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

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)> tryptamine (12:1) >tyramine (3.3:1) > dopamine (2.4:1) > benzylamine (1:23). No comparable ratio was determined for β-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 K m values for MAO A and B showed that with the exception of benzylamine and β-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.