- Email: [email protected]
Fibrin Zymography: A Direct Analysis of Fibrinolytic Enzymes on Gels
115
NOTES & TIPS
were dimeric, a second site in a DR3 element would have the nonconsensus sequence GGAGGT which differs from the inhibitory dimeric VDRE identified in the chicken PTH gene (GGGTCA gga GGGTGT) (10) and in the rat parathyroid hormone-related peptide gene (GGGTGGA gag GGGTGA) (11). It is also possible that the overexpression of the VDR in our experimental system increases the likelihood that VDR binds as a monomer to elements resembling that found in the human PTH sequence (9). A similar inhibition of luciferase expression has been reported by thyroid hormones in CV1 cells using a different vector (12). This was independent of the promoter and was suggested to occur through an interaction of the thyroid hormone receptor with the LUC coding region. There are clearly parallels between that study and ours. Perhaps most importantly they stress the need to perform the appropriate control experiments when studying hormone response elements in transfected cells. Fortunately we found no interaction with the pGL3 vectors in Caco-2 cells which leads us to suggest that these may be preferable to the pGL2 vectors for studies of 1,25(OH)2D3 actions. In summary, potential inhibitory VDRE resembling that in the human PTH gene are present in the pGL2 vector but have been altered in pGL3. Acknowledgments. This work was supported by grants from the UK Medical Research Council and the Wellcome Trust.
REFERENCES 1. Umesono, K., Murakami, K. K., Thompson, C. C., and Evans, R. M. (1991) Cell 65, 1255–1266. 2. Demay, M. B., Kiernan, M. S., DeLuca, H. F., and Kronenberg, H. M. (1992) Proc. Natl. Acad. Sci. USA 89, 8097– 8101. 3. Carlberg, C. (1995) Eur. J. Biochem. 231, 517–527. 4. Schra¨der, M., Nayeri, S., Kahlen, J.-P., Mu¨ller, K. M., and Carlberg, C. (1995) Mol. Cell. Biol. 15, 1154 –1161. 5. Darwish, H. M., and DeLuca, H. F. (1996) Arch. Biochem. Biophys. 334, 223–234. 6. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745– 2752. 7. Giuliano, A. R., and Wood, R. J. (1991) Am. J. Physiol. 260, G207–G212. 8. Groskreutz, D. J., Sherf, B. A., Wood, K. V., and Schenborn, E. T. (1994) Promega Notes 50, 2– 8. 9. Mackey, S. L., Heymont, J. L., Kronenberg, H. M., and Demay, M. B. (1996) Mol. Endocrinol. 10, 298 –305. 10. Liu, S. M., Koszewski, N., Lupez, M., Malluche, H. H., Olivera, A., and Russell, J. (1996) Mol. Endocrinol. 10, 206 –215. 11. Kremer, R., Sebag, M., Champigny, C., Meerovitch, K., Hendy, G. N., White, J., and Goltzman, D. (1996) J. Biol. Chem. 271, 16310 –16316. 12. Tillman, J. B., Crone, D. E., Kim, H.-S., Sprung, C. N., and Spindler, S. R. (1993) Mol. Cell. Endocrinol. 95, 101–109.
Fibrin Zymography: A Direct Analysis of Fibrinolytic Enzymes on Gels Seung-Ho Kim,1 Nack-Shick Choi, and Woo-Yiel Lee Protein Function Research Unit, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon 305-600, Korea Received May 8, 1998
For the assay of fibrinolytic activity, the reverse fibrin autographic technique is used routinely; samples containing fibrinolytic enzymes are electrophoresed in SDS–polyacrylamide gels and then placed on the surface of the fibrin/agar indicator gel (1–3). The major drawback to reverse fibrin autography is that it is time-consuming and has low sensitivity for enzymatic activity as well. Zymographic techniques, which are based on a SDS–polyacrylamide gel copolymerized with a protein substrate that is degraded by the proteases restored during the incubation period after the electrophoretic separation, have been recently reported for the quantitation of proteases such as metalloproteinases (4, 5). Casein zymography (6) has also been used as an alternate assay for fibrinolytic enzymes because of the ability of plasmin to degrade casein. In this short communication we report a direct zymographic assay (fibrin zymography) for fibrinolytic enzymes which is more convenient, sensitive, and specific for fibrinolytic activity than the previous methods. Resolving gel solution (11%) containing 0.12% (w/v) fibrinogen was prepared in a total volume of 10 mL and centrifuged to remove insoluble impurities which were induced when SDS stock solution was mixed. Thrombin (1 unit/mL) solution and N,N,N9,N9-tetramethylethylenediamine were added to the gel solution in final concentrations of 0.1 munit/mL and 0.028% (v/v), respectively. Various amounts (0.25 to 200 ng) of plasmin were electrophoresed into the fibrin gel, and subsequently washed in 2.5% Triton X-100 solution and incubated in a bath containing 0.1 M glycine-NaOH at 37°C for overnight as previously described (7). Figure 1 shows the fibrin zymogram in which plasmin appears as single clear bands of fibrinolysis against a blue background of undigested fibrin substrate. The enzyme amounts could be detected as low as 6.3 ng with an overnight incubation. This detection limit is comparable to those of gelatin and casein zymography (6) and Leber and Balkwill’s zymography for the quantitation of proteolytic activity on substrate gels (8). We have examined the possibility of using fibrin zymography to detect fibrinolytic enzymes from the 1
To whom correspondence should be addressed. Fax: 82-42-8604593. E-mail: [email protected]. ANALYTICAL BIOCHEMISTRY 263, 115–116 (1998) ARTICLE NO. AB982816
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
116
NOTES AND TIPS
culture media of Bacillus strains isolated from DoenJang, the Korean traditional fermented food. The reverse fibrin autography (Fig. 2A) shows two broad bands barely visualized on the fibrin/agar indicator gel (Fig. 2B). On the other hand, several polypeptides possessing fibrinolytic activity are detected as sharp, clear bands on the fibrin zymogram (Fig. 2B). Molecular markers are also perceptible by eye; they do not photograph well, though. It has been demonstrated that at least four fibrinolytic enzymes with apparent molecular sizes of approximately 64, 53, 38, and 29 kDa are secreted from each of two Bacillus strains isolated from Doen-Jang. The purification and characterization of the proteases will be described elsewhere. Even though gelatin and casein are satisfactory substrates for plasmin (7, 8), all fibrinolytic enzymes are not sensitive for the substrates as much as plasmin is. Apparently fibrin is an in vivo substrate for plasmin. We took advantage of this fact to explore the possibility of assaying plasmin-like enzymes with fibrin zymography, an electrophoretic technique in which fibrin is copolymerized with the acrylamide during casting of the separating gel. We have demonstrated that the zymographic technique can be used reliably for the rapid qualitative evaluation of plasmin-like enzymes, which have the ability to restore enzymatic activity on the gel copolymerized with fibrin upon removal of the SDS, with a sensitivity level comparable to that of other zymographic techniques.
FIG. 2. Fibrin zymogram (A) and reverse fibrin autography (B) of the culture media of two Bacillus strains (designated as DJ-4 and DJ-8) isolated from Doem-Jang, a Korean traditional fermented food; 10 ml of the crude enzyme solution, which was obtained by centrifugation of cells at 10,000g for 10 min, was applied for each assay. Fibrin autography was carried out as previously described (1). Lanes 1 and 2 for each assay are the crude enzyme solutions from DJ-4 and -8, respectively.
REFERENCES 1. Granelli-Piperno, A., and Reich, E. (1978) J. Exp. Med. 148, 223–234. 2. Sprengers, E. D., Verheijen, J. U., van Hinshergh, V. W. M., and Emeis, J. J. (1984) Biochim. Biophys. Acta 801, 163–170. 3. Loskutoff, D. J., van Mourik, J. A., Erickson, L. A., and Lawrence, D. (1983) Proc. Natl. Acad. Sci. USA 80, 2956 –2960. 4. Paech, C., Christianson, T., and Maurer, K.-H. (1993) Anal. Biochem. 208, 249 –254. 5. Kleiner, D. E., and Stetler-Stevenson, W. G. (1994) Anal. Biochem. 218, 325–329. 6. Raser, K. J., Posner, A., and Wang, K. K. W. (1995) Arch. Biochem. Biophys. 319, 211–216. 7. Heussen, C., and Dowdle, E. B. (1980) Anal. Biochem. 102, 196 –202. 8. Leber, T. M., and Balkwill, F. R. (1997) Anal. Biochem. 249, 24 –28.
Effect of a Polyhistidine Terminal Extension on Eglin c Stability Jennifer C. Waldner,* Steven J. Lahr,† Marshall Hall Edgell,‡ and Gary J. Pielak*,1 FIG. 1. Fibrin zymogram of plasmin. Various amounts (6.3 to 200 ng, as indicated) of plasmin were electrophoresed into a fibrin-copolymerized gel at 10 mA constant currency and subsequently washed with 2.5% Triton X containing Tris (50 mM) buffer (pH 7.4) to remove SDS. After washing the gel in distillation water for 30 min, the gel was incubated in 30 mM Tris buffer (pH 7.4) containing 200 mM NaCl, 10 mM CaCl2, and 0.02% NaN3 at 37°C for 16 h. The zymogram shown here was a Coomassie blue-stained fibrin gel. As little as 6.3 ng (lane 7) of plasmin can be visualized in the present study. Minor bands in lanes 2 and 3 are believed to be due to self-digestion and aggregation of plasmin during sample preparation. Lane 1, protein markers.
*Department of Chemistry, †Department of Biochemistry & Biophysics, and ‡Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Received May 20, 1998
Polyhistidine terminal extensions (his tags) are attached to recombinant proteins to facilitate purifica1
To whom correspondence should be addressed. Fax: 966-3675. E-mail: [email protected]. ANALYTICAL BIOCHEMISTRY 263, 116 –118 (1998) ARTICLE NO. AB982808
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
NOTES & TIPS
were dimeric, a second site in a DR3 element would have the nonconsensus sequence GGAGGT which differs from the inhibitory dimeric VDRE identified in the chicken PTH gene (GGGTCA gga GGGTGT) (10) and in the rat parathyroid hormone-related peptide gene (GGGTGGA gag GGGTGA) (11). It is also possible that the overexpression of the VDR in our experimental system increases the likelihood that VDR binds as a monomer to elements resembling that found in the human PTH sequence (9). A similar inhibition of luciferase expression has been reported by thyroid hormones in CV1 cells using a different vector (12). This was independent of the promoter and was suggested to occur through an interaction of the thyroid hormone receptor with the LUC coding region. There are clearly parallels between that study and ours. Perhaps most importantly they stress the need to perform the appropriate control experiments when studying hormone response elements in transfected cells. Fortunately we found no interaction with the pGL3 vectors in Caco-2 cells which leads us to suggest that these may be preferable to the pGL2 vectors for studies of 1,25(OH)2D3 actions. In summary, potential inhibitory VDRE resembling that in the human PTH gene are present in the pGL2 vector but have been altered in pGL3. Acknowledgments. This work was supported by grants from the UK Medical Research Council and the Wellcome Trust.
REFERENCES 1. Umesono, K., Murakami, K. K., Thompson, C. C., and Evans, R. M. (1991) Cell 65, 1255–1266. 2. Demay, M. B., Kiernan, M. S., DeLuca, H. F., and Kronenberg, H. M. (1992) Proc. Natl. Acad. Sci. USA 89, 8097– 8101. 3. Carlberg, C. (1995) Eur. J. Biochem. 231, 517–527. 4. Schra¨der, M., Nayeri, S., Kahlen, J.-P., Mu¨ller, K. M., and Carlberg, C. (1995) Mol. Cell. Biol. 15, 1154 –1161. 5. Darwish, H. M., and DeLuca, H. F. (1996) Arch. Biochem. Biophys. 334, 223–234. 6. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745– 2752. 7. Giuliano, A. R., and Wood, R. J. (1991) Am. J. Physiol. 260, G207–G212. 8. Groskreutz, D. J., Sherf, B. A., Wood, K. V., and Schenborn, E. T. (1994) Promega Notes 50, 2– 8. 9. Mackey, S. L., Heymont, J. L., Kronenberg, H. M., and Demay, M. B. (1996) Mol. Endocrinol. 10, 298 –305. 10. Liu, S. M., Koszewski, N., Lupez, M., Malluche, H. H., Olivera, A., and Russell, J. (1996) Mol. Endocrinol. 10, 206 –215. 11. Kremer, R., Sebag, M., Champigny, C., Meerovitch, K., Hendy, G. N., White, J., and Goltzman, D. (1996) J. Biol. Chem. 271, 16310 –16316. 12. Tillman, J. B., Crone, D. E., Kim, H.-S., Sprung, C. N., and Spindler, S. R. (1993) Mol. Cell. Endocrinol. 95, 101–109.
Fibrin Zymography: A Direct Analysis of Fibrinolytic Enzymes on Gels Seung-Ho Kim,1 Nack-Shick Choi, and Woo-Yiel Lee Protein Function Research Unit, Korea Research Institute of Bioscience and Biotechnology, Yusong, Taejon 305-600, Korea Received May 8, 1998
For the assay of fibrinolytic activity, the reverse fibrin autographic technique is used routinely; samples containing fibrinolytic enzymes are electrophoresed in SDS–polyacrylamide gels and then placed on the surface of the fibrin/agar indicator gel (1–3). The major drawback to reverse fibrin autography is that it is time-consuming and has low sensitivity for enzymatic activity as well. Zymographic techniques, which are based on a SDS–polyacrylamide gel copolymerized with a protein substrate that is degraded by the proteases restored during the incubation period after the electrophoretic separation, have been recently reported for the quantitation of proteases such as metalloproteinases (4, 5). Casein zymography (6) has also been used as an alternate assay for fibrinolytic enzymes because of the ability of plasmin to degrade casein. In this short communication we report a direct zymographic assay (fibrin zymography) for fibrinolytic enzymes which is more convenient, sensitive, and specific for fibrinolytic activity than the previous methods. Resolving gel solution (11%) containing 0.12% (w/v) fibrinogen was prepared in a total volume of 10 mL and centrifuged to remove insoluble impurities which were induced when SDS stock solution was mixed. Thrombin (1 unit/mL) solution and N,N,N9,N9-tetramethylethylenediamine were added to the gel solution in final concentrations of 0.1 munit/mL and 0.028% (v/v), respectively. Various amounts (0.25 to 200 ng) of plasmin were electrophoresed into the fibrin gel, and subsequently washed in 2.5% Triton X-100 solution and incubated in a bath containing 0.1 M glycine-NaOH at 37°C for overnight as previously described (7). Figure 1 shows the fibrin zymogram in which plasmin appears as single clear bands of fibrinolysis against a blue background of undigested fibrin substrate. The enzyme amounts could be detected as low as 6.3 ng with an overnight incubation. This detection limit is comparable to those of gelatin and casein zymography (6) and Leber and Balkwill’s zymography for the quantitation of proteolytic activity on substrate gels (8). We have examined the possibility of using fibrin zymography to detect fibrinolytic enzymes from the 1
To whom correspondence should be addressed. Fax: 82-42-8604593. E-mail: [email protected]. ANALYTICAL BIOCHEMISTRY 263, 115–116 (1998) ARTICLE NO. AB982816
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
116
NOTES AND TIPS
culture media of Bacillus strains isolated from DoenJang, the Korean traditional fermented food. The reverse fibrin autography (Fig. 2A) shows two broad bands barely visualized on the fibrin/agar indicator gel (Fig. 2B). On the other hand, several polypeptides possessing fibrinolytic activity are detected as sharp, clear bands on the fibrin zymogram (Fig. 2B). Molecular markers are also perceptible by eye; they do not photograph well, though. It has been demonstrated that at least four fibrinolytic enzymes with apparent molecular sizes of approximately 64, 53, 38, and 29 kDa are secreted from each of two Bacillus strains isolated from Doen-Jang. The purification and characterization of the proteases will be described elsewhere. Even though gelatin and casein are satisfactory substrates for plasmin (7, 8), all fibrinolytic enzymes are not sensitive for the substrates as much as plasmin is. Apparently fibrin is an in vivo substrate for plasmin. We took advantage of this fact to explore the possibility of assaying plasmin-like enzymes with fibrin zymography, an electrophoretic technique in which fibrin is copolymerized with the acrylamide during casting of the separating gel. We have demonstrated that the zymographic technique can be used reliably for the rapid qualitative evaluation of plasmin-like enzymes, which have the ability to restore enzymatic activity on the gel copolymerized with fibrin upon removal of the SDS, with a sensitivity level comparable to that of other zymographic techniques.
FIG. 2. Fibrin zymogram (A) and reverse fibrin autography (B) of the culture media of two Bacillus strains (designated as DJ-4 and DJ-8) isolated from Doem-Jang, a Korean traditional fermented food; 10 ml of the crude enzyme solution, which was obtained by centrifugation of cells at 10,000g for 10 min, was applied for each assay. Fibrin autography was carried out as previously described (1). Lanes 1 and 2 for each assay are the crude enzyme solutions from DJ-4 and -8, respectively.
REFERENCES 1. Granelli-Piperno, A., and Reich, E. (1978) J. Exp. Med. 148, 223–234. 2. Sprengers, E. D., Verheijen, J. U., van Hinshergh, V. W. M., and Emeis, J. J. (1984) Biochim. Biophys. Acta 801, 163–170. 3. Loskutoff, D. J., van Mourik, J. A., Erickson, L. A., and Lawrence, D. (1983) Proc. Natl. Acad. Sci. USA 80, 2956 –2960. 4. Paech, C., Christianson, T., and Maurer, K.-H. (1993) Anal. Biochem. 208, 249 –254. 5. Kleiner, D. E., and Stetler-Stevenson, W. G. (1994) Anal. Biochem. 218, 325–329. 6. Raser, K. J., Posner, A., and Wang, K. K. W. (1995) Arch. Biochem. Biophys. 319, 211–216. 7. Heussen, C., and Dowdle, E. B. (1980) Anal. Biochem. 102, 196 –202. 8. Leber, T. M., and Balkwill, F. R. (1997) Anal. Biochem. 249, 24 –28.
Effect of a Polyhistidine Terminal Extension on Eglin c Stability Jennifer C. Waldner,* Steven J. Lahr,† Marshall Hall Edgell,‡ and Gary J. Pielak*,1 FIG. 1. Fibrin zymogram of plasmin. Various amounts (6.3 to 200 ng, as indicated) of plasmin were electrophoresed into a fibrin-copolymerized gel at 10 mA constant currency and subsequently washed with 2.5% Triton X containing Tris (50 mM) buffer (pH 7.4) to remove SDS. After washing the gel in distillation water for 30 min, the gel was incubated in 30 mM Tris buffer (pH 7.4) containing 200 mM NaCl, 10 mM CaCl2, and 0.02% NaN3 at 37°C for 16 h. The zymogram shown here was a Coomassie blue-stained fibrin gel. As little as 6.3 ng (lane 7) of plasmin can be visualized in the present study. Minor bands in lanes 2 and 3 are believed to be due to self-digestion and aggregation of plasmin during sample preparation. Lane 1, protein markers.
*Department of Chemistry, †Department of Biochemistry & Biophysics, and ‡Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Received May 20, 1998
Polyhistidine terminal extensions (his tags) are attached to recombinant proteins to facilitate purifica1
To whom correspondence should be addressed. Fax: 966-3675. E-mail: [email protected]. ANALYTICAL BIOCHEMISTRY 263, 116 –118 (1998) ARTICLE NO. AB982808
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.