Zymography of extracellular matrix proteases

170 and incubated overnight at 37°C. Finally, the gel was stained with a fixing mixture containing Coomassie Blue R for 30 min and destained with the same mixture without the dye.

Experimental Section 0307-4412(95)00165-4 Zymography of extraceilular matrix proteases ANA R QUESADA, I G N A C I O FAJARDO, D A N I E L R O D R [ G U E Z - A G U D O , JOSI~ MIGUEL ANGEL MEDINA

MANUEL

PACHON,

Laboratorio de Bioquimica y Biologia Molecular Facultad de Ciencias Universidad de M~laga E-29071 M~laga, Spain Introduction Electrophoretic techniques are widely used in biochemistry laboratories. Most Experimental Biochemistry courses for undergraduate students devote some effort to training students in electrophoresis. They are mostly taught to perform denaturating SDS-PAGE or submarine agarose electrophoresis to separate proteins or nucleic acids by weight. However, electrophoretic techniques are also potent and convenient for detecting specific enzyme activities by the technique referred to as zymography. Proteolytic activities, and specifically those of extracellular matrix proteases, are easily and conveniently detected by the use of zymographic techniques 1,2 Zymographic techniques are extensively used for the detection of proteinase activities because they display several advantages, including their simplicity and high sensitivity. Moreover, they allow the identification of the enzyme of interest in a complex mixture of proteinases, and the determination of the molecular weight of latent and active forms of the enzymes due to the processes of denaturation-renaturation carried out in the gel. Samples to be tested are submitted to a non-reducing SDS-PAGE; afterwards, protein activities are reconstituted in situ by washing with buffers containing the zwitterionic detergent Triton X-IO0. Finally, specific activities are detected in situ or with the use of an overlaid gel. In this report, we describe the use of zymography to detect type IV collagenases and other matrix metalloproteases, as well as plasminogen activators. We also offer some cheap alternatives to train students in zymography.

Experimental Conditioned media and cell extracts To prepare conditioned media, cells such as fibroblasts are grown in six-well plates. When subconfluent, medium was aspirated, cells were washed twice with phosphate-buffered saline and each well received 1 ml of culture medium without serum and with 200 KIU of Trasylol. Cells were washed twice with phosphate-buffered saline and extracted by using 0.2% Triton X-100 in 0.2 M TrisHCI. Both conditioned media and cell extracts were centrifuged for 10 min at 1000 g and supernatants were stored at -20°C until used. In duplicate samples, cells were trypsinized and counted.

Gelatin zymography Conditioned media and cell extracts were submitted without previous heating to SDS-PAGE in the cool room, as described by Laemmli, 3 with the following modifications. Reducing agent (DTI' or mercaptoethanol) was absent and the resolving gel contained gelatin (1 mg/ml). After electrophoresis, the gel was washed twice in 50 mM Tris-HCl, pH 7.4, supplemented with 2% Triton X-100, and twice with 50 nM TrisHCI (pH 7.4). Each washing step was carried out for 10 min at room temperature and with agitation. Afterwards, the resolving gel was covered with a substrate buffer (composition: 50 mM Tris-HCl, 5 mM CaC12, 1% Triton X-100, 0.902% Na3N, pH 7.4) BIOCHEMICAL EDUCATION 24(3) 1996

Casein zymography The procedure was as described for the gelatin zymography but with casein (0.5 mg/ml) instead of gelatin in the resolving gel. As previously shown, 4 a pre-run of the casein gel is required.

Plasminogen activator zymography

In this case, there was no addition to the resolving gel. After electrophoresis and washing, a substrate gel (composition: 2.7% skimmed dry milk, 0.8% agar, 0.0005% Na3N and plasminogen 0.44 KUI/ml in phosphate buffered saline) was laid over the resolving gel. The system was left overnight in a humidified chamber at 4°C, and then it was incubated in the humidified chamber at 37°C until lytic bands were detectable on a dark field,

Activation with APMA

Extracellular matrix proteases are activable by treatment with 1 mM p-aminophenyl mercuric acetate (APMA) for lh at 37°C.

Suggestions for a practical on zymography for undergraduated students. The procedures described are easy to carry out and can be performed by students in a two-day session. In the first day, students are provided with the theoretical background, and they carry out the electrophoresis, and washing. In the second day, they carry out the staining and destaining, they observe the results and discuss them. In those labs with access to culture cell facilities, students should be provided with conditioned media and/or cell extracts from one or several cell lines. However, this requirement is not a limitation and some cheap alternatives are possible for those labs without access to culture cell facilities. In fact, some inexpensive commercially available proteases could substitute for the conditioned media. They include trypsin, pepsin or the collagenase extracted from Clostridum histo~yticum (reference C0-7926 in the Sigma catalogue). When students are provided with commercial proteases, they could be asked to prepare successive dilutions from a stock in order to determine an optimal concentration for the easy observation of the proteolytic activity. For instance, in the case of Clostridum histolyticum collagenase a dilution series between 100 /~g/ml and 100 pg/mol would be convenient. In the same way, an alternative to conditioned media for plasminogen zymography could be a serial dilution of the commercially available urokinase.

Results In both casein and gelatin zymographies, proteolytic bands are observed as unstained bands in an otherwise stained resolving gel. In plasminogen zymography, the lysis band is observed as a dark band surrounded by clear casein gel when observed on a dark field. We have successfully used these zymographic techniques in our studies of extracellular proteases produced by cancer cells, 5 including neuroblastoma, rhadomyosarcoma, osteosarcoma, and mammary carcinoma cells. A convenient, easily maintained and non-hazardous cell line is Ehrlich ascites carcinoma. Clearly observable bands are obtained with media conditioned by Ehrlich cells and Ehrlich cell extracts in both gelatin-zymograms (two main bands, corresponding to 92 and 72 kDa gelatinases; see Figure 1) and casein-zymograms (a band, corresponding to the 92 kDa gelatinase, which also exhibits

171

Figure 1 Gelatin-zymography of media conditioned by Ehrlich cells and Ehrlich cell extracts. Lanes 1-3 and 7." 151d of medium conditioned by Ehrlich cells. Lanes 4-6." 15 /11 of Ehrlich cell extracts. Lane 9: Molecular weight markers. Apparent molecular weight of the three observed bands: 94, 67 and 43 kDa

Figure 3 Gelatin-zymography of Clostridium histolyticum collagenase. Samples containing ten-fold dilutions between 15 ng and 1.5 pg of this commercially available collagenase were applied to lanes 1 to 5 Treatment of proteases with APMA produces a cut, giving raise to an active fragment of lesser molecular weight. In the gel, the activated protease is observed as a band lower than the native band (see Figure 2). Figure 3 shows the result obtained when a serial dilution of Clostridum histolyticum collagenase was applied to a gel containing gelatin. Two of the authors (DRA and JMP) were undergraduate students in a course on General Biochemistry for Biologists during the optimisation of the procedures here described and they were actively involved in the project.

Acknowledgements Supported by a Grant from the UMA (University of M~tlaga).

Figure 2 Gelatin-zymography of diluted media conditioned by Ehrlich cells pre-treated or not with APMA. Lanes 1 and 3: Two dilutions of control conditioned media not pre-treated. Lane 2: A diluted, conditioned medium pre-treated with APMA as described in the Experimental section caseinolytic activity; results not shown). A very well defined band is obtained for the plasminogen zymography with media conditioned by A-204 human rhabdomyosarcoma cells (results not shown).

BIOCHEMICAL EDUCATION 24(3) 1996

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