Adenylyl and Guanylyl Cyclase Assays

John J. Kelly1, Troy Stevens1, W. Joseph Thompson2, Roland Seifert3

1 University of South Alabama College of Medicine, Mobile, Alabama, 2 Cell Pathways, Horsham, Pennsylvania, 3 Universität Regensburg, Regensburg
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 2.2
DOI:  10.1002/0471141755.ph0202s30
Online Posting Date:  October, 2005
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Abstract

This unit presents two basic protocols to determine adenylyl cyclase and guanylyl cyclase activity in tissue and cell homogenates, permeabilized cells, or subcellular fractions. Each method is divided into two parts: the enzyme reaction that causes the formation of the labeled cyclic nucleotide, and the separation of cyclic nucleotide products from unreacted nucleotide triphosphates and metabolites using Dowex 50 resin and aluminum oxide chromatographies. In the case of guanylyl cyclase, alternative separation protocols are also provided. Additionally, protocols are provided that describe preparation of both the columns used in the assays and the tissue or cells to be assayed.

Keywords: cyclase; cAMP; cGMP; G proteins; G protein-coupled receptors; cyclic nucleotide

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of Adenylyl Cyclase Activity in Vitro
  • Support Protocol 1: Preparation of Dowex Columns
  • Support Protocol 2: Preparation of Alumina Columns
  • Support Protocol 3: Tissue Preparation
  • Support Protocol 4: Cell Preparation
  • Basic Protocol 2: Measurement of Guanylyl Cyclase Activity in Vitro
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Adenylyl Cyclase Activity in Vitro

  Materials
  • 4× adenylyl cyclase reaction mix (see recipe)
  • 5 mM ATP (see recipe)
  • 108 cpm/ml [α‐32P]ATP (see recipe)
  • 1 M Tris–acetate, pH 7.6 (see recipe), with or without agonist/test compound
  • 100 mM imidazole (see recipe)
  • Scintillation fluid with high H 2O capacity (Ready Safe, Beckman)
  • Enzyme preparation (see protocol 4 or protocol 54)
  • cAMP stop solution (see recipe)
  • Dowex columns (see protocol 2)
  • Alumina columns (see protocol 3)
  • 20‐ml scintillation vials
  • 30° and 100°C water baths
  • Lucite racks, stackable with 50‐ to 100‐column capacity (Kontes Glass; alternatively, design and make with Plexiglas)
  • Beta scintillation counter with a dual‐label (3H/32P) setting
  • Spreadsheet software (e.g., Lotus 123 or Microsoft Excel)

Support Protocol 1: Preparation of Dowex Columns

  Materials
  • Dowex 50‐X4 or AG 50W resin (200‐ to 400‐mesh, hydrogen form; Aldrich)
  • 0.05 N and 1.0 N HCl
  • Calibration solution (see recipe)
  • ∼8 × 150–mm glass or plastic columns (9‐ml volume) with cup reservoir at top, plugged with glass wool (e.g., Kontes Glass)
  • 20‐ml scintillation vials
  • Beta scintillation counter with a dual‐label (3H/32P) setting

Support Protocol 2: Preparation of Alumina Columns

  Materials
  • Aluminum oxide (alumina; e.g., neutral alumina from E.M. Labs, or WN3 from Sigma)
  • 5% trichloroacetic acid (TCA; Sigma)
  • 100 mM and 1 M imidazole, pH 7.3 (see recipe)
  • 100 mM Tris–acetate, pH 7.6 (see recipe)
  • [3H]cAMP eluted from Dowex column (see protocol 2, step )
  • ∼8 × 150‐mm glass or plastic columns (9‐ml volume) with a cup reservoir at top, plugged with glass wool (e.g., Kontes Glass)

Support Protocol 3: Tissue Preparation

  Materials
  • Brain tissue
  • Homogenization buffer (0.32 M sucrose in resuspension buffer)
  • Resuspension buffer (see recipe)
  • HEPES buffer (see recipe)
  • Glass homogenizer with Teflon pestle
  • Refrigerated centrifuge
  • Additional reagents and equipment for determination of protein concentration ( appendix 3A)

Support Protocol 4: Cell Preparation

  Materials
  • Cultured cells (e.g., BHK)
  • Complete DMEM‐10 (see recipe)
  • 0.25% (w/v) trypsin
  • 0.32 M sucrose in 50 mM Tris⋅Cl, pH 7.4 (see appendix 2A for Tris⋅Cl; adjust pH at 4°C)
  • 150‐cm2 tissue culture flasks
  • 50‐ml conical polypropylene tube
  • Microprobe‐fitted sonicator (Biosonic IV, from Bronwill, or equivalent)
  • Additional reagents and equipment for cell counting with a hemacytometer (Phelan, ) and determination of protein concentration ( appendix 3A)

Basic Protocol 2: Measurement of Guanylyl Cyclase Activity in Vitro

  Materials
  • 4× guanylyl cyclase reaction mix (see recipe)
  • 0.2 mM GTP (see recipe)
  • 1 M Tris–acetate, pH 7.6 (see recipe), with or without agonist/test compound
  • 50 mM magnesium acetate (dilute from 1 M stock solution; see recipe)
  • 200 mM ammonium formate
  • 0.05 N and 3 N HCl
  • Enzyme preparation (see protocol 4 or protocol 54)
  • cGMP stop solution (see recipe)
  • Alumina columns (see protocol 3)
  • Dowex columns (see protocol 2)
  • 5% trichloroacetic acid (TCA; Sigma)
  • Scintillation fluid with high H 2O capacity (Ready Safe, Beckman)
  • 200 mM zinc acetate/cGMP stop solution (see recipe for cGMP stop solution)
  • 200 mM Na 2CO 3
  • 1.0 N perchloric acid
  • 30° and 100°C water baths
  • Lucite racks, stackable with 50‐column capacity (Kontes Glass or construct in‐house from Plexiglas)
  • Beta scintillation counter with a dual‐label (3H/32P) setting
  • Spreadsheet software (e.g., Lotus 123 or Microsoft Excel)
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Figures

Videos

Literature Cited

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