Problems with the measurement of monoamine oxidase A protein concentration in mitochondrial preparations. Revised molecular activities and implications for estimating ratios of MAO A:MAO B molecules from radiochemical assay data

Biochem Pharmacol. 1991 Oct 24;42(10):1953-9. doi: 10.1016/0006-2952(91)90595-v.

Abstract

There are significant discrepancies in the literature concerning the concentration of monoamine oxidase A (MAO A) from a number of tissue sources. Therefore, we compared the two principal techniques that have been used for quantitation of MAO A protein concentration: (1) titration of the enzyme with the MAO A-selective inhibitor clorgyline, and (2) saturation of the enzyme with [3H]-pargyline followed by immunoprecipitation with an MAO A-specific monoclonal antibody. To determine which of the two techniques was likely to yield more reliable values for MAO A, MAO A protein concentrations in the same preparations were determined by quantitative immunoblotting. [3H]Pargyline binding and quantitative immunoblotting yielded comparable values which were markedly lower than those obtained by titration of MAO A with unlabeled clorgyline. Therefore, clorgyline titration can seriously overestimate the concentration of MAO A protein in mitochondrial preparations. Since many literature values for the molecular activity of MAO A have relied upon enzyme concentrations determined by clorgyline binding, we reevaluated the molecular activities of MAO A and B for five important substrates. The ratio, MAO A molecular activity:MAO B molecular activity decreased in the order: serotonin (35:1) greater than tryptamine (12:1) greater than tyramine (3.3:1) greater than dopamine (2.4:1) greater than benzylamine (1:23). No comparable ratio was determined for beta-phenylethylamine because of its previously described substrate inhibition of MAO B, although it is oxidized faster by MAO B over a wide range of concentrations. Comparison of molecular activities and Km values for MAO A and B showed that with the exception of benzylamine and beta-phenylethylamine, MAO A oxidizes the other tested substrates faster than MAO B over a wide range of concentrations. Therefore, measured ratios of MAO A:MAO B activity are generally greater than the ratios of MAO A:MAO B molecules in the preparations.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Binding Sites
  • Blood Platelets / enzymology
  • Clorgyline*
  • False Positive Reactions
  • Immunoblotting
  • Kinetics
  • Liver / enzymology
  • Mitochondria / enzymology*
  • Monoamine Oxidase / analysis*
  • Monoamine Oxidase / chemistry
  • Pargyline*
  • Placenta / enzymology
  • Tritium

Substances

  • Tritium
  • Pargyline
  • Monoamine Oxidase
  • Clorgyline