Purification and properties of mitochondrial monoamine oxidase type A from human placenta.
A high yield purification scheme for monoamine oxidase A from human placental mitochondria is described. The enzyme is solubilized by a combination of treatment with phospholipase A and C and extraction with Triton X-100 and further purified by partitioning between dextran and polyethylene glycol polymers. The enzyme was obtained in 35% yield and high purity on DEAE-Sepharose CL-6B chromatography. This product, 90% catalytically active, showed a single major and several minor bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further purification could be achieved by additional chromatography using Bio-Gel HTP, but concomitant loss of catalytic activity occurred (enzyme remained about 60% active). The difference extinction coefficient for flavinox--flavinred at 456 nm was 10,800 +/- 350 m-1 cm-1. A sulfhydryl to flavin ratio of 7.5 was obtained when enzyme was denatured with sodium dodecyl sulfate, reduced with 2-mercaptoethanol, and titrated with 2,2'-dipyridyl disulfide. Anaerobic titration with 0.5 eq of sodium dithionite gave rise to the red anionic flavin radical, and full reduction was observed on further addition of reagent. The Km value for kynuramine was essentially the same for mitochondria (0.12 mM) and enzyme after DEAE-Sepharose CL-6B chromatography (0.17 mM). The concentration of clorgyline and deprenyl required for 50% inactivation also remained essentially unchanged. Incubation of the enzyme with 2,2'-dipyridyl disulfide caused inactivation in a biphasic manner with apparent second-order rate constants of 1230 M-1 min-1 and 235 M-1 min-1 for the rapid and slow phase, respectively. This inactivation was largely abolished by the inclusion of the competitive inhibitor amphetamine (Ki = 20 microM) in the incubation mixture. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a subunit molecular mass of 60-64 kDa, about 1.5-2.5 kDa higher than human liver monoamine oxidase B.
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000494:pharmacology; D000661:Amphetamine / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002845:Chromatography; D003010:Clorgyline / Q000494:pharmacology; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005415:Flavins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D008995:Monoamine Oxidase / Q000302:isolation & purification / Q000378:metabolism*; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology; D010084:Oxidation-Reduction; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D012642:Selegiline / Q000494:pharmacology; D012995:Solubility; D013438:Sulfhydryl Compounds / Q000378:metabolism
D004220:Disulfides; D005415:Flavins; D008996:Monoamine Oxidase Inhibitors; D013438:Sulfhydryl Compounds; C001105:2,2'-dipyridyl disulfide; D012642:Selegiline; D015082:2,2'-Dipyridyl; D000661:Amphetamine; D008995:Monoamine Oxidase; D003010:Clorgyline