Assaying Proteases in Cellular Environments

Kevin K.W. Wang1

1 Pfizer Global Research and Development, Ann Arbor, Michigan
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.12
DOI:  10.1002/0471140864.ps2112s27
Online Posting Date:  May, 2002
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Abstract

To better study protease activation, regulation, and inhibition physiologically or pharmacologically, it is often advantageous to use cell‐based systems. The introduction of the cellular environment poses additional challenges for the assay of the protease of interest. In this unit, various methods for monitoring protease activity in cell‐based systems are discussedctivity is discussed. The scope of the unit is restricted to mammalian cell system. It is also stressed that specific considerations or requirements for a particular protease or peptidase are often needed in order to optimize the assay.

     
 
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Table of Contents

  • Basic Protocol 1: Monitoring Intracellular Protease Activity In Situ with Cell‐Permeable Peptide Substrate
  • Basic Protocol 2: Monitoring Intracellular Protease Activity In Situ by Autolytic Activation and Endogenous Substrate Protein Degradation
  • Basic Protocol 3: Monitoring Protease Activity Levels as Reflected in Cell Lysate
  • Basic Protocol 4: Monitoring Activity of Secreted Protease In Situ
  • Basic Protocol 5: Using Zymography to Measure Activity of Cell‐Secreted MMPs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Monitoring Intracellular Protease Activity In Situ with Cell‐Permeable Peptide Substrate

  Materials
  • Cell line or primary cell culture with demonstrated expression of calpain I or II, e.g., human neuroblastoma SH‐SY5Y cells (Wang et al., )
  • DMEM medium, serum‐free (Sigma), containing 0.8 mM CaCl 2
  • 40 mM stock solution of succinyl‐Leu‐Leu‐Val‐Tyr‐7‐amido‐4‐methylcoumarin (SLLVY‐AMC; Sigma) in DMSO
  • Calcium channel opener: 1 µM maitotoxin (MTX) or 10 mM calcium ionophore A 23187 (Calbiochem) in DMSO
  • Cell culture medium, serum‐free, containing 5 mM EDTA
  • Calpain inhibitor I (Ac‐Leu‐Leu‐nLeu‐CHO; Calbiochem)
  • 12‐well plates (tissue–culture treated; Corning)
  • Fluorescence plate reader (e.g., Millipore Cytofluor 2300)
  • Additional reagents and equipment for cell culture ( appendix 3C)

Basic Protocol 2: Monitoring Intracellular Protease Activity In Situ by Autolytic Activation and Endogenous Substrate Protein Degradation

  Materials
  • Cell type expressing protease of interest
  • Serum‐free DMEM medium (Sigma), containing 0.8 mM CaCl 2
  • Calcium channel opener: maitotoxin (MTX) or calcium ionophore (Calbiochem)
  • recipeLysis buffer I (see recipe)
  • recipeSDS sample buffer (see recipe)
  • 4% to 20% polyacrylamide gradient Tris‐glycine mini‐gel (Novex)
  • Rainbow‐colored molecular weight markers (RPN756: Amersham)
  • recipeRunning buffer (see recipe)
  • recipeTBST (see recipe)
  • 5% (w/v) nonfat milk in recipeTBST (see recipe for recipeTBST)
  • Primary antibody: Monoclonal anti‐calpain I antibody diluted 1:500 in recipeTBSTor monoclonal anti‐α‐spectrin antibody diluted 1:3000 in recipeTBST (both antibodies available from Chemicon; see recipe for TBST)
  • Secondary antibody: biotinylated anti‐mouse IgG diluted 1:1000 in TBST (see recipe for TBST)
  • Alkaline phosphatase–conjugated avidin diluted 1:3000 in recipeTBST (see recipe for recipeTBST)
  • 12‐well plates (tissue culture–treated; Corning)
  • PVDF membrane (Novex), presoaked with 100% methanol
  • Semidry electrotransfer unit (e.g., Bio‐Rad)
  • Additional reagents and equipment for protein assay (unit 3.4), SDS‐PAGE (unit 10.1), electroblotting onto PVDF membranes (unit 10.7), and immunoblot detection (unit 10.10)

Basic Protocol 3: Monitoring Protease Activity Levels as Reflected in Cell Lysate

  Materials
  • Human neuroblastoma SH‐SY5Y cells growing in 12‐well plates
  • Serum‐free DMEM medium (Sigma) containing 0.8 mM CaCl 2
  • Staurosporine (Calbiochem)
  • recipeTBS‐EDTA (see recipe), room temperature
  • recipeLysis buffer II (see recipe), 4°C
  • Glycerol
  • recipeSubstrate solution for caspase‐3 (see recipe)
  • Caspase inhibitor: carbobenzoxy‐Asp‐CH 2OC(=O)‐2,6‐dichlorobenzene (Z‐D‐DCB; Bachem)
  • 12‐well plates (tissue culture–treated; Corning)
  • Fluorescence plate reader (e.g., Millipore Cytofluor 2300)

Basic Protocol 4: Monitoring Activity of Secreted Protease In Situ

  Materials
  • Human mast cells or other cells that express chymase/tryptase, growing in culture
  • Serum‐free RPMI medium
  • Immunoglobulin for activation and degranulation of mast cells, e.g., IgE
  • 1 M HEPES, pH 7.4
  • recipeSubstrate solution for tryptase/chymase (see recipe)
  • Serine protease inhibitors (unit 21.7):
  •  Diisopropyl fluorophosphate (for both serine proteases; Sigma)
  •  Tosyllysine chloromethyl ketone (TLCK, for tryptase; Sigma)
  •  Tosylphenylalanine chloromethyl ketone (TPCK, for chymase; Sigma)
  • 12‐well plates (tissue culture–treated; Corning)
  • 96‐well microtiter plates
  • UV‐visible spectrophotometer with microtiter plate reader (e.g., Spectramax; Molecular Devices)

Basic Protocol 5: Using Zymography to Measure Activity of Cell‐Secreted MMPs

  Materials
  • Human fibroblasts growing in cell culture
  • DMEM medium (Sigma), serum free
  • Phorbol ester: e.g., phorbol‐12‐myristate‐13‐acetate to induce MMP‐2 and MMP‐9
  • 100 mM EGTA
  • recipeSDS sample buffer (see recipe) without 2‐mercaptoethanol
  • Precast gelatin PAGE gel (Novex)
  • recipeRunning buffer (see recipe)
  • SDS‐PAGE molecular‐weight standards (broad‐range, Bio‐Rad)
  • Reactivation buffer: 2.5% (v/v) Triton X‐100/100 mM glycine, pH 8.3
  • Proteolysis buffer: 100 mM glycine, pH 8.3
  • Fixing/destaining solution: 5:4:1 methanol:water:glacial acetic acid
  • 0.25% (w/v) Coomassie blue R‐250 in fixing/destaining solution
  • 2.5% (v/v) acetic acid
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1)
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Figures

Videos

Literature Cited

Literature Cited
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   Caughey, G.H., Viro, N.F., Lazarus, S.C., and Nadel, J.A. 1988. Purification and characterization of dog mastocytoma chymase: Identification of an octapeptide conserved in chymotryptic leukocyte proteinases. Biochim. Biophys. Acta. 952:142‐149.
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