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.
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.
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DOI：
10.1016/j.ab.2009.05.025
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发表时间：
2009-08-15
影响因子：
2.9
通讯作者：
Cooper AJ
中科院分区：
文献类型：
--
作者：
Krasnikov BF;Nostramo R;Pinto JT;Cooper AJ
从大鼠肝细胞质中分离出的ω-酰胺酶[ω-酰胺二羧酸酰胺水解酶，E.C. 3.5.1.3]是一种多功能酶，可催化大量酰胺酶、转酰胺酶和酯水解反应。 α-酮戊二酸和 α-酮琥珀酸（分别是谷氨酰胺和天冬酰胺的 α-酮酸类似物）的 ω-酰胺酶活性存在于哺乳动物组织、肿瘤、植物、细菌和真菌中。尽管其多功能性、广泛存在和高比活性，但对该酶的研究很少，可能是因为以前的测定程序需要一种无法市售的底物（α-酮戊二酸）。在这里，我们报告了一种制备 α-酮戊二酸的简化方法，以及以 96 孔板形式测量 α-酮戊二酸形成 α-酮戊二酸的测定程序。我们还描述了一种 96 孔板测定程序，用于测量市售琥珀酸的 ω-酰胺酶催化的羟氨解作用。该产品琥珀酰异羟肟酸酯在酸性氯化铁存在下会产生稳定的棕色，可以通过分光光度法进行定量，背景干扰可忽略不计。两种测定程序（即 α-酮戊二酸的水解和琥珀酸的羟氨解）用于从大鼠肝细胞质中纯化约 3,600 倍的 ω-酰胺酶。 α-酮戊二酸水解与琥珀酸羟氨分解的比率在纯化过程中保持恒定。最近已证明 ω-酰胺酶与 Nit2（一种假定的肿瘤抑制蛋白）相同。预计这些新的检测程序将有助于表征 ω-酰胺酶/Nit2 在肿瘤抑制中的功能，为寻找 ω-酰胺酶的有效抑制剂和增强剂的高通量程序提供基础，并有助于识别氮代谢与肿瘤生物学之间的生物相互作用。
ω-Amidase [ω-amidodicarboxylate amidohydrolase, E.C. 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. ω-Amidase activity toward α-ketoglutaramate and α-ketosuccinamate (the α-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 (α-ketoglutaramate) that is not commercially available. Here we report a simplified method for preparing α-ketoglutaramate and an assay procedure that measures α-ketoglutarate formation from α-ketoglutaramate in a 96-well-plate format. We also describe a 96-well plate assay procedure that measures ω-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 spectrophometrically with negligible background interference. The two assay procedures (i.e. hydrolysis of α-ketoglutaramate and hydroxaminolysis of succinamate) were employed in purifying ω-amidase about ∼3,600-fold from rat liver cytosol. The ratio of α-ketoglutaramate hydrolysis to succinamate hydroxaminolysis remained constant during the purification. ω-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 characterize the function of ω-amidase/Nit2 in tumor suppression, will provide the basis of high-throughput procedures to search for potent inhibitors and enhancers of ω-amidase, and will assist in identifying biological interactions between nitrogen metabolism and tumor biology.
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影响因子：
9.2
作者：
Pace, HC;Hodawadekar, SC;Brenner, C
通讯作者：
Brenner, C
影响因子：
4.3
作者：
Krasnikov, BF;Zorov, DB;Brown, AM
通讯作者：
Brown, AM
影响因子：
2.9
作者：
HERSH, LB
通讯作者：
HERSH, LB
影响因子：
4.9
作者：
LUCHER, LA;CHEN, YM;WALKER, JB
通讯作者：
WALKER, JB
影响因子：
7.4
作者：
TA, TC;JOY, KW;IRELAND, RJ
通讯作者：
IRELAND, RJ