Binding of [ 14C] allylamine to isolated mitochondria from rat heart and aorta

Abstract

This study demonstrates specific and saturable binding [ 14C] allylamine to mitochondria derived from rat aorta and heart. Specific binding is linear with respect to mitochondrial concentration and has a pH optimum of 7.0. Saturation isotherms reveal anomolus kinetics of specific binding on heart mitochondria with a high affinity site ( K D 16 nM) and a lower affinity site ( K D 80 nM); Scatchard plots have a common intercept. Exhaustive flow dialysis in the presence of SDS demonstrates that as much as 23.5% of bound radioactive moieties in aorta mitochondria are covalently bound, and as much as 42.6% are covalently bound in heart mitochondria. Hydrolysis of heart mitochondria with phospholipase C markedly enhances saturation of [ 14C]allylamine, and greatly increases the quantity of covalently bound radioactive ligand. Phospholipase C hydrolysis of heart mitochondria increased monoamine oxidase B activities and unmasked a small amount of benzylamine oxidase activity, whereas hydrolysis of mitochondria with phospholipases A 2 and D diminish MAO-B activity. The monoamine oxidase B inhibitor, deprenyl, significantly reduced both specific and covalent binding of the 14C-activity from [ 14C]allylamine to phospholipase hydrolyzed mitochondria. The benzylamine oxidase inhibitor, phenelzine, significantly decreased specific binding but had no effect on the degree of covalent binding of [ 14C]allylamine to phospholipase C hydrolyzed mitochondria. The benzylamine oxidase inhibitor, semicarbazide, had no effect in inhibiting [ 14C]allylamine binding. Covalent binding of 14-moiety from [ 14C]allylamine to mitochondria — which express specific sites for the [ 14C]allylamine—and inhibition of binding by monoamine oxidase inhibitors, suggest the formation of highly reactive intermediates.