An examination of the reliability of the radiochemical assay for monoamine oxidases A and B.
The radiochemical assay for MAO A has been compared with the polarographic and alcohol dehydrogenase-coupled assays, using 5-hydroxytryptamine (5-HT) and tyramine and the homogeneous human liver enzyme expressed in yeast, and rat liver mitochondria. Despite efforts to measure true initial rates and to avoid known sources of error in the radiochemical procedure, significantly higher rates (Vmax) and lower Km values for the substrate were obtained with the polarographic than with the radiochemical method for 5-HT and tyramine, using either highly purified enzyme or mitochondria. The rate of tyramine oxidation measured by the polarographic method and the coupled assay agreed well, however. Consequently, for kinetic and inhibition studies we recommend the polarographic method for MAO A substrates. Comparison of the radiochemical, polarographic, spectrophotometric, and coupled assays for MAO B from beef liver (mitochondria and homogeneous enzyme) showed polarography to be the method of choice on all substrates tested, although, if all known sources of error are carefully controlled, the radiochemical assay gives the same Vmax and Km values. We also report that with each substrate studied the apparent Km is much lower when mitochondria are used than when the highly purified, virtually lipid-free preparations of MAO A are studied.
Krueger
M J
MJ
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Singer
T P
TP
D000818:Animals; D002250:Carbon Radioisotopes; D002417:Cattle; D007527:Isoenzymes / Q000032:analysis* / Q000378:metabolism; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008995:Monoamine Oxidase / Q000032:analysis* / Q000378:metabolism; D011048:Polarography / Q000379:methods; D011865:Radioisotope Dilution Technique; D051381:Rats; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity
D002250:Carbon Radioisotopes; D007527:Isoenzymes; D008995:Monoamine Oxidase