Some Studies on the Purification and the Properties of Monoamine Oxidase

Attempts were made to purify rat liver mitochondrial MAO in a single step by affinity chromatography. Except for tryptamine and serotonin-Sepharose, which irreversibly bound the enzyme, most adsorbents bound little if any of the enzyme. This was true for both detergent (triton X-100 and digitonin) and non-detergent (sonication and perchlorate) soluble enzyme preparations. However, the non-bound enzyme that eluted from either organomecurial or DTNB-Sepharose was purified 4 fold. Treatment of the triton X-100 or sonicated soluble MAO preparation with phospholipase A resulted in the binding of the enzyme to the DTNB-Sepharose but no increase in specific activity was observed on elution. Binding also occurred with the AB-15 absorbent but the enzyme could not be eluted in an active form. Using the triton X-100 solubilized enzyme, ammonium sulphate fractionation, DINB-Sepharose and DEAE-Sephadex, a 15 fold purification was obtained with a 30% recovery of enzyme activity in under 3 hours. The rat liver mitochondrial MAO could be differentiated into an A and B form with the irreversible substrate selective inhibitors Clorgyline, Deprenil and pargyline. The Clorgyline derivative Lilly 51618 was found to be reversible and to have a greatly reduced selectivity. In addition propargylamine, an acetylenic irreversible MAO inhibitor that lacks an aromatic moiety also had a greatly reduced substrate selectivity. Clorgyline was also shown to perturb the mitochondrial membrane structure through hydrophobic interactions unrelated to the inhibition of MAO. Ferrous ion induced lipid peroxidation in mitochondrial suspensions inactivates MAO possibly by the interaction of lipid peroxides with protein SH groups. The altered membrane environment from lipid peroxidation however, does not affect the heat stability or the Deprenil inhibition pattern of MAO A or MAO B. Treatment of the membrane bound or soluble MAO with phospholipase C had no effect on the enzyme activity or on the heat stability, tryptic digestibility, Arrhenius plots or the Deprenil inhibition pattern of MAO A and MAO B. Treatment of the membrane bound enzyme with phospholipase A decreased both the MAO activity by 23% and the overall heat stability of the enzyme; however the tryptic digestibility, Arrhenius plots and Deprenil inhibition patterns were unchanged. After lipid depletion of the soluble enzyme with phospholipase A, a similar loss of activity was observed as with the membrane bound enzyme but this preparation was more sensitive to inactivation by heat and trypsin. However, the different rates of inactivation of MAO A and MAO B were still observed and the Arrhenius plots and Deprenil inhibition patterns were similar to those of the membrane bound enzyme. By labelling MAO with[14C] pargyline the inactivated enzyme could be assayed, 30% of the enzyme was found in the mitochondrial structural protein fraction. In addition, when mild sonication was used to resolve the mitochondrial membranes, the MAO protein was found still associated with the membranes. The use of proteolytic digestion and DINB-Sepharose showed a similar accessibility to the enzyme from either side of the membrane. The results are discussed in terms of the role of the enzyme environment and the relationship of the enzyme protein to the outer mitochondrial membrane.

Name dc:type.qualificationname

Ph.D.

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

Aston University

Year dc:date.issued

1976

Author dc:creator

• Baker, S.P.

DOI dc:identifier.doi

https://doi.org/10.48780/publications.aston.ac.uk.00010690

OAI identifier oai:identifier

oai:publications.aston.ac.uk:10690