-Hep-1에서 분리된 matrix metalloproteinase-9(MMP-9)의 zymography에서 저해능을 나타내었다. 분리 정제한 물질의 분자량은 GC-MS spectrum에서 $M^+=281$인 것으로 나타났고 MMP-9의 활성은 $314.7{\mu}g/g$에서 47% 억제되는 것으로 나타났다. 그리고 SK-Hep-1 세포주는 $31.47{\mu}g/g$에서 60%의 생존율을 나타났다.

Identification of Total Extracellular Fibrinase from Bacillus sp. DJ Using One-or Two-Dimensional Fibrin Zymography for Proteomic Approach

  • CHOI, NACK-SHICK;JIN-YOUNG LEE;KAB-SEOG YOON;KYOUNG-YOEN HAN;SEUNG-HO KIM
    • Journal of Microbiology and Biotechnology
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    • v.11 no.6
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    • pp.1111-1114
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    • 2001
  • An extracellular fibrinolytic-enzyme-producing bacterium was isolated from Doen-Jang, a Korean traditional fermented flood, and identified as Bacillus sp. DJ based on its morphology and cellular fatty acid composition. The total extracellular fibrinase (EF) from Bacillus sp. DJ was analyzed using three fibrin zymographic techniques, SDS-fibrin zymography (SDS-FZ), isoelectrofocucing-fibrin zymographs(IEF-FZ), and a two-dimensional SDS-fibrin zymographic analysis (2D SDS-FZ). As a result, the EP map of Bacillus sp. DJ was established. The results suggest that the 2D SDS-FZ method will be a useful tool for the proteomic approach for many other bacterial pretenses.

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Relationship Between Acrylamide Concentration and Enzymatic Activity in An Improved Single Fibrin Zymogram Gel System

  • Choi, Nack-Shick;Kim, Byoung-Young;Lee, Jin-Young;Yoon, Kab-Seog;Han, Kyoung-Yoen;Kim, Seung-Ho
    • BMB Reports
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    • v.35 no.2
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    • pp.236-238
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    • 2002
  • Based on the zymography analysis, Bacillus sp. DJ-4 (screened from Doen-Jang, a Korean traditional fermented food) secretes seven extracellular fibrinolytic enzymes (EFEs; 68, 64, 55, 45, 33, 27, and 13 kDa) in culture broth. These seven EFEs were analyzed by newly applied SDS-fibrin zymography combined with gradient polyacrylamide (SDS-FZGP). This improved gel system was used with a 5-20% acrylamide gradient in a fibrin zymogram gel for the separation of proteins with molecular masses from below 10kDa to over 100kDa on one gel plate. Using this system, high molecular weight bands (HMWBs) were clearly and sharply resolved. We also examined the relationship between an acrylamide concentration and the enzymatic activity of EFE using densitometric analysis.

Nano-scale Proteomics Approach Using Two-dimensional Fibrin Zymography Combined with Fluorescent SYPRO Ruby Dye

  • Choi, Nack-Shick;Yoo, Ki-Hyun;Yoon, Kab-Seog;Maeng, Pil-Jae;Kim, Seung-Ho
    • BMB Reports
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    • v.37 no.3
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    • pp.298-303
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    • 2004
  • In general, a SYPRO Ruby dye is well known as a sensitive fluorescence-based method for detecting proteins by one-or two-dimensional SDS-PAGE (1-DE or 2-DE). Based on the SYPRO Ruby dye system, the combined two-dimensional fibrin zymography (2-D FZ) with SYPRO Ruby staining was newly developed to identify the Bacillus sp. proteases. Namely, complex protein mixtures from Bacillus sp. DJ-4, which were screened from Doen-Jang (Korean traditional fermented food), showed activity on the zymogram gel. The gel spots on the SYPRO Ruby gel, which corresponded to the active spots showing on the 2-D FZ gel, were analyzed by a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analysis. Five intracellular fibrinolytic enzymes of Bacillus sp. DJ-4 were detected through 2-D FZ. The gel spots on the SYPRO Ruby dye stained 2-D gel corresponding to 2-D FZ were then analyzed by MALID TOF MS. Three of the five gel spots proved to be quite similar to the ATP-dependent protease, extracellular neutral metalloprotease, and protease of Bacillus subtilis. Also, the extracellular proteases of Bacillus sp. DJ-4 employing this combined system were identified on three gels (e.g., casein, fibrin, and gelatin) and the proteolytic maps were established. This combined system of 2-D zymography and SYPRO Ruby dye should be useful for searching the specific protease from complex protein mixtures of many other sources (e.g., yeast and cancer cell lines).

골감소증 환자의 혈청중 Insulin-Like Growth Factor Binding Protein-5의 변화 (Changes in Serum Insulin-Like Growth Factor Binding Protein-5 of Osteopenia)

  • 김영;남택정
    • 한국식품영양과학회지
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    • v.29 no.3
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    • pp.493-499
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    • 2000
  • 골감소증 환자의 혈청중에 존재하는 IGFBP-5의 존재와 변화를 검토한 결과, 골감소즈 환자에서는 정상 대조군에 비해 IGFBP-5가 감소하였는데 이 변화는 IGFBP-5의 분해효소가 작용하기 때문인 것으로 나타났다. IGFBP-5에 작용하는 단백질분해효소 저해제중 metallo계인 EDTA 및 1,10-phenanthroline과 seriner계인 aprotinin, heparin, heparin cofactor 2(HC2), heparine+HC2가 IGFBP-5에 대해 저해효과가 크므로 metalloprotease이면서 serine protease의 성질을 가지는 효소들이 IGFBP-5에 작용하였다. IGF-I과 IGF-II 그리고 insulin은 효소 활성에 아무런 영향이 없었다. IGFBP-5의 zymography에서 정상인과 골감소증 환제어서 180 kDa 크기의 band가 나타났고, gelatin zymography에서 정상 대조군의 경우 66 kDa과 97 kDa 정도의 band가 확인되었고 골감소증 환자의 경우는 69 kDa의 band가 확인되었다.

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가는갯능쟁이(Atriplex gmelinii) 추출물과 용매분획물의 MMP-2와 MMP-9 활성 저해효과 (Inhibition of MMP-2 and -9 by Crude Extracts and Their Solvent-partitioned Fractions from the Halophyte Atriplex gmelinii)

  • 박민정;김준세;공창숙;서영완
    • Ocean and Polar Research
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    • v.41 no.2
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    • pp.79-88
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    • 2019
  • In this study, the inhibitory effect of Atriplex gmelinii C. A. Mey. against the activity of MMP-2 and MMP-9 secreted from phorbol-12-myristate-13-acetate (PMA)-stimulated HT-1080 cells was evaluated by gelatin zymography and enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase-chain reaction (RT-PCR), and Western blot assay. Specimens of the halophyte A. gmelinii were extracted twice for 24 hr with methylene chloride ($CH_2Cl_2$), and then twice with methanol (MeOH), in turn. Each extract significantly inhibited the enzymatic activities in gelatin zymography and MMP ELISA kit, and expression of MMP-2 and 9 in mRNA and protein levels. Two crude extracts were combined and then the combined crude extracts were fractionated into n-hexane, 85% aqueous methanol (85% aq.MeOH), n-butanol (n-BuOH), and water ($H_2O$) fractions, according to solvent polarity. Among solvent-partitioned fractions, the 85% aq.MeOH fraction showed the strongest inhibitory effect against MMP-2 and -9 in gelatin zymography and MMP ELISA kit. In RT-PCR, all solvent-partitioned fractions significantly suppressed mRNA expression of MMP-2 and -9. On the other hand, in Western blot assay, all solvent-partitioned fractions except $H_2O$ significantly reduced expression levels of protein. HT 1080 cell migration was most significantly inhibited by the n-BuOH fraction followed by the 85% aq.MeOH and $H_2O$ fractions. These results suggest that A. gmelinii could be used as a potential source to inhibit tumor cell metastasis.

Identification of Three Extracellular Proteases from Bacillus subtilis KCTC 3014

  • Choi Nack-Shick;Chung Dong-Min;Ryu Chung-Hun;Yoon Kab-Seog;Maeng Pil-Jae;Kim Seung-Ho
    • Journal of Microbiology and Biotechnology
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    • v.16 no.3
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    • pp.457-464
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    • 2006
  • Three extracellular proteases (Vpr, peptidase T, and subtilisin) were identified from the culture supernatant of Bacillus subtilis KCTC 3014. All the proteins were partially purified as a mature form by using a DEAE-cellulose ion-exchange column chromatography. Their activities were determined by using zymography and densitometry. The relative molecular masses of Vpr and peptidase T (PepT) were determined to be 68 and 48 kDa by SDS-PAGE and zymography, respectively. However, subtilisin formed a 'binding mode' at the top of the separating gel. After denaturation by boiling at $100^{\circ}C$ for 5 min, its molecular mass was determined to be 29 kDa, whereas its activity was lost. The optimal pH of Vpr, PepT, and subtilisin were 9.0, 6.0-7.0, and 7.0-8.0, respectively. The optimal temperature of Vpr, PepT, and subtilisin was 40, 50, and $40^{\circ}C$, respectively. Inhibitor test revealed that Vpr and subtilisin were serine proteases and that PepT was a metalloprotease. Interestingly, we found that Vpr showed no enzyme activity on a 2DE zymogram gel. Three genes, vpr, pepT, and apr (encoding subtilisin protein), were cloned and their nucleotide and deduced amino acid sequences were determined.