• Title, Summary, Keyword: zymography

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

  • Kang, Dae-Ook;Choi, Nack-Shick
    • Journal of Life Science
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    • v.29 no.12
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    • pp.1408-1414
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    • 2019
  • Enzymes are widely used in industrial applications such as detergents, food, feed production, pharmaceuticals and medical applications and major contributors to clean industrial products and processes. To screen, identify, and characterize the enzymes the zymography techniques are routinely used. The zymography technique is a simple, sensitive, and quantifiable technique that is widely used to detect functional enzymes following electrophoretic separation in sodium dodecyl sulfate (SDS)-polyacrylamide gels. The method is a versatile two-stage technique involving protein separation by electrophoresis followed by the detection of enzyme activity in polyacrylamide gels under non-reducing conditions. It is based on SDS-polyacrylamide gel (PAG) copolymerization with substrates, which are degraded by the hydrolytic enzymes restored in enzyme reaction buffer after the electrophoretic separation. Any kind of biological sample can be applied and analyzed on zymography, including culture supernatants of microbes, plants extracts, blood, tissue culture fluids, enzymes in foods extracts and metaproteome. The advantage of zymography is that it is possible to directly detect the protein with activity on the electrophoretic gel as well as confirm the activity at the nanogram level. Thus, this zymography technology can be applied in various fields. However, these advantages are rather disadvantageous and can often lead to experimental errors. In this review, the advantages, disadvantages, and problem solving of zymography technique are described.

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.

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

  • Son, Jaemin;Kim, Hojun;Kong, Chang-Suk;Seo, Youngwan
    • Journal of Life Science
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    • v.28 no.3
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    • pp.293-299
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    • 2018
  • Viticis fructus (fruits of Vitex rotundifolia) is the dried fruit from Vitex rotundifolia; is a traditional medicine for treating inflammation, migraines, chronic bronchitis, headaches, eye pain, and gastrointestinal infections; and demonstrates various bioactivities, including anti-allergic, anti-cancer, and anti-inflammatory effects, which are partly due to its phenolic compound content. This study examines the inhibitory effects of viticis fructus (fruits of Vitex rotundifolia) on MMP-2 and MMP-9 expression using gelatin zymography and RT-PCR in phorbol-12-myristate-13-acetate (PMA)-induced HT-1080 fibro-sarcoma cells. Fruits of Vitex rotundifolia were extracted twice using dichloromethane ($CH_2Cl_2$) and methanol (MeOH). The combined crude extracts ($CH_2Cl_2$ and MeOH) significantly inhibited MMP-2 and MMP-9 activities in gelatin zymography. The combined extracts were fractionated into n-hexane, 85% aqueous methanol (85% aq. MeOH), n-butanol, and water, successively according to polarity. Among all solvent-partitioned fractions, 85% aq. MeOH fractions showed the strongest inhibition on the activation of MMP-2 and MMP-9 in gelatin zymography. In PMA-stimulated HT-1080 cells, the expression levels of MMP-2 and MMP-9 mRNA were also greatly inhibited by the 85% aq. MeOH fraction. These results suggest that viticis fructus can be used as an excellent source for anti-invasive agents.

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

  • Han, Kee-Jung;Lee, Kwang-Soo;Kong, Kwang-Hoon;Cho, Sung-Hye
    • Analytical Science and Technology
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    • v.16 no.3
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    • pp.179-184
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    • 2003
  • Root bark of Ulmus davidiana Plancn. var. japonica Nakai was extracted by using several solvents with different polarities. Each extract was treated on the MMPs obtained from SK-Hep-1 in order to investigate inhibition effect. Zymography of MMPs showed that MeOH extract has significant inhibition effect. On the GC-MS analysis the highest mass to charge ratio (m/z) of the purified substance was 281. Also, on zymography of MMPs the substance showed 47% inhibition effect at the concentration of $314.7{\mu}g/g$. Cell viability of SK-Hep-1 was 60% at $31.47{\mu}g/g$.

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).

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

  • Kim, Young;Nam, Taek-Jeong
    • Journal of the Korean Society of Food Science and Nutrition
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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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Inhibition of MMP-2 and -9 by Crude Extracts and Their Solvent-partitioned Fractions from the Halophyte Atriplex gmelinii (가는갯능쟁이(Atriplex gmelinii) 추출물과 용매분획물의 MMP-2와 MMP-9 활성 저해효과)

  • Park, Min Jeong;Kim, Junse;Kong, Chang-Suk;Seo, Youngwan
    • 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.