Aldehyde Dehydrogenases: Measurement of Activities and Protein Levels

Lakshmaiah Sreerama1, Norman E. Sládek2

1 St. Cloud State University, Saint Cloud, Minnesota, 2 University of Minnesota, Minneapolis, Minnesota
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.18
DOI:  10.1002/0471140856.tx0418s26
Online Posting Date:  December, 2005
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Abstract

Seventeen enzymes are currently viewed as belonging to the human aldehyde dehydrogenase superfamily, and all of them catalyze the pyridine nucleotide–dependent oxidation of aldehydes to acids. Depending on the specific aldehyde dehydrogenase, lack of sufficient catalytic activity (1) results in a gross pathological phenotype in the absence of any insult, or (2) is ordinarily of no consequence with respect to gross phenotype, but is of consequence when the organism is subjected to a relevant insult. Described in this unit are eight assays that can be used to (semi)quantify various in vitro aldehyde dehydrogenase protein and/or catalytic activity levels, and two that can be used to semiquantify various in situ aldehyde dehydrogenase protein and/or catalytic activity levels. Aldehyde dehydrogenases also catalyze the hydrolysis of esters; this unit includes an assay that can be used to quantify that catalytic activity as well. Preparation of test materials and of antibodies to the aldehyde dehydrogenases are described in three support protocols.

Keywords: Aldehyde dehydrogenases; ALDH bioassays; substrate specificity; spectrophotometry; HPLC; ester hydrolysis; ALDH2 purification; ELISA; anti‐ALDH1A1; immunoblotting; cytochemical/histochemical localization of ALDHs

     
 
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Table of Contents

  • Basic Protocol 1: Spectrophotometric Quantification of in vitro NAD‐Linked Aldehyde Dehydrogenase–Catalyzed Octanal Oxidation
  • Alternate Protocol 1: Spectrophotometric Quantification of in vitro Aldehyde Dehydrogenase–Catalyzed Oxidation and Reduction of Isozyme‐Specific Substrates and Proton Acceptors
  • Alternate Protocol 2: Microtiter Plate–Based Spectrophotometric Quantification of in vitro NAD(P)‐Linked Aldehyde Dehydrogenase–Catalyzed Oxidation of Relatively Isozyme‐Specific and Isozyme‐Nonspecific Substrates
  • Alternate Protocol 3: HPLC‐Based Spectrophotometric Quantification of in vitro NAD(P)‐Linked Aldehyde Dehydrogenase–Catalyzed All‐‐Retinaldehyde Oxidation
  • Alternate Protocol 4: HPLC‐Based Spectrophotometric Quantification of in vitro NAD(P)‐Linked Aldehyde Dehydrogenase–Catalyzed Oxidation of Aldehydes that Contain Nitro or Amino Groups
  • Basic Protocol 2: Spectrophotometric Quantification of Aldehyde Dehydrogenase–Catalyzed Ester Hydrolysis with Purified Enzyme
  • Preparation and Long‐Term Storage of Samples for Aldehyde Dehydrogenase Assays
  • Support Protocol 1: Preparation and Storage of Samples from Cultured Cells
  • Support Protocol 2: Preparation and Storage of Homogenates from E. coli Engineered to Express an Aldehyde Dehydrogenase
  • Support Protocol 3: Preparation and Storage of Saliva Samples for Aldehyde Dehydrogenase Assays
  • Support Protocol 4: Preparation and Storage of Aldehyde Dehydrogenase from Tissues
  • Support Protocol 5: Purification of Hepatic and Recombinant E. coli ALDH2
  • Basic Protocol 3: Quantification of in vitro Aldehyde Dehydrogenase (ALDH1A1) Levels by ELISA
  • Support Protocol 6: Preparation of ALDH1A1 Antibodies
  • Basic Protocol 4: Gel‐Based Resolution, Visualization, and Semiquantification of Aldehyde Dehydrogenase Levels
  • Basic Protocol 5: Immunoblot Analysis of Aldehyde Dehydrogenase Levels
  • Basic Protocol 6: Immunocytochemical Visualization and Semiquantification of ALDH1A1 and ALDH3A1 in situ
  • Alternate Protocol 5: Histochemical Localization of ALDH1A1 and ALDH3A1
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Spectrophotometric Quantification of in vitro NAD‐Linked Aldehyde Dehydrogenase–Catalyzed Octanal Oxidation

  Materials
  • Assay buffer 1 (see recipe)
  • 80 mM NAD (see recipe)
  • 100 mM reduced glutathione (GSH; see recipe)
  • 2 mM pyrazole (see recipe)
  • 80 mM octanal (see recipe)
  • Test preparations (tissue, cells, sub‐cellular fraction, purified enzyme; Support Protocols protocol 71 to protocol 104)
  • UV/Vis recording spectrophotometer
  • 37°C water bath and water circulator or other device for maintaining constant temperature of 37°C in the cuvette holder
  • 5‐ml glass tubes
  • 37°C water bath or constant‐temperature dry bath for preincubating reaction mixtures and substrate
  • 1‐ml quartz cuvettes
  • Concentrators (optional; MWCO ≤30 kDa; see appendix 3H): e.g., Amicon fluid concentrators, Amicon spinal‐fluid concentrators, Amicon Centriprep concentrators (Millipore)
  • Additional reagents and equipment for determining concentration of protein solutions ( appendix 3H)

Alternate Protocol 1: Spectrophotometric Quantification of in vitro Aldehyde Dehydrogenase–Catalyzed Oxidation and Reduction of Isozyme‐Specific Substrates and Proton Acceptors

  • 80 mM NADP (see recipe)
  • Microtiter plate reader capable of readings at 340 nm, preferably equipped with a thermocontroller (Molecular Devices)
  • 50‐ml conical polypropylene centrifuge tubes
  • ELISA dispenser trays or 150‐mm petri dishes
  • 8‐ or 12‐channel automatic pipettors
  • 96‐well flat‐bottom microtiter plates (Falcon, Becton Dickinson)
  • Microtiter plate seals, or foil wrap

Alternate Protocol 2: Microtiter Plate–Based Spectrophotometric Quantification of in vitro NAD(P)‐Linked Aldehyde Dehydrogenase–Catalyzed Oxidation of Relatively Isozyme‐Specific and Isozyme‐Nonspecific Substrates

  • 1 mM retinoid working solutions: all‐trans‐retinaldehyde (all‐trans‐retinal) and all‐trans retinoic acid (see recipe)
  • Extraction solvent with internal standard (see recipe)
  • Potassium phosphate, saturated (see recipe)
  • Mobile phase elution solvent I (see recipe)
  • Test preparations (tissue, cells, subcellular fraction, purified enzyme; Support Protocols protocol 71 to protocol 104)
  • Ultrasphere‐ODS HPLC column (4.6‐mm i.d. × 25 cm)
  • 5‐µm Adsorbosphere ODS guard column
  • Beckman Model 322 HPLC system with Model 160 UV detector and Hewlett‐Packard 3390A integrator, or equivalent (also see unit 6.2)
NOTE: All stock solutions are prepared with HPLC‐grade solvents, degassed, and filtered through a 0.45‐µm membrane prior to use.NOTE: All steps involving the handling of retinoids should be performed under dim light.

Alternate Protocol 3: HPLC‐Based Spectrophotometric Quantification of in vitro NAD(P)‐Linked Aldehyde Dehydrogenase–Catalyzed All‐‐Retinaldehyde Oxidation

  • 1 mM NADH (see recipe)
  • 1 mM NADPH (see recipe)
  • Mobile phase elution solvent II (see recipe)
  • Working solutions of 4‐(diethylamino)benzaldehyde, nitrobenzaldehyde, or aminobenzaldehyde (see recipe)
  • Ultrasphere‐ODS HPLC column (4.6‐mm i.d. × 25 cm)
  • 5‐µm Adsorbosphere ODS guard column
  • Beckman Model 322 HPLC system with Model 160 UV detector and Hewlett‐Packard 3390A integrator, or equivalent (also see unit 6.2)
NOTE: All stock solutions are prepared with HPLC‐grade solvents, degassed, and filtered through a 0.45‐µm membrane prior to use.

Alternate Protocol 4: HPLC‐Based Spectrophotometric Quantification of in vitro NAD(P)‐Linked Aldehyde Dehydrogenase–Catalyzed Oxidation of Aldehydes that Contain Nitro or Amino Groups

  Materials
  • Assay buffer 2 (see recipe)
  • 1 mM p‐nitrophenol (see recipe)
  • 10 mM p‐nitrophenyl acetate (see recipe)
  • Test preparation: purified aldehyde dehydrogenase
  • UV/Vis recording spectrophotometer
  • 37°C water bath and water circulator or other device for maintaining constant temperature of 37°C in the cuvette holder
  • 5‐ml glass tubes
  • Concentrators (optional; MWCO ≤30 kDa; see appendix 3H): e.g., Amicon fluid concentrators, Amicon spinal‐fluid concentrators, Amicon Centriprep concentrators (Millipore)
  • Additional reagents and equipment for concentration of protein solutions ( appendix 3H)

Basic Protocol 2: Spectrophotometric Quantification of Aldehyde Dehydrogenase–Catalyzed Ester Hydrolysis with Purified Enzyme

  Materials
  • Cultured cells of interest
  • Cell lysis solution (see recipe)
  • 3% (v/v) Lubrol or Triton X‐100
  • Homogenization/storage buffer (see recipe)
  • Probe sonicator
  • Refrigerated centrifuge and ultracentrifuge
  • PD‐10 (Sephadex G‐25) columns (Amersham Biosciences)

Support Protocol 1: Preparation and Storage of Samples from Cultured Cells

  Materials
  • Recombinant E. coli growing in culture (∼12 to 14 hr)
  • E. coli lysis buffer (see recipe)
  • Homogenization/storage buffer (see recipe)
  • Probe sonicator
  • Refrigerated centrifuge
  • PD‐10 (Sephadex G‐25) columns (Amersham Biosciences)

Support Protocol 2: Preparation and Storage of Homogenates from E. coli Engineered to Express an Aldehyde Dehydrogenase

  Materials
  • Human donor
  • 505 mM DTT (see recipe)
  • Homogenization/storage buffer (see recipe)
  • 15‐ml graduated tube
  • Refrigerated centrifuge

Support Protocol 3: Preparation and Storage of Saliva Samples for Aldehyde Dehydrogenase Assays

  Materials
  • Fresh or frozen tissue
  • Homogenization/storage buffer (see recipe), 4°C
  • 3% (v/v) Lubrol or Triton X‐100
  • Balance
  • Scalpel
  • Tissue tearer or blender
  • Mortar and pestle or grater
  • Dounce homogenizer with type B pestle

Support Protocol 4: Preparation and Storage of Aldehyde Dehydrogenase from Tissues

  Materials
  • Glycerol stock of E. coli (BL21[DE3]pLysS) transfected with ALDH2 cDNA ligated to a pT7‐7 vector (prepared from single colony) or solubilized whole‐cell homogenate of human liver (see protocol 10, steps to ; human liver may be obtained from any number of sources including the National Cancer Institute's Cooperative Human Tissue Network, http://www‐chtn.ims.nci.nih.gov/)
  • Culture medium 1 and 2 (see recipes)
  • 0.9% (w/v) NaCl
  • Buffers A, B, C, D, and E (see recipes)
  • 0.2 M NaCl in buffer A
  • 1 mM acetaldehyde
  • 250‐ml Erlenmeyer flask
  • Orbital shaker
  • Spectrophotometer
  • Refrigerated centrifuge
  • PD‐10 (Sephadex G‐25) column (Amersham Biosciences)
  • 1.5 × 30–cm DEAE Sephacel column (Amersham Biosciences)
  • Gradient maker
  • Amicon Diaflow concentrator with YM‐30 membrane (both available from Millipore)
  • 1.5 × 30–cm CM Sepharose CL 6B column (Amersham Pharmacia Biotech)
  • 1 × 30–cm 5′‐AMP‐Sepharose CL 6B column (Amersham Pharmacia Biotech)
  • 1.0 × 15–cm Reactive Blue 2‐Sepharose CL 6B column (Amersham Pharmacia Biotech)
  • Additional reagents and equipment for assay of ALDH2 catalytic activity (see protocol 1) and SDS‐PAGE ( appendix 3F)

Support Protocol 5: Purification of Hepatic and Recombinant E. coli ALDH2

  Materials
  • Test preparation (tissue, cells, subcellular fraction, or purified enzyme; see Support Protocols protocol 71 to protocol 104)
  • Coating buffer (see recipe)
  • Wash buffer (see recipe)
  • Blocking buffer for ELISA (see recipe)
  • Primary antibody (titrated): chicken anti‐ALDH1A1 IgY ( protocol 13)
  • Preimmune IgY ( protocol 13)
  • Secondary antibody: anti‐chicken IgG conjugated to alkaline phosphatase (Sigma)
  • Tris‐buffered saline with Mg (see recipe)
  • 0.3 mM p‐nitrophenyl phosphate (see recipe)
  • Purified human liver ALDH1A1 (Dockham et al., )
  • 96‐well microtiter plate
  • Adhesive microtiter plate sealer
  • Humidified chamber (plastic box containing moist tissue paper at bottom) or humidified incubator
  • Microtiter plate reader capable of readings at 340 nm, preferably equipped with a thermocontroller (Molecular Devices)
  • Spreadsheet program capable of linear regression analysis (e.g., Microsoft Excel)

Basic Protocol 3: Quantification of in vitro Aldehyde Dehydrogenase (ALDH1A1) Levels by ELISA

  Materials
  • White Leghorn egg‐laying hens (∼1 year old; purchase from poultry farm or local Poultry Teaching and Research Facility)
  • Human liver ALDH1A1 (Dockham et al., )
  • Buffer F (see recipe)
  • Freund's complete and incomplete adjuvants (Sigma)
  • 7% and 24% (w/v) PEG 6000 (see recipe)
  • PEG 6000 powder (Sigma)
  • Buffer G (see recipe)
  • 15 and 200 mM NaCl in buffer G
  • Sodium azide powder (Sigma)
  • Bath sonicator
  • 100‐ml graduated cylinder
  • Glass stirring rods
  • Refrigerated centrifuge
  • Cheese cloth
  • 2.5 × 30–cm DEAE‐Sephacel column (Amersham Biosciences)
  • Gradient maker
  • Additional reagents and equipment for dialysis ( appendix 3H) and ELISA for aldehyde dehydrogenases (see protocol 12)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Support Protocol 6: Preparation of ALDH1A1 Antibodies

  Materials
  • Anode buffer (see recipe)
  • Cathode buffer: 1 M NaOH (store up to 1 year at room temperature)
  • pI marker proteins (Sigma; reconstitute according to manufacturer's instructions)
  • Purified aldehyde dehydrogenase (e.g., ALDH1A1; Dockham et al., ), dialyzed ( appendix 3H) against 10 mM sodium phosphate buffer, pH 7.2 ( appendix 2A)
  • Samples: tissue/cellular/subcellular fractions (see Support Protocols protocol 71 to protocol 104)
  • Destaining solution (see recipe)
  • Protein staining solution (see recipe)
  • Aldehyde dehydrogenase staining solution for gels (see recipe)
  • Flatbed IEF system (Amersham Biosciences)
  • Temperature‐controlled water circulator (Fisher)
  • EC 4000 electrophoresis power supply (4000 V output; Fisher)
  • Nitrogen tank and regulator
  • Ampholine PAGplates (precast IEF gels; 25 × 11.5 × 0.01–cm; pH 3.5–9.5; Amersham Biosciences)
  • Scalpel
  • Gel roller (Fisher)
  • Buffer loading strips, 25 × 0.5 × 0.05–cm (highly adsorbent filter paper strips; Amersham Biosciences)
  • Gel loading templates: 3 cm wide × 25 cm long × 0.1 mm thick plastic strips with 0.5 × 1–cm holes, 0.5 cm apart (Amersham Biosciences)
  • Gel‐loading wicks, 0.5 × 1 cm (highly adsorbent filter paper pieces; Amersham Biosciences)
  • Plastic boxes for staining and destaining gel
  • Platform shaker
  • 37°C shaking water bath
  • Imaging system: digital camera, digital imaging system, or computer with flatbed scanner
  • Imaging software: NIH Image for Macintosh or NIH ImageJ for Windows (available free at http://rsb.info.nih.gov/nih‐image/)
  • Additional reagents and equipment for dialysis ( appendix 3H)

Basic Protocol 4: Gel‐Based Resolution, Visualization, and Semiquantification of Aldehyde Dehydrogenase Levels

  Materials
  • Samples: tissue/cellular/subcellular fractions (see Support Protocols protocol 71 to protocol 104)
  • 50 µg/ml ALDH3A1, purified (Sreerama and Sládek, )
  • 2× sample loading buffer (see recipe)
  • Running buffer: 1.5 M Tris⋅Cl, pH 8.3 ( appendix 2A)
  • 40% (19:1) acrylamide/bisacrylamide (Bio‐Rad)
  • 10% (w/v) sodium dodecyl sulfate (SDS; appendix 2A)
  • TEMED
  • 10% (w/v) ammonium persulfate (prepare fresh; use within 30 min of preparation)
  • H 2O‐saturated isobutanol (optional)
  • Stacking gel buffer: 0.5 M Tris⋅Cl, pH 6.8 ( appendix 2A)
  • 5× electrophoresis reservoir buffer (see recipe)
  • Prestained protein markers (e.g., Bio‐Rad Kaleidoscope)
  • Transfer buffer (see recipe)
  • Methanol
  • Protein staining solution (see recipe)
  • Destaining solution (see recipe)
  • Membrane blocking buffer: dissolve 1 g nonfat dry milk in 100 ml (final volume) PBS and store at 4°C
  • Primary antibody: anti‐ALDH3A1 IgY ( protocol 11)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Secondary antibody: rabbit anti–chicken IgG alkaline phosphatase conjugate
  • Tris‐buffered saline with Mg (see recipe)
  • BCIP/NBT solution (see recipe)
  • Amicon Diaflow or spin concentrators (also see appendix 3H)
  • Boiling water bath
  • PAGE system (Protean II or III minigel unit, Bio‐Rad Laboratories) including:
    • Glass plates
    • Gel‐casting stand
    • Spacers
    • Combs
    • Electrophoresis cell
  • Electrophoresis power source
  • 10‐ and 25‐ml beakers
  • Filter paper (Whatman)
  • Long gel‐loading tips or Hamilton syringes
  • Plastic trays to accomodate gel(s)
  • Gel cutter or scalpel
  • Nylon or PVDF transfer membrane (e.g., Millipore)
  • Tweezers
  • Semidry electroblotting apparatus (Bio‐Rad)
  • Platform shaker
  • Imaging system: digital camera, digital imaging system, or computer with flatbed scanner
  • Additional reagents and equipment for protein assay ( appendix 3I) and concentration of proteins ( appendix 3H)

Basic Protocol 5: Immunoblot Analysis of Aldehyde Dehydrogenase Levels

  Materials
  • Tissue or cultured cells of interest
  • Optimal cutting temperature (OCT) compound (Fisher)
  • Dry ice/ethanol bath
  • Acetone
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 4 mM sodium deoxycholate (optional)
  • Methanol, ethanol, or industrial methylated spirits
  • PBS ( appendix 2A) containing 0.025% (v/v) Triton X‐100
  • 0.3% (v/v) hydrogen peroxide in methanol: mix 0.1 ml 30% H 2O 2 with 9.9 ml methanol and use immediately
  • Blocking solution 1 (see recipe)
  • Blocking solution 2 (see recipe) containing 10 µg/ml avidin (prepare fresh and use immediately)
  • Blocking solution 2 (see recipe) supplemented with 2 µg/ml biotin (prepare fresh and use immediately)
  • Anti‐ALDH3A1 IgY (Sreerama and Sládek, )
  • Preimmune IgY ( protocol 13) for negative control
  • Positive controls: Cells or tissue sections containing know aldehyde dehydrogenase isozymes (e.g., human liver sections for ALDH1A1 and ALDH2 or human breast adenocarcinoma MCF‐7 cells cultured in the presence of 30 µM catechol for 5 days or human stomach mucosa for ALDH3A1)
  • Blocking solution 2 (see recipe)
  • Biotinylated goat anti‐chicken IgG (Vector Laboratories)
  • ABC‐HRP system (Vector Laboratories)
  • DAB staining solution (see recipe)
  • Hematoxylin (Harris Modified; Fisher)
  • 70% and 100% ethanol
  • Xylene
  • Methanol
  • Permount mounting medium (Fisher)
  • Microtome (see Gerfen, )
  • 3‐aminopropyltriethoxysilane (APES)‐coated slides (Fisher)
  • Slide boxes
  • Coplin jars
  • Humidified chambers: 110 × 50 × 40–mm plastic boxes with lids (Fisher) containing PBS‐soaked tissue paper at the bottom
  • Glass coverslips (Fisher)

Basic Protocol 6: Immunocytochemical Visualization and Semiquantification of ALDH1A1 and ALDH3A1 in situ

  • Aldehyde dehydrogenase staining solution for histochemistry (see recipe)
  • Stop solution: 4:5:1 (v/v/v) methanol/water/glacial acetic acid
  • 37°C to 40°C slide warmer or oven
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Figures

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Literature Cited

Literature Cited
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