Hydralazine is a potent irreversible inhibitor of the semicarbazide-sensitive amine oxidase (SSAO) found in brown fat. Initially it may act on the enzyme as a competitive inhibitor, but irreversible inhibition occurs rapidly in a concentration- and temperature-dependent manner. The presence of primary amines known to be substrates for the enzyme, but not of secondary amines, which are not metabolized by SSAO, diminishes this rate of inactivation, whereas removal of O2 is without effect. The kinetic pattern of inactivation of SSAO by hydralazine is consistent with an active-site-directed site-saturable binding followed by the development of an irreversible enzyme-inhibitor complex. [3H]Hydralazine, used as an affinity label for SSAO, shows saturable binding to brown-fat membranes. This binding is inhibited by other inhibitors of SSAO. The rate of binding to membrane pellets containing SSAO is not affected by substrates for the enzyme. However, if solubilized partially purified SSAO preparations are used instead, the rate of binding is lowered in the presence of the SSAO substrate benzylamine. 3H-labelled material solubilized from [3H]hydralazine-treated membrane pellets by Triton X-100 at that detergent/protein ratio which releases SSAO from membranes shows the same gel-filtration characteristics as SSAO and appears by lentil lectin-agarose affinity chromatography to contain similar carbohydrate moieties. 3H-labelled material, partially purified by gel filtration and affinity chromatography, produces predominantly a single band of radioactivity on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The position of this band corresponds to an Mr of about 94 000, almost exactly half the Mr already estimated for the functional unit of SSAO. Radiolabelled hydralazine may thus be used as a label for purified SSAO, but it is not specific enough to be suitable as a ligand in vivo.

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