Danson Jedidah W, Trawick Mary Lynn, Cooper Arthur J L
Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, New York, 10021, USA.
Anal Biochem. 2002 Apr 15;303(2):120-30. doi: 10.1006/abio.2002.5587.
A new assay for l-lysine alpha-oxidase is described. In this assay, the oxidized product generated from l-lysine is reacted with semicarbazide to form alpha-keto-epsilon-aminocaproate semicarbazone. Formation of the alpha-keto acid semicarbazone is continuously monitored spectrophotometrically at 248 nm (epsilon 10,160 +/- 240 M(-1) cm(-1)). The method was adapted to provide a new assay for gamma-glutamylamine cyclotransferase. This enzyme catalyzes the conversion of many l-gamma-glutamylamines to 5-oxo-l-proline and free amine. A biologically important substrate is N(epsilon)-(gamma-l-glutamyl)-l-lysine, which is converted to 5-oxo-l-proline and l-lysine by the action of gamma-glutamylamine cyclotransferase. The l-lysine generated from N(epsilon)-(gamma-l-glutamyl)-l-lysine in an endpoint assay is converted to alpha-keto epsilon-aminocaproate semicarbazone in the presence of semicarbazide, excess l-lysine alpha-oxidase, and catalase. The methods were applied to the determination of gamma-glutamylamine cyclotransferase activity of partially purified preparations of the bovine kidney enzyme and to detect gamma-glutamylamine cyclotransferase activity in rat kidney and liver homogenates.
本文描述了一种新的L-赖氨酸α-氧化酶测定方法。在该测定中，L-赖氨酸生成的氧化产物与氨基脲反应形成α-酮基-ε-氨基己酸氨基脲。通过在248nm处进行分光光度法连续监测α-酮酸氨基脲的形成（ε10,160±240M⁻¹cm⁻¹）。该方法经过调整，可用于γ-谷氨酰胺环转移酶的新测定。这种酶催化许多L-γ-谷氨酰胺转化为5-氧代-L-脯氨酸和游离胺。一种具有生物学重要性的底物是N（ε）-（γ-L-谷氨酰基）-L-赖氨酸，它在γ-谷氨酰胺环转移酶的作用下转化为5-氧代-L-脯氨酸和L-赖氨酸。在终点测定中，由N（ε）-（γ-L-谷氨酰基）-L-赖氨酸生成的L-赖氨酸在氨基脲、过量的L-赖氨酸α-氧化酶和过氧化氢酶存在的情况下转化为α-酮基ε-氨基己酸氨基脲。这些方法被应用于测定牛肾酶部分纯化制剂的γ-谷氨酰胺环转移酶活性，并检测大鼠肾和肝匀浆中的γ-谷氨酰胺环转移酶活性。
