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.
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.
Riley
L A
LA
Department of Human Biological Chemistry and Genetics, Graduate School of Biomedical Sciences, University of Texas Medical Branch, Galveston 77550.
Denney
R M
RM
D001665:Binding Sites; D001792:Blood Platelets / Q000201:enzymology; D003010:Clorgyline; D005189:False Positive Reactions; D015151:Immunoblotting; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology*; D008995:Monoamine Oxidase / Q000032:analysis* / Q000737:chemistry; D010293:Pargyline; D010920:Placenta / Q000201:enzymology; D014316:Tritium
D014316:Tritium; D010293:Pargyline; D008995:Monoamine Oxidase; D003010:Clorgyline