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Human brain monoamine oxidase: solubilization and kinetics of inhibition by octylglucoside
Abstract:
Complete solubilization of both the A and B forms of human brain monoamine oxidase (MAO) occurred when crude mitochondria were incubated in the presence of 50 mM octylglucoside (OG). Upon removal of this nonionic detergent by dialysis, approximately 100% of the starting activity was present in the dialysate. The effects of solubilization were examined by comparison of several properties of the membrane-bound and OG-treated oxidases. The percentage inhibition of phenylethylamine (PEA) and the 5-hydroxytryptamine (5-HT) deamination by deprenyl and clorgyline were identical. The Km values obtained for the deamination of PEA, a B-selective substrate, 5-HT, an A-selective substrate, and tyramine (TYR), a nonselective substrate, were also comparable. OG was found to inhibit type A (I50 = 8.1 mM) and B (I50 = 4.7 mM) MAO activities at concentrations at least 10-fold below those used to solubilize the oxidases. Kinetic studies revealed that OG was an apparent competitive inhibitor of PEA deamination whereas OG produced a mixed-type pattern of inhibition when 5-HT was the variable substrate. Inhibition of TYR deamination by either the A or B form of MAO produced a mixed pattern of inhibition. The findings herein suggest that solubilization of the A and B forms of MAO by OG does not significantly alter the substrate and inhibitor specificity of the oxidases following removal of detergent. However, in the presence of concentrations of OG 50 times less than the critical micellar concentration of this detergent, marked inhibition of deamination by both forms of human brain MAO is observed. Accordingly, the usefulness of OG is limited to situations where the detergent is completely removed before quantitation of MAO activity.
Insights
Octylglucoside (OG) effectively solubilizes human brain monoamine oxidase (MAO) A and B forms without altering their activity or specificity after detergent removal. However, low OG concentrations inhibit MAO activity, limiting its use in assays.
Area of Science:
- Biochemistry
- Neuroscience
- Enzymology
Background:
- Monoamine oxidase (MAO) is crucial for neurotransmitter metabolism.
- MAO exists as two forms, A and B, with distinct substrate specificities.
- Solubilization is necessary to study membrane-bound enzymes like MAO.
Purpose of the Study:
- To investigate the effect of octylglucoside (OG) on human brain MAO A and B forms.
- To determine if OG affects MAO activity and specificity after solubilization and removal.
- To assess the inhibitory potential of OG on MAO activity.
Main Methods:
- Solubilization of crude mitochondria using 50 mM octylglucoside (OG).
- Detergent removal by dialysis.
- Enzyme activity and kinetic assays using selective substrates (PEA, 5-HT, TYR).
- Inhibitor (deprenyl, clorgyline) sensitivity testing.
- Determination of inhibition constants (IC50, Km) and inhibition patterns.
Main Results:
- Complete solubilization of MAO A and B was achieved with 50 mM OG, with full activity recovery after dialysis.
- Solubilized MAO retained identical substrate and inhibitor specificities compared to membrane-bound MAO.
- Low OG concentrations (50-fold below CMC) exhibited significant inhibition of both MAO A and B.
- OG acted as a competitive inhibitor for PEA deamination and mixed-type inhibitor for 5-HT and TYR deamination.
Conclusions:
- Octylglucoside effectively solubilizes human brain MAO A and B without compromising enzyme integrity or specificity post-removal.
- The inhibitory effects of OG at sub-solubilizing concentrations necessitate complete detergent removal for accurate MAO activity measurements.
- OG's utility in MAO research is confirmed, provided experimental conditions preclude its inhibitory action.