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Choi, Nack-Shick,Kim, Seung-Ho Korean Society for Biochemistry and Molecular Biol 2001 Journal of biochemistry and molecular biology Vol.34 No.2
By adding sodium chloride (2.5%) into a Bacillus amyloliquefaciens DJ-4 culture broth, two serine-type fibrinolytic proteases with a molecular weight of 29 (subtilisin DJ-4) and 38-kDa were stimulated on the SDS-fibrin zymogram or inhibitor gels. B. amyloliquefaciens DJ-4 showed the highest proteolytic activity (5.52 plasmin NIH unit/ml) on the fibrin plate based on the molar ratio when cells were subjected to the 2.5% NaCl. Using a fibrin plate, the secreted protease from this strain in the presence of 5% NaCl showed that about 49% of the enzyme's activity remained after incubation at $60^{\circ}C$ for 30 min, but as the salt concentration was increased (10% NaCl) the activity nearly disappeared (0.14 plasmin NIH unit/ml). However, through a fibrin zymography assay, three fibrinolytic enzymes (38, 53 and 80-kDa) from the cells in the presence of 10% NaCl were detected. Also, two salt-activated serine-type fibrinolytic professes (29 and 38kDa) showed thermostability from 65 to $70^{\circ}C$ for 30 min. Furthermore, these professes also showed stability, pH 6-11. In particular, 29-kDa (subtilisin DJ-4) was very stable in the pH range of 4-11 at $4^{\circ}C$ for 48 h.
Choi, Nack-Shick,Choi, Jong Hyun,Kim, Bo-Hye,Han, Yun-Jon,Kim, Joong Su,Lee, Seung-Goo,Song, Jae Jun WILEY-VCH Verlag 2009 ELECTROPHORESIS Vol.30 No.12
<P>A new zymography method for simultaneous detection of two different enzymatic activities (lipolytic and proteolytic) using a single SDS-containing or native-conformation gel and a mixed-substrate (glycerol tributyrate and fibrin) (MS)<SUP>1</SUP> gel was developed. After routine electrophoresis, SDS in the gel was removed by treatment with Triton X-100. Gel proteins were electrotransferred to the MS gel. To visualize lipolytic activity, the MS gel was incubated at 37°C (for 6 or 24 h) until clear bands against an opaque background were observed. To detect proteolytic activity, the same MS gel was stained with Coomassie brilliant blue. Using this method, we show that six lipolytic enzymes from Staphylococcus pasteuri NJ-1 and four proteolytic enzymes from two Bacillus strains, B. licheniformis DJ-2 and B. licheniformis NJ-5, isolated from soil, can be simultaneously detected.</P>
Choi, Nack-Shick,Chung, Dong-Min,Park, Chan-Sun,Ahn, Keug-Hyun,Kim, Joong-Su,Song, Jae-Jun,Kim, Seung-Ho,Yoon, Byung-Dae,Kim, Min-Soo 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.3
Previously, three extracellular proteases, Vpr, PepT, and subtilisin were identified from Bacillus subtilis KCTC 3014. To confirm the activity of Vpr, two recombinant Vpr proteins, full Vpr with TTG (pGST-fTTG-Vpr) and full Vpr with ATG (pGST-fATG-Vpr) as an initiation codon were expressed using a pGEX-2T vector encoding glutathione S-transferase (GST) in Escherichia coli. Vpr was produced in two forms, occurring as four spots on a 2-DE gel, 68 and 75 kDa proteins with similar pI values (4.0 ~ 4.5). Activity was detected in a fibrin zymography at the expected molecular size of 68 kDa (mature form) processed from full Vpr. However, the recombinant 75 kDa of GST-fVpr did not exhibit activity. Replacement of the TTG codon with ATG led to 1.9-fold increased enzyme activity in 68 kDa. Interestingly, the expression of GST-Vpr resulted in the proteolytic degradation of the protein and no GST fusion Vpr protein was detected.
Comparison of Three Substrates (Casein, Fibrin, and Gelatin) in Zymographic Gel
Choi, Nack-Shick,Yoon, Kab-Seog,Lee, Jin-Young,Han, Kyoung-Yoen,Kim, Seung-Ho Korean Society for Biochemistry and Molecular Biol 2001 Journal of biochemistry and molecular biology Vol.34 No.6
Three zymographic techniques using casein, fibrin, and gelatin as substrates in SDS-PAGE were compared based on three aspects: (1) The proteolytic pattern of extracellular enzymes from the three bacterial strains, Bacillus sp. DJ-1, DJ-2, and DJ-3. (2) The enzymatic sensitivity of their activity on zymogram gels. (3) The stability of stained zymogram gels with Coomassie brilliant blue in the destaining solution. There was no significant difference on the pattern of extracellular enzymes from the three strains. The bands in the fibrin gel were clearer and more distinct from the extensive destaining process. It was also shown that the gelatin gel revealed the highest enzymatic sensitivity among the three gels, based on the densitometric analysis. In the casein gel, a trace that could be mistaken as a proteolytic band appeared around 40-50 kDa.
( Nack Shick Choi ),( Jeung Ho Hahm ),( Pil Jae Maeng ),( Seung Ho Kim ) 생화학분자생물학회 2005 BMB Reports Vol.38 No.2
Effects of common electrophoretic reagents, reducing agents (β-mercaptoethanol [BME] and DTT), denaturants (SDS and urea), and non-ionic detergent (Triton X-100), on the activity and stability of bovine plasmin (b-pin) and human plasmin (h-pin) were compared. In the presence of 0.1% SDS (w/v), all reagents completely inhibited two pins, whereas SDS (1%) and urea (1 M) denatured pins recovered their activities after removal of SDS by treatment of 2.5% Triton X-100 (v/v). However, reducing agents (0.1 M of BME and DTT) treated pins did not restore their activities. Based on a fibrin zymogram gel, five (from b-pin) and four (from h-pin) active fragments were resolved. Two pins exhibited unusual stability in concentrated SDS and Triton X-100 (final 10%) and urea (final 6 M) solutions. Two bands, heavy chain-2 (HC-2) and cleaved heavy chain-2 (CHC-2), of b-pin were completely inhibited in 0.5% SDS or 3 M urea, whereas no significant difference was found in h-pin. Interestingly, 50 kDa (cleaved heavy chain-1, CHC-1) of b-pin and two fragments, 26 kDa (light chain, LC) and 29 kDa (microplasmin, MP), of h-pin were increased by SDS in a concentration dependent manner. We also found that the inhibition of SDS against both pins was reversible.
Choi, Nack-Shick,Chung, Dong-Min,Yoon, Kab-Seog,Maeng, Pil-Jae,Kim, Seung-Ho Korean Society for Biochemistry and Molecular Biol 2005 Journal of biochemistry and molecular biology Vol.38 No.6
A new simplified procedure for identifying human plasmin was developed using a DTT copolymerized agarose stacking gel (ASG) system. Agarose (1%) was used for the stacking gel because DTT inhibits the polymerization of acrylamide. Human plasmin showed the lowest activity at pH 9.0. There was a similar catalytically active pattern observed under acidic conditions (pH 3.0) to that observed under alkaline conditions (pH 10.0 or 11.0). Using the ASG system, the primary structure of the heavy chain could be established at pH 3.0. This protein was found to consist of three fragments, 45 kDa, 23 kDa, and 13 kDa. These results showed that the heavy chain has a similar structure to the autolysed plasmin (Wu et al., 1987b) but there is a different start amino acid sequence of the N-termini.