Measurement of cGMP and Soluble Guanylyl Cyclase Activity

Eric Southam1

1 Glaxo Wellcome Medicines Research Center, Stevenage, United Kingdom
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 10.5
DOI:  10.1002/0471140856.tx1005s04
Online Posting Date:  May, 2001
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Abstract

Soluble guanylyl cyclase is responsible for synthesis of guanosine 3'5'‐cyclic monophosphate. The enzyme is activated by NO, so measurement of tissue levels of cGMP and the activity of guanylyl cyclase is important in studies of NO‐cGMP signal transduction. The method of choice for measuring tissue levels of cGMP is a radioimmunoassay, but it can also be measured using a scintillation proximity assay. Guanylyl cyclase activity is determined by measuring the conversion of GTP to cGMP using a radioimmunoassay or by following the conversion of [32P]GTP to [32P]cGMP, with separation of the product by precipitation and chromatography on an alumina column.

     
 
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Table of Contents

  • Basic Protocol 1: Determination of Tissue cGMP Levels by RIA
  • Alternate Protocol 1: Determination of Tissue cGMP Levels by Scintillation Proximity Assay
  • Basic Protocol 2: Soluble Guanylyl Cyclase Assay Using RIA to Quantify cGMP Product
  • Alternate Protocol 2: Soluble Guanylyl Cyclase Assay Using [α‐32P] GTP as Substrate
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Determination of Tissue cGMP Levels by RIA

  Materials
  • Tissue to be analyzed
  • Tris/EDTA buffer: 50 mM Tris/4 mM EDTA, pH 7.6 (prepare from Tris base and EDTA, and adjust pH with 1 M HCl)
  • ∼5.9 kBq/ml [3H]cGMP (Amersham)
  • Rabbit antiserum specific for cGMP (raised according to the method of Steiner et al., )
  • 80, 40, 20, 10, and 5 nM unlabeled cGMP (standards)
  • 10 µM cGMP (for determining nonspecific binding)
  • 0.39 g/ml ammonium sulfate [(NH 4) 2SO 4], ice cold
  • Water‐compatible scintillation fluid
  • Probe sonicator
  • Aspirator (a narrow‐gauge syringe needle attached to a vacuum line works well)

Alternate Protocol 1: Determination of Tissue cGMP Levels by Scintillation Proximity Assay

  • Anti‐rabbit SPA antibody binding beads (fluomicrospheres, Amersham)
  • 96‐well microtiter plate and sealer (e.g., T‐tray, Wallac)
  • β‐plate scintillation counter

Basic Protocol 2: Soluble Guanylyl Cyclase Assay Using RIA to Quantify cGMP Product

  Materials
  • Animals whose tissues are to be analyzed
  • Tris/DTT buffer, ice cold: 10 mM Tris/1 mM dithiothreitol, pH 7.4 (prepare with Tris base and adjust pH with 1 M HCl; appendix 2A)
  • Reaction buffer containing unlabeled GTP (see recipe)
  • 0.1 M EDTA ( appendix 2A)
  • Motorized homogenizer, 4°C
  • Refrigerated centrifuge capable of 48,000 × g

Alternate Protocol 2: Soluble Guanylyl Cyclase Assay Using [α‐32P] GTP as Substrate

  • Reaction buffer containing [α‐32P]GTP (see recipe)
  • 125 mM zinc acetate [Zn(C 2H 3O 2) 2⋅2H 2O]
  • 125 mM sodium carbonate (Na 2CO 3⋅10H 2O)
  • Neutral alumina column (see recipe)
  • 0.1 M Tris⋅Cl, pH 7.5 ( appendix 2A)
  • [3H]cGMP containing ∼5.9 kBq/ml (Amersham)
  • Water‐miscible scintillation fluid
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Figures

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Literature Cited

Literature Cited
   Garthwaite, J., Woodhams, P.L., Collins, M.J., and Balazs, R. 1979. On the preparation of brain slices: Morphology and cyclic nucleotides. Brain Res. 173:373‐377.
   Garthwaite, J., Charles, S.L., and Chess‐Williams, R. 1988. Endothelium‐derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain. Nature 336:385‐388.
   Harper, J.F. and Brooker, G. 1975. Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2′0 acetylation by acetic anhydride in aqueous solution. J. Cyclic Nucleotide Res. 1:207‐218.
   Humbert, P., Niroomand, F., Fischer, G., Mayer, B., Koesling, D., Hinsch, K.‐D., Gausepohl, H., Frank, R., Schultz, G., and Böhme, E. 1990. Purification of soluble guanylyl cyclase from bovine lung by a new immunoaffinity chromatographic method. Eur. J. Biochem. 190:273‐278.
   Mayer, B., Koesling, D., and Böhme, E. 1993. Characterization of nitric oxide synthase, soluble guanylyl cyclase, and Ca2+/calmodulin‐stimulated cGMP phosphodiesterase as components of neuronal signal transduction. In Advances in Second Messenger and Phosphoprotein Research, Vol. 28 (B.L. Brown and P.R.M. Dobson, eds.) pp. 111‐119. Raven Press, New York
   Mülsch, A. and Gerzer, R. 1991. Purification of heme‐containing soluble guanylyl cyclase. Methods Enzymol. 195:377‐383.
   Schultz, G. and Böhme, E. 1984. Guanylate cyclase. In Methods of Enzymatic Analysis, Vol. 4 (H.U. Bergmeyer, J. Bergmeyer, and M. Grassl, eds.) pp. 379‐389. Verlag Chemie Weinheim, Germany,
   Southam, E. and Garthwaite, J. 1991. Comparative effects of some nitric oxide donors on cyclic GMP levels in rat cerebellar slices. Neurosci. Lett. 130:107‐111.
   Southam, E., East, S.J., and Garthwaite, J. 1991. Excitatory amino acid receptors coupled to the nitric oxide/cyclic GMP pathway in rat cerebellum during development. J. Neurochem. 56:2072‐2081.
   Steiner, A.L., Parker, C.W., and Kipnis, D.M. 1972. Radioimmunoassay for cyclic nucleotides. I. Preparation of antibodies and iodinated cyclic nucleotides. J. Biol. Chem. 247:1106‐1113.
   Troyer, E.W., Hall, I.A., and Ferrendelli, J.A. 1978. Guanylate cyclase in CNS: Enzymatic characteristics of soluble and particulate enzymes from mouse cerebellum and retina. J. Neurochem. 31:825‐833.
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   Wood, P.L. 1991. Pharmacology of the second messenger, cyclic guanosine 3′,5′‐monophosphate, in the cerebellum. Pharmacol. Rev. 43:1‐25.
Key References
   Mayer et al., 1993. See .
  Short review describing the NO‐cGMP pathway at the molecular level.
   Johnson, R.A. and Corbin, J.D. (eds.) 1991 Methods in Enzymology, Vol. 195. Adenyl Cyclase, G. Proteins, and Guanylyl Cyclases. Academic Press, San Diego.
  A relatively recent volume that is an excellent source of practical information.
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