Handling Metalloproteinases

Sven Fridrich1, Konstantin Karmilin1, Walter Stöcker1

1 Johannes Gutenberg University Mainz, Institute of Zoology, Cell and Matrix Biology
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.16
DOI:  10.1002/0471140864.ps2116s83
Online Posting Date:  February, 2016
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Substrate cleavage by metalloproteinases involves nucleophilic attack on the scissile peptide bond by a water molecule that is polarized by a catalytic metal, usually a zinc ion, and a general base, usually the carboxyl group of a glutamic acid side chain. The zinc ion is most often complexed by imidazole nitrogens of histidine side chains. This arrangement suggests that the physiological pH optimum of most metalloproteinases is in the neutral range. In addition to their catalytic metal ion, many metalloproteinases contain additional transition metal or alkaline earth ions, which are structurally important or modulate the catalytic activity. As a consequence, these enzymes are generally sensitive to metal chelators. Moreover, the catalytic metal can be displaced by adventitious metal ions from buffers or biological fluids, which may fundamentally alter the catalytic function. Therefore, handling, purification, and assaying of metalloproteinases require specific precautions to warrant their stability. © 2016 by John Wiley & Sons, Inc.

Keywords: metzincin; MMP; ADAM; ADAMTS; astacin; meprin; tolloid

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Table of Contents

  • Introduction
  • Basic Protocol 1: Purification of Meprin α from Transfected Sf21 Insect Cells
  • Support Protocol 1: Cultivation and Infection of Insect Cells
  • Basic Protocol 2: Activation of Meprin α and FRET Assay
  • Basic Protocol 3: Using Azocasein to Measure Proteinase Activity
  • Alternate Protocol 1: Zymography to Measure Proteinase Activity
  • Basic Protocol 4: Activation of pro‐MMPs with Amino‐Phenyl‐Mercuric Acetate (APMA)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Purification of Meprin α from Transfected Sf21 Insect Cells

  • Ammonium sulfate
  • Promeprin α conditioned medium from infected insect cells (Köhler et al., ; see protocol 6Support Protocol)
  • 50 mM HEPES/NaOH, pH 7.5, pH 3.5, and pH 10.4
  • Sephacryl S‐300 (GE Healthcare)
  • Pro‐Leu‐Gly‐hydroxamate (PLG‐NHOH)
  • CH‐Sepharose‐4B (GE Healthcare)
  • Centrifuge (cooled)
  • 10,000 MWCO dialysis membrane
  • 240‐ml, 3.5‐cm chromatography column
  • Chromatography system (e.g., ÄKTA Prime, GE Healthcare)
  • 10 ml polypropylene column (Thermo Scientific, cat. no. 29924)
  • Additional reagents and equipment for dialysis ( appendix 3B; Zumstein, ), activation of promeprin α ( protocol 2, step 1), measurement of proteolytic activity using FRET substrate ( protocol 2, step 3) or azocasein ( protocol 3), immunoblotting (unit 10.10; Gallagher, 1996; also see Dumermuth et al., ), and SDS‐PAGE (unit 10.1; Gallagher, )

Support Protocol 1: Cultivation and Infection of Insect Cells

  • BTI‐TN‐5B1‐4 insect cells (High Five; Thermo Fisher, cat. no. B855‐02)
  • Express Five serum‐free medium (Thermo Fisher, cat. no. 10486‐025)
  • Penicillin/streptomycin mix (Thermo Fisher, cat. no. 15140‐122)
  • Recombinant baculovirus containing DNA construct of interest
  • Incubator‐shaker
  • Spinner flask (250 ml)
  • Fernbach flask (2800 ml)
  • Centrifuge
  • Additional reagents and equipment for counting cells ( appendix 2E; Phelan, )

Basic Protocol 2: Activation of Meprin α and FRET Assay

  • 1 μM recombinant promeprin α or β (meprin α, R&D Systems, cat. no. 3220‐ZN‐010; meprin β, R&D Systems, cat. no. 2895‐ZN‐010) in 50 mM HEPES/NaOH, pH 7.5
  • 0.54 μM bovine trypsin in 50 mM HEPES/NaOH, pH 7.5
  • 400 mM Pefabloc (e.g., Sigma‐Aldrich) in distilled H 2O (since Pefabloc has a limited half‐life of only 2 hr in aqueous solutions, it must be prepared fresh each time before use.)
  • Dimethylsulfoxide (DMSO)
  • Fluorogenic substrate: Ac‐R‐E(Edans)‐DR‐Nle‐VGDDPY‐K(Dabcyl)‐amide [Ac = acetyl; EDANS = (5‐((2‐aminoethyl)amino)naphthalene‐1‐sulfonic acid); Dabcyl = 4‐((4‐(dimethylamino)phenyl)azo)benzoic acid)]; store protected from light at up to several months at –20°C
  • 50 mM HEPES/NaOH, pH 7.5 (Biosyntan)
  • 25 mg/ml proteinase K in 50 mM HEPES/NaOH, pH 7.5, for complete substrate turnover
  • 96‐well black microtiter plate (Thermo Scientific, cat. no. 237105)
  • Varioskan Flash 3001 spectral plate reader (Thermo Scientific)

Basic Protocol 3: Using Azocasein to Measure Proteinase Activity

  • 12 mg/ml azocasein (sulfanilamide‐azocasein; Sigma‐Aldrich, cat. no. A2765) in a suitable buffer (azocasein can be dissolved in a broad variety of different buffers; for accurate kinetic measurements, make sure that all azocasein is dissolved; if not, heat the solution to 65°C; centrifuge for 5 min at maximum speed to remove non‐dissolved protein)
  • Suitable buffer for the particular proteinase under study (see recipes in Reagents and Solutions)
  • Proteinase of interest
  • 10% glacial acetic acid: 50 g of glacial acetic acid in 227 ml of H 2O
  • Thermostatted water bath
  • Centrifuge (cooled)
  • Spectrophotometer

Alternate Protocol 1: Zymography to Measure Proteinase Activity

Basic Protocol 4: Activation of pro‐MMPs with Amino‐Phenyl‐Mercuric Acetate (APMA)

  • Amino‐phenyl‐mercuric acetate (APMA; Sigma‐Aldrich, cat. no. A9563)
  • Dimethyl sulfoxide (DMSO)
  • MMP in suitable buffer, e.g., MMP buffer, BTP buffer, ADAM buffer, or ovastacin buffer (see recipes for these buffers in Reagents and Solutions)
  • Thermostatted water bath
NOTE: Since APMA has a limited half‐life, it has to be prepared fresh each time before use. Due to its poor solubility in water, addition of any kind of salt will lead to precipitation of APMA in the stock solution.
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