omega-Amidase (omega-amidodicarboxylate
amidohydrolase, EC 3.5.1.3) isolated from rat liver cytosol is a versatile
enzyme that catalyzes a large number of
amidase, transamidase, and
ester hydrolysis reactions.
omega-Amidase activity toward
alpha-ketoglutaramate and alpha-ketosuccinamate (the alpha-keto
acid analogues of
glutamine and
asparagine, respectively) is present in mammalian tissues,
tumors, plants, bacteria, and fungi. Despite its versatility, widespread occurrence, and high specific activity, the
enzyme has been little studied, possibly because the assay procedure previously required a substrate (
alpha-ketoglutaramate) that is not commercially available. Here we report a simplified method for preparing
alpha-ketoglutaramate and an assay procedure that measures
alpha-ketoglutarate formation from
alpha-ketoglutaramate in a 96-well plate format. We also describe a 96-well plate assay procedure that measures
omega-amidase-catalyzed hydroxaminolysis of commercially available succinamic
acid. The product, succinyl hydroxamate, yields a stable brown color in the presence of acidic
ferric chloride that can be quantitated spectrophotometrically with negligible background interference. The two assay procedures (hydrolysis of
alpha-ketoglutaramate and hydroxaminolysis of succinamate) were employed in purifying
omega-amidase approximately 3600-fold from rat liver cytosol. The ratio of
alpha-ketoglutaramate hydrolysis to succinamate hydroxaminolysis remained constant during the purification.
omega-Amidase has recently been shown to be identical to Nit2, a putative
tumor suppressor protein. It is anticipated that these new assay procedures will help to characterize the function of
omega-amidase/Nit2 in
tumor suppression, will provide the basis of high-throughput procedures to search for potent inhibitors and enhancers of
omega-amidase, and will assist in identifying
biological interactions between
nitrogen metabolism and
tumor biology.