A new mass spectrometric assay of N8-acetylspermidine deacetylase and partial purification of the enzyme

<p>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.</p><p>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 &#956;M to 100 &#956;M, 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.</p><p>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 K_m value of 80.6 &#956;M for acetylputrescine and a V_max 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.</p><p>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.</p>

Name thesis:degree_name

Master of Science (M.S.)

Level thesis:degree_level

Thesis - Pacific Access Restricted

Discipline thesis:degree_discipline

Chemistry

Year dc:date.available

2007

Author dc:creator

• Zhao, YongYuan

Contributors dc:contributor

• Patrick R. Jones

• Enzyme-linked immunosorbent assay
• Enzymatic analysis
• Mass spectrometry
• Chemistry
• Medicinal-Pharmaceutical Chemistry
• Physical Sciences and Mathematics

Licence

• http://rightsstatements.org/vocab/InC/1.0/

Repository record dc:identifier

https://scholarlycommons.pacific.edu/uop_etds/683

OAI identifier oai:identifier

oai:scholarlycommons.pacific.edu:uop_etds-1682