Measurement of ALA Synthase Activity

Peter R. Sinclair1, Nadia Gorman1, Neal W. Cornell2

1 VA Medical Center, White River Junction, Vermont, 2 Marine Biology Laboratory, Woods Hole, Massachusetts
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 8.2
DOI:  10.1002/0471140856.tx0802s00
Online Posting Date:  May, 2001
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In most cells, δ‐aminolevulinate (ALA) synthase is the rate‐limiting enzyme in heme synthesis. It is inducible by drugs and toxins and is feedback regulated by heme. This unit describes a radiometric assay using [14C]succinate as a substrate and a colorimetric assay based on the conversion of ALA to a pyrrole.

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Table of Contents

  • Basic Protocol 1: Radiometric Assay for ALA Synthase
  • Basic Protocol 2: Colorimetric Assay for ALA Synthase
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Radiometric Assay for ALA Synthase

  • Control (C) and reaction (R) buffers (see reciperecipes)
  • Cofactor solution (see recipe)
  • [2,314C]succinate (see recipe)
  • Cell or tissue sample
  • Succinyl‐CoA synthase (see recipe)
  • 0.5 M NaOH
  • 8.7 M acetic acid
  • Stop solution: 10% (w/v) SDS containing 10 mM sodium succinate and 1 mM δ‐aminolevulinic acid
  • Dowex 50W‐X8 cation exchange resin, 100‐200 mesh (see recipe)
  • 0.1 M sodium acetate, pH 3.9
  • Methanol/0.1 M sodium acetate, pH 3.9 (2:1, v/v)
  • 0.01 M HCl
  • 1.0 M Tris base (aqueous solution with unadjusted pH of ∼10.5)
  • Aquasol II or other liquid scintillation fluid compatible with aqueous samples
  • 2‐ml screw‐cap tubes (e.g., Sarstedt)
  • Heating blocks, 37° and 65°C
  • 10‐ml chromatography columns (e.g., Bio‐Rad)
  • Stacking cap (e.g., Bio‐Rad #731‐1555), optional

Basic Protocol 2: Colorimetric Assay for ALA Synthase

  • Cultured hepatocytes or liver
  • Glycine medium (GM) assay buffer (no glycine; see recipe)
  • 0.25 M glycine solution (see recipe)
  • 15% (v/v) trichloroacetic acid (TCA) prepared from 100% (w/v) TCA
  • 2 M sodium acetate in H 2O
  • 8% acetylacetone (2,4‐pentanedione) in 2 M sodium acetate(see recipe)
  • NaOH‐phosphate solution (see recipe)
  • Dichloromethane
  • Modified Ehrlich reagent (see recipe)
  • 12 × 75–mm culture tubes
  • Heating block or water bath, 85° to 95°C
  • Spectrophotometer
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Literature Cited

   Bishop, D.F. and Wood, W.A. 1977. An assay for δ‐ALA synthetase based on a specific, semi‐automatic determination of picomole quantities of δ‐[14C]aminolevulinate. Anal. Biochem. 80:466‐482.
   Bonkowsky, H.L. and Pomeroy, J.S. 1978. Assay of δ‐aminolevulinic acid synthetase in homogenates of mouse, rat and human liver: Species differences in requirement for and exogenous succinyl‐CoA‐generating system. Anal. Biochem. 91:82‐91.
   Brooker, J.D., Srivastava, G., May, B.K., and Elliot, W.H. 1982. Radiochemical assay for δ‐aminolevulinate synthase. Enzyme 28:109‐119.
   Cornell, N.W., Hahn, M.E., and Martin, H.A. 1995. Characterization and use of isolated toadfish hepatocytes for studies of heme synthesis and utilization. Biol. Bull. 189:227‐228.
   Ebert, P.S., Tschudy, D.P., Choudhry, J.N., and Chirigos, M.A. 1970. A simple micro method for the direct determination of δ‐amino[14C]levulinic acid production in murine spleen and liver homogenates. Biochim. Biophys. Acta. 208:236‐250.
   Hamilton, J.W., Bement, W.J., Sinclair, P.R., Sinclair, J.F., and Wetterhahn, K.E. 1988. Expression of 5‐aminolevulinate synthase and cytochrome P450 mRNAs in chicken embryo hepatocytes in vivo and in culture. Biochem. J. 255:267‐275.
   Hunter, G.A. and Ferreira, G.C. 1995. A continuous spectrophotometric assay for 5‐aminolevulinate synthase that utilizes substrate cycling. Anal. Biochem. 226:221‐224.
   Lien, L.‐F. and Beattie, D.S. 1982. Comparisons and modifications of the colorimetric assay for delta‐aminolevulinic acid synthase. Enzyme 28:120‐132.
   Minaga, T., Sharma, M.L., Kun, E., and Piper, W.N. 1978. Enzymatic degradation of succinyl‐coenzyme A by rat liver homogenates. Biochim. Biophys. Acta 538:417‐425.
   Riddle, R.D., Yamamto, M., and Engel, J.D. 1989. Expression of delta‐aminolevulinate synthase in avaian cells: Separate genes encode erythroid‐specific and nonspecific isozymes. Proc. Natl. Acad. Sci. U.S.A. 86:792‐796.
   Sassa, S. and Kappas, A. 1977. Induction of aminolevulinate synthase and porphyrins in cultured liver cells maintained in chemically defined medium. J. Biol. Chem. 252:2428‐2436.
   Scotto, A.W., Chang, L.‐F.L., and Beattie, D.S. 1983. The characterization and submitochondrial localization of δ‐aminolevulinic acid synthase and an associated amidase in rat liver mitochondria using an improved assay for both enzymes. J. Biol. Chem. 258:81‐90.
   Shoolingin‐Jordan, P.M., LeLean, J.E., and Lloyd, A.J. 1997. Continuous coupled assay for 5‐aminolevulinate synthase. Methods. Enzymol. 281:309‐316.
   Sinclair, P.R. and Granick, S. 1977. Two methods for determining the activity of aminolevulinate synthetase within intact liver cells in culture. Anal. Biochem. 79:380‐393.
   Strand, L.J., Swanson, A.L., Manning, J., Branch, S., and Marver, H.S. 1972. Radiochemical microassay of δ‐aminolevulinic acid synthetase in hepatic and erythroid tissues. Anal. Biochem. 47:457‐470.
   Wolfson, S.J., Allen, R.M., and Bloomer, J.R. 1980. Effect of an exogenous succinyl‐CoA‐generating system on the measurement of δ‐aminolevulinic acid synthase activity in rat liver tissue by a radiochemical assay. Biochim. Biophys. Acta. 611:72‐78.
   Yoda, B., Schacter, B.A., and Israels, L.G. 1975. δ‐Aminolevulinic acid synthetase assay in chicken liver homogenates and particulate fractions. Anal. Biochem. 66:221‐233.
Key References
   Ebert et al., 1970. See above.
   Provides a detailed description of the chromatographic isolation of aminolevulinate with a cation‐exchange resin.
   Scotto et al., 1983. See above.
   Describes the development and application of the SDS procedure for stopping incubations.
   Sinclair and Granick, 1977. See above.
   Describes the colorimetric method and the adaptation for direct assay of activity in cultured hepatocytes.
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