Elsevier

Methods in Enzymology

Volume 89, 1982, Pages 326-335
Methods in Enzymology

[57] Glyceraldehyde-3-phosphate dehydrogenase from yeast

https://doi.org/10.1016/S0076-6879(82)89059-9Get rights and content

Publisher Summary

Glyceraldehyde-3-phosphate dehydrogenase is present in high concentrations in most organisms. This chapter describes the assay method, purification procedure, and properties of glyceraldehyde-3-phosphate dehydrogenase isolated from yeast. The most commonly used procedures for monitoring the reversible oxidative phosphorylation reaction are the Ferdinand assay for the forward reaction and the coupled assay of Kirschner and Voigt for the backward reaction. Other assay conditions, which involve in situ generation of substrates and/or the removal of products, are also described in the chapter. The arsenolysis reaction provides a convenient assay for glyceraldehyde-3-phosphate dehydrogenase activity because the immediate product, 1-arseno-3-phosphoglycerate, is rapidly hydrolyzed. The traditional arsenolysis assay is carried out at low levels of substrates. The use of higher substrate concentrations improves the linearity and enhances the sensitivity of the traditional standard assay. The production of nicotinamide adenine dinucleotide dehydrogenase (NADH) is monitored spectrophotometrically. Alternatively, the reaction is monitored by (a) fluorometric detection of NADH, either directly or indirectly; (b) bioluminescent detection of NADH using the luciferase coupling reaction; and (c) continuous potentiometric titration by means of a pH-stat.

Reference (65)

  • AtkinsonM.R. et al.
  • AragónJ.J. et al.

    Biochem. Biophys. Res. Commun.

    (1978)
  • StallcupW.B. et al.

    J. Biol. Chem.

    (1972)
  • HoreckerB.L. et al.

    J. Biol. Chem.

    (1948)
  • CantarowW. et al.

    Anal. Biochem.

    (1976)
  • OgilvieJ.W. et al.

    Biochim. Biophys. Acta

    (1976)
  • HarrisJ.I. et al.
  • KrebsE.G.

    J. Biol. Chem.

    (1953)
  • KrebsE.G. et al.

    J. Biol. Chem.

    (1953)
  • ByersL.D.

    Arch. Biochem. Biophys.

    (1978)
  • BöhmeH.J. et al.

    J. Chromatogr.

    (1972)
  • KirschnerK. et al.

    J. Mol. Biol.

    (1971)
  • ShibataY. et al.

    Arch. Biochem. Biophys.

    (1967)
  • ConstantinidesS.M. et al.

    J. Biol. Chem.

    (1969)
  • JonesG.M.T. et al.

    FEBS Lett.

    (1972)
  • MorasD. et al.

    J. Biol. Chem.

    (1975)
  • StallcupW.B. et al.

    J. Mol. Biol.

    (1973)
  • StockellA.

    J. Biol. Chem.

    (1959)
  • KaplanN.O. et al.

    J. Biol. Chem.

    (1956)
  • ByersL.D. et al.

    Biochemistry

    (1979)
  • FerdinandW.

    Biochem. J.

    (1964)
  • KirschnerK. et al.

    Hoppe Seyler's Z. Physiol. Chem.

    (1968)
  • RossnerG.

    Arch. Klin. Exp. Dermatol.

    (1965)
  • TrenthamD.R. et al.

    Biochem. J.

    (1969)
  • OvádiJ. et al.

    Eur. J. Biochem.

    (1978)
  • WarburgO. et al.

    Biochem. Z.

    (1939)
  • S. F. Velick, this series, Vol. 1, p. 401...
  • E. G. Krebs, this series, Vol. 1, p. 407...
  • DealW.C.

    Biochemistry

    (1969)
  • McCombR.B. et al.

    Clin. Chem.

    (1976)
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