Two continuous coupled assays for ornithine-δ-aminotransferase

Jose I. Juncosa, Hyunbeom Lee, Richard B. Silverman*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

We have developed two new continuous coupled assays for ornithine-d-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multiwell plates for convenience and high throughput. The first assay is based on the reduction of Δ1-pyrroline-5- carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1 (PYCR1), which results in the concomitant oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) to NAD+ (nicotinamide adenine dinucleotide, oxidized form). This procedure was found to be three times more sensitive than previous methods and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of L-glutamate, produced during the regeneration of the cofactor pyridoxal 5'-phosphate (PLP) of OAT by an unamplified modification of the commercially available Amplex Red L-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT; measuring the transformation of L-ornithine at high concentrations by this assay is complicated by the fact that it also acts as a substrate for the L-glutamate oxidase (GluOx) reporter enzyme.

Original languageEnglish (US)
Pages (from-to)145-149
Number of pages5
JournalAnalytical Biochemistry
Volume440
Issue number2
DOIs
StatePublished - 2013

Keywords

  • Amplex Red
  • Continuous assay
  • Coupled assay
  • D1-Pyrroline-5-carboxylate reductase 1
  • L-Glutamate oxidase
  • Ornithine-d-aminotransferase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Two continuous coupled assays for ornithine-δ-aminotransferase'. Together they form a unique fingerprint.

Cite this