Spectrophotometric Assays for l-Lysine α-Oxidase and γ-Glutamylamine Cyclotransferase

A new assay for l-lysine α-oxidase is described. In this assay, the oxidized product generated from l-lysine is reacted with semicarbazide to form α-keto-ϵ-aminocaproate semicarbazone. Formation of the α-keto acid semicarbazone is continuously monitored spectrophotometrically at 248 nm (ϵ 10,160 ± 2...

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Bibliographic Details
Published in:Analytical biochemistry 2002-04, Vol.303 (2), p.120-130
Main Authors: Danson, Jedidah W., Trawick, Mary Lynn, Cooper, Arthur J.L.
Format: Article
Language:English
Subjects:
Amino Acid Oxidoreductases - analysis
Amino Acid Oxidoreductases - antagonists & inhibitors
Aminocaproates - chemistry
Animals
Carbazoles - chemistry
Carcinogens - pharmacology
Cattle
gamma-Glutamylcyclotransferase - analysis
gamma-Glutamyltransferase - metabolism
Kidney - enzymology
l-lysine α-oxidase
Liver - enzymology
Lysine - metabolism
Male
Neurodegenerative Diseases - enzymology
Rats
Rats, Inbred F344
Semicarbazides - pharmacology
Spectrophotometry - methods
transglutaminase
γ-glutamyl transpeptidase
γ-glutamylamine cyclotransferase
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Summary:A new assay for l-lysine α-oxidase is described. In this assay, the oxidized product generated from l-lysine is reacted with semicarbazide to form α-keto-ϵ-aminocaproate semicarbazone. Formation of the α-keto acid semicarbazone is continuously monitored spectrophotometrically at 248 nm (ϵ 10,160 ± 240 M−1 cm−1). The method was adapted to provide a new assay for γ-glutamylamine cyclotransferase. This enzyme catalyzes the conversion of many l-γ-glutamylamines to 5-oxo-l-proline and free amine. A biologically important substrate is Nϵ-(γ-l-glutamyl)-l-lysine, which is converted to 5-oxo-l-proline and l-lysine by the action of γ-glutamylamine cyclotransferase. The l-lysine generated from Nϵ-(γ-l-glutamyl)-l-lysine in an endpoint assay is converted to α-keto ϵ-aminocaproate semicarbazone in the presence of semicarbazide, excess l-lysine α-oxidase, and catalase. The methods were applied to the determination of γ-glutamylamine cyclotransferase activity of partially purified preparations of the bovine kidney enzyme and to detect γ-glutamylamine cyclotransferase activity in rat kidney and liver homogenates.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.2002.5587