Rapid and simultaneous determination of monoamine oxidase A and monoamine oxidase B activities in mouse brain homogenates by liquid chromatography with electrochemical detection.
Sensitive and rapid enzymatic assays have been developed and optimized to measure the separate and combined activities of monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) in mouse brain tissue homogenates using liquid chromatography with electrochemical detection (LCEC). The selectivity for the two isozymes is primarily afforded by use of selective substrates, 5-hydroxytryptamine (5-HT) for MAO-A and 3-methoxy-4-hydroxybenzylamine (MHBA) for MAO-B. The selectivity of the separate assays is further enhanced by the use of inhibitors, deprenyl to block MAO-B and clorgyline to block MAO-A. The dual assay procedure, which employs no inhibitors, shows remarkably enhanced selectivity for each of the isozymes through the use of the two substrates; the preferred substrate for one isozyme acts as an effective competitive inhibitor of the nontargeted substrate for that isozyme, leading to substantially decreased activity for the latter substrate. Using the dual assay, kinetic constants determined for MAO-A (mean +/- SD) were: Km,5-HT = 39 +/- 7 microM, Vmax,5-HT = 37.6 +/- 2.1 pmol/mg wet tissue/min, Km,MHBA = 341 +/- 75 microM, and Vmax,MHBA = 27.7 +/- 2.3 pmol/mg wet tissue/min; those for MAO-B were: Km,MHBA = 108 +/- 11 microM, Vmax,MHBA = 44.3 +/- 1.2 pmol/mg wet tissue/min, Km,5-HT = 1704 +/- 122 microM, and Vmax,5-HT = 12.0 +/- 0.3 pmol/mg wet tissue/min. The separate isozyme procedures, when used without selective inhibitors, reflect only 88.3% of the MAO-A activity using the 5-HT velocity and only 66.0% of the MAO-B activity using the MHBA velocity. On the other hand, when the dual assay is employed, 98% of the observed 5-HT velocity can be directly attributed to MAO-A, and 94% of the observed MHBA velocity can be directly attributed to MAO-B. The dual assay was employed to demonstrate the relative change in the activity of these two enzymes in whole mouse brain between 27 and 74 days of age. During this time, the MAO-B activity increased from approximately 40 to approximately 60% of the total MAO activity. Under typical conditions, results can be easily obtained from any of the three procedures outlined for 100 samples in less than 2 working days, including only 3.5 h for the LCEC portion.
Freeman
K B
KB
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019.
Bulawa
M C
MC
Zeng
Q
Q
Blank
C L
CL
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002853:Chromatography, Liquid / Q000379:methods* / Q000706:statistics & numerical data; D004563:Electrochemistry; D005069:Evaluation Studies as Topic; D005260:Female; D007527:Isoenzymes / Q000032:analysis*; D007700:Kinetics; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008995:Monoamine Oxidase / Q000032:analysis*; D012680:Sensitivity and Specificity
D007527:Isoenzymes; D008995:Monoamine Oxidase