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Kazushi Imai

Yasunori Okada

Assays for matrix metalloprotein activity

To detect activities of MMP-1, MMP-2, MMP-3, MMP-7 and MMP-9, use radio-labeled fibrillar collagen \(for MMP-1), gelatin \(for MMP-2 and MMP-9) and carboxymethylated transferrin \(for MMP-3 and MMP-7) substrates for the assays 1, although these can be substituted with commercially available FITC-labeled substrates and fluorogenic synthetic peptides for the MMPs. Zymography using gelatin or casein substrate gels is also useful to monitor gelatinolytic activity of MMP-2 and MMP-9 or caseinolytic activity of MMP-3 and MMP-7 in each column fraction, although zymography is not suitable for calculation of purification fold and recovery of proteinases.

**Collagenase assay for MMP-1** 


1. Mix a small amount of \[14C]-labeled collagen \(23 ml) with unlabeled collagen in 3% \(vol/vol) acetic acid \(45 ml; 3 mg/ml) to make the count 3000 c.p.m. in 50 &#x3BC;l. 
 2. Dialyze the mixture against 0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.02% \(wt/vol) NaN3 and then 0.01 M Tris-HCl, pH 7.5, 0.2 M NaCl, 0.02% \(wt/vol) NaN3 at 4&#x2DA;C, and store the collagen at 4&#x2DA;C. 
 3. For collagen fibril assay, take 50 &#x3BC;l of the labeled collagen solution into 400 &#x3BC;l long centrifuge tubes \(150 &#x3BC;g/tube), and incubate with 10 &#x3BC;l of each or combined fraction in TNC or TNC+B buffer, pH 7.5 \(225 &#x3BC;l) in the presence or absence of 1 mM APMA \(15 &#x3BC;l of 20 mM APMA) at 37&#xB0;C \(total volume of 300 &#x3BC;l). 
 4. After incubation, spin the tubes at 10,000 g for 15 min using Beckman microcentrifuge and take 200 &#x3BC;l of the supernatant for counting using a scintillation counter. 
 &#x25B2;CRITICAL For the controls, add 250 &#x3BC;l of TNC or TNC+B buffer, pH 7.5 \(blank), 10 &#x3BC;l of 100 &#x3BC;g/ml trypsin and 240 &#x3BC;l of the buffer \(negative control) or 10 &#x3BC;l of 4 mg/ml bacterial collagenase and 240 &#x3BC;l of the buffer \(positive control) in the assay. Activity is calculated as follows: \(count of enzyme solution digestion &#x2013; count of trypsin digestion) / \(count of bacterial collagenase digestion &#x2013; count of trypsin digestion) x 150 &#x3BC;g/min. Although we have used \[14C]-labeled collagen as a substrate, collagenase assays using FITC-labeled type I collagen \(collagenase assay kit for collagenase, Chondrex, "http://www.chondrex.com":http://www.chondrex.com), fluorogenic peptide substrate \(MMP-1 Substrate I, Fluorogenic, MERC, "http://www.merckbioscience.com/":http://www.merckbioscience.com/) or FRET substrate \(SensoLyte Plus&#x2122; 520 MMP-1 assay kit, AnaSpec, "http://www.anaspec.com":http://www.anaspec.com) are commercially available and can be used to detect MMP-1 activity. 





**Gelatinase assay for MMP-2 and MMP-9** 


1. Prepare heat-denatured gelatin by incubation of the \[14C]-labeled collagen at 60&#x2DA;C for ~30 min. 
 2. Take 50 &#x3BC;g of the gelatin solution into 400 &#x3BC;l long centrifuge tubes, and incubate with enzyme solution in TNC or TNC+B buffer, pH 7.5 \(135 &#x3BC;l) in the presence or absence of 1 mM APMA \(15 &#x3BC;l of 20 mM APMA) at 37&#x2DA;C \(total volume of 200 &#x3BC;l). 
 3. After incubation, add 100 &#x3BC;l of cold 45% \(vol/vol) trichloroacetic acid. Keep samples in ice for at least 10 min and spin at 10,000 g for 15 min. 
 4. Take 100 &#x3BC;l of the supernatant for counting using a scintillation counter. 
 &#x25B2;CRITICAL As for controls, add 150 &#x3BC;l of TNC or TNC+B buffer, pH 7.5 \(blank) or 10 &#x3BC;l of 100 &#x3BC;g/ml trypsin and 140 &#x3BC;l of the buffer \(positive control) in the assay. Activity is calculated as follows: \(count of enzyme solution digestion &#x2013; count of blank) / \(count of trypsin digestion &#x2013; count of blank) x 150 &#x3BC;g/min. Although we have used \[14C]-labeled gelatin as a substrate, quenched fluorescent peptide substrate \(EnzChek for gelatinases, Invitrogen, "http://www.invitrogen.com/":http://www.invitrogen.com/) is commercially available and can be used to detect MMP-2 and MMP-9 activities. 





**Carboxymethylated transferrin \(Cm-Tf) assay for MMP-3 and MMP-7** 


1. Reduce human transferrin \(Tf) \(20 mg) by incubation with 20 mM dithiothreitol in 20 ml of 0.2 M Tris-HCl, pH 8.6 containing 8 M urea for 4 h at 37&#x2DA;C. 
 2. Apply the reduced Tf to a column of Sephadex G-50 \(&#x3A6; 2.5 cm x 20 cm) equilibrated in 0.2 M Tris-HCl, pH 8.6 containing 8 M urea at room temperature to remove dithiothreitol, and combine the void volume. 
 3. Incubate the pooled sample with 1 mCi of \[3H]iodoacetic acid for 30 min at 23&#x2DA;C, and then with 30 mM iodoacetic acid for 30 min at 23&#x2DA;C to complete carboxymethylation. 
 4. After the reactions, add the same volume of cold 10% \(vol/vol) trichloroacetic acid to the radiolabeled Tf and obtain the precipitate by centrifugation at 21,000 g for 5 min at 4&#x2DA;C using a benchtop centrifuge. Repeat this precipitation step three times. 
 5. Dissolve the precipitate in 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl at a concentration of 3 mg/ml. Store the substrate at -20&#x2DA;C in 1 ml portions. 
 6. For the assay, take 10 &#x3BC;l of \[3H]Cm-Tf into 1.5 ml Eppendorf tubes, and incubate with 10 &#x3BC;l of each or combined fraction in TNC or TNC+B buffer, pH 7.5 in the presence or absence of 1 mM APMA \(10 &#x3BC;l of 3 mM APMA) at 37&#x2DA;C \(total volume of 30 &#x3BC;l). After incubation, add 200 &#x3BC;l of cold 3.3% \(vol/vol) trichloroacetic acid. Keep samples at room temperature for 15 min and spin at 10,000 g for 5 min. Take 100 &#x3BC;l of the supernatant for counting by a scintillation counter. 
 &#x25B2;CRITICAL As for controls, add 10 &#x3BC;l of TNC or TNC+B buffer, pH 7.5 \(negative control) or 10 &#x3BC;l of 100 &#x3BC;g/ml trypsin in TNC buffer, pH 7.5 \(positive control) in the assay. Activity is calculated as follows: \(count of enzyme solution digestion &#x2013; count of blank) / \(count of trypsin digestion &#x2013; count of blank) x 30 &#x3BC;g/min. Although we have used \[3H]-labeled Cm-Tf as a substrate, fluorogenic peptide substrates \(Oncogene, "http://www.apoptosis.com":http://www.apoptosis.com or MERCK, "http://www.merckbioscience.com":http://www.merckbioscience.com) are commercially available and can be used to detect MMP-3 and MMP-7 activities.





**Zymography** 


1. Make SDS-PAGE gels \(1 or 2 mm in thickness) containing 0.2% \(wt/vol) gelatin or 0.1% \(wt/vol) casein. 
 2. Take 2040 &#xB5;l of samples from column fractions, and incubate them with equal volume of SDS-PAGE sample buffer without 2-mercaptoethanol for 30 min at 37&#x2DA;C. Subject the samples to SDS-PAGE at 4&#x2DA;C. 
 3. After electrophoresis, wash the gels in 2.5% \(vol/vol) Triton X-100 in TNC buffer, pH 7.5 for 15 min twice and then incubate in the incubation buffer at 37&#x2DA;C for 12 - 24 h under shaking. 
 4. After incubation, stain with Coomassie Brilliant Blue R and destain the gels. 
 &#x25B2;CRITICAL Rinsing the gels with 2.5% \(vol/vol) Triton X in TNC buffer, pH 7.5 is necessary to remove SDS from the gels. 


&#x25B2;CRITICAL Activation of proMMPs with APMA is not necessary for zymography, since proMMPs are spontaneously activated during SDS-PAGE probably because of their conformational changes. Proteolytic bands corresponding to proMMPs and active MMPs are seen as transparent bands within dark blue background. 


&#x25B2;CRITICAL Inhibitor studies can be done by adding appropriate inhibitors \(e.g., 15 mM EDTA for MMPs, 2 mM phenylmethane sulfonyl fluoride for serine proteinases, 5 mM N-ethylamaleimide for cysteine proteinases and 0.5 mM pepstatin A for aspartic acid proteinases) into the incubation buffer during incubation of the gels.

1. Okada, Y., Nagase, H. &amp; Harris, E.D., Jr. A metalloproteinase from human rheumatoid synovial fibroblasts that digests connective tissue matrix components. Purification and characterization. _J Biol Chem_ **261**, 14245-55 \(1986).

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Biochemistry

Computational biology and bioinformatics

Method Article

Kazushi Imai, Yasunori Okada

Kazushi Imai

Department of Biochemistry, Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan

Yasunori Okada

Department of Pathology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-0016, Japan

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10.1038/nprot.2008.85

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Kazushi Imai, Yasunori Okada

Kazushi Imai

Department of Biochemistry, Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan

Yasunori Okada

Department of Pathology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-0016, Japan

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