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Purification and characterization of two distinct aldehyde-oxidizing enzymes from the liver of black seabream.

Authors
  • Fong, Wing-Ping
  • Cheng, Christopher H K
  • Tang, Wai-Kwan
Type
Published Article
Journal
Chemico-Biological Interactions
Publisher
Elsevier
Publication Date
Feb 01, 2003
Volume
143-144
Pages
159–167
Identifiers
PMID: 12604200
Source
Medline
License
Unknown

Abstract

Two aldehyde dehydrogenases (ALDH) were purified from the liver of black seabream (Acanthopagrus schlegeli). Chromatography of the liver homogenate on an alpha-cyanocinnamate-Sepharose affinity column results in two activity peaks using acetaldehyde as the substrate. The eluate was subjected to another affinity chromatography on p-hydroxyacetophenone-Sepharose. The final preparation showed a single band on SDS-PAGE with a subunit M.W. of 56,000. N-terminal amino acid sequencing of the first 29 residues followed by blastp analysis on the NCBI database revealed this protein as ALDH-2, as it exhibited 69% identity with human mitochondrial ALDH-2. Chromatography of the alpha-cyanocinnamate-Sepharose column flow-through fractions on Affi-gel Blue agarose yielded another ALDH. The purified protein, with a subunit M.W. of 57,500, was identified as antiquitin (turgor ALDH) by its first 18 N-terminal amino acid residues, which showed 83% identity with the deduced amino acid sequence of human antiquitin. Kinetically, both ALDHs showed maximal activity at pH around 8.5-9.0. They differed, however, in their catalytic efficiency towards the oxidation of acetaldehyde. Antiquitin had much lower affinity towards acetaldehyde; the Km value being approximately 220-fold higher than that of ALDH-2. The Vmax of antiquitin was only approximately 12% of ALDH-2. Antiquitin is believed to be involved in the regulation of cellular turgor pressure. However, all previous studies on antiquitin have been confined to the nucleotide level and the protein has never been isolated from any source. The development of an effective purification procedure and the demonstration that this protein is an enzyme with aldehyde-oxidizing activity would be highly valuable for further investigations on the physiological significance of this evolutionarily conserved protein.

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