Solubilization and some properties of a semicarbazide-sensitive amine oxidase in brown adipose tissue of the rat.
A semicarbazide-sensitive clorgyline-resistant amine oxidase (SSAO) was solubilized from membrane fractions of rat brown adipose tissue by the non-ionic detergent, Triton X-100. Alteration of ionic strength or addition of chelating agents alone failed to release the enzyme from its membrane. Lipid-depletion led to loss of enzyme activity and alteration of substrate affinity. Over 80% of the activity of the solubilized enzyme was found in gel filtration fractions corresponding to an Mr of between 160 000 and 180 000. The glycoprotein nature of SSAO was established from affinity chromatography with either immobilized concanavalin A or Lens culinaris lectin. Elution of over 50% SSAO activity from the lentil lectin was achieved with 0.25M-alpha-methyl D-mannoside to give 80-90-fold purification of the enzyme. Irradiation inactivation gave a value for Mr of around 183 000 for both soluble and membrane-bound SSAO. Substrate affinity and inhibitor sensitivity of the enzyme were unaltered by solubilization. The copper-chelating agent, diethyldithiocarbamate, did not affect the enzyme, shedding doubt on the suggestion that SSAO is a copper-requiring enzyme. The significance of these findings in relation to the nature of SSAO and to its disposition within the cell membrane is discussed.
D002001:Adipose Tissue, Brown / Q000201:enzymology* / Q000528:radiation effects; D006631:Amine Oxidase (Copper-Containing); D000818:Animals; D001596:Benzylamines / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D017830:Octoxynol; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D012663:Semicarbazides / Q000494:pharmacology; D012995:Solubility
D001596:Benzylamines; D012663:Semicarbazides; D011092:Polyethylene Glycols; D017830:Octoxynol; C030796:benzylamine; D006631:Amine Oxidase (Copper-Containing); D000587:Oxidoreductases Acting on CH-NH Group Donors