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.
","abstract_html":"<p>A new enzyme activity assay has been developed for the target N<sup>8</sup>-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 μM to 100 μM, with R<sup>2</sup> > 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 N<sup>8</sup>-acetylspermidine deacetylase in lieu of N<sup>8</sup> -acetylspermidine. The crude enzyme extracted from rat liver had an apparent K<sub>m</sub> value of 80.6 μM for acetylputrescine and a V<sub>max</sub> of 1.1 nmol mg<sup>-1 </sup> min<sup>-1</sup>. 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<sup>-1</sup> min<sup>-1</sup>.</p><p>N<sup>8</sup>-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>","abstract_has_math":false,"creators":["Zhao, YongYuan"],"institution":null,"degree_name":"Master of Science (M.S.)","degree_level":"Thesis - Pacific Access Restricted","degree_discipline":"Chemistry","degree_department":null,"school":null,"contributors":["Patrick R. Jones"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2007,"date_issued":"2007-01-01T08:00:00Z","date_published":"2007-01-01T08:00:00Z","updated_at":"2026-07-24T05:36:35Z","subjects":["Enzyme-linked immunosorbent assay","Enzymatic analysis","Mass spectrometry","Chemistry","Medicinal-Pharmaceutical Chemistry","Physical Sciences and Mathematics"],"languages":[],"rights":[],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[]},"links":{"outbound_url":"https://scholarlycommons.pacific.edu/uop_etds/683","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Patrick R. Jones"]},{"key":"dc:creator","label":"Author","values":["Zhao, YongYuan"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2007-01-01T08:00:00Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Thesis - Pacific Access Restricted"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science (M.S.)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Enzyme-linked immunosorbent assay","Enzymatic analysis","Mass spectrometry","Chemistry","Medicinal-Pharmaceutical Chemistry","Physical Sciences and Mathematics"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["http://rightsstatements.org/vocab/InC/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://scholarlycommons.pacific.edu/uop_etds/683"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.
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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.
"],"dc:identifier":["https://scholarlycommons.pacific.edu/uop_etds/683"],"dc:rights":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:source":["108"],"dc:subject":["Enzyme-linked immunosorbent assay","Enzymatic analysis","Mass spectrometry","Chemistry","Medicinal-Pharmaceutical Chemistry","Physical Sciences and Mathematics"],"dc:title":["A new mass spectrometric assay of N8-acetylspermidine deacetylase and partial purification of the enzyme"],"thesis:degree_discipline":["Chemistry"],"thesis:degree_level":["Thesis - Pacific Access Restricted"],"thesis:degree_name":["Master of Science (M.S.)"]},"updated_at":"2026-07-24T05:36:35Z"}