A new enzyme activity assay has been developed for the target N8-acetylspermidine deacetylase, a not-well-studied but essential enzyme in the polyamine interconversion and reutilization pathway.
The enzyme assay, based on mass spectrometric detection of a specific reaction product following sample introduction by flow injection, was shown to have a sensitivity of smaller than 1 micromolar and typical RSD of 3-10%. The linear range for analyte was from 1 μM to 100 μM, with R2 > 0.992. The new assay avoids the use of radio labels. Sample preparation is straightforward, and high specificity is provided by the selected reaction monitoring, SRM, using a triple quadrupole mass spectrometer.
Acetylputrescine was used for the first time as the substrate for the assay of N8-acetylspermidine deacetylase in lieu of N8 -acetylspermidine. The crude enzyme extracted from rat liver had an apparent Km value of 80.6 μM for acetylputrescine and a Vmax of 1.1 nmol mg-1 min-1. Enzyme extracted from frozen rat liver was compared with that from fresh rat liver. Frozen rat liver extraction had similar kinetics parameters with the fresh preparation and had a specific activity of 0.8 nmol mg-1 min-1.
N8-acetylspermidine deacetylase was partially purified by protein precipitation and gell filtration chromatography. Affinity chromatography was tentatively applied for further isolation of the enzyme, but was not yet successful.
| Identifer | oai:union.ndltd.org:pacific.edu/oai:scholarlycommons.pacific.edu:uop_etds-1682 |
| Date | 01 January 2007 |
| Creators | Zhao, YongYuan |
| Publisher | Scholarly Commons |
| Source Sets | University of the Pacific |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | University of the Pacific Theses and Dissertations |
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