Assay and purification of omega-amidase/Nit2, a ubiquitously expressed putative tumor suppressor, that catalyzes the deamidation of the alpha-keto acid analogues of glutamine and asparagine.
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.
Krasnikov
Boris F
BF
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA. boris_krasnikov@nymc.edu
Nostramo
Regina
R
Pinto
John T
JT
Cooper
Arthur J L
AJ
D000581:Amidohydrolases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001216:Asparagine / Q000031:analogs & derivatives* / Q000378:metabolism; D002384:Catalysis; D003641:Deamination; D005973:Glutamine / Q000031:analogs & derivatives* / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D051381:Rats; D013053:Spectrophotometry
D007656:Ketoglutaric Acids; D005973:Glutamine; C008230:alpha-ketoglutaramate; D001216:Asparagine; D000581:Amidohydrolases; C047679:omega-amidase