First purification of the antiquitin protein and demonstration of its enzymatic activity.
Antiquitin is an evolutionarily conserved protein believed to play a role in the regulation of cellular turgor. Based on sequence analysis, this protein is classified as a member of the aldehyde dehydrogenase superfamily. All previous studies on antiquitin have been confined to the nucleotide level, and the protein has never been purified and characterized. In the present investigation, the antiquitin protein was purified for the first time. An acetaldehyde-oxidizing protein was isolated from the liver of black seabream (Mylio macrocephalus) by chromatographies on alpha-cyanocinnamate Sepharose and Affi-gel Blue agarose, followed by ammonium sulfate precipitation. The purified protein was identified as antiquitin by the first 18 N-terminal amino acid residues which showed 83.3% identity with the deduced sequence of human antiquitin. Electrophoretic mobility studies indicated that black seabream antiquitin is a tetramer with a subunit molecular mass of 57.5 kDa. Kinetic analysis of the purified protein indicated that it catalyzes the oxidation of acetaldehyde with K(m) and V(max) values of 2.0 mM and 1.3 U/mg, respectively. The longer aliphatic propionaldehyde and the aromatic benzaldehyde are also substrates of the purified enzyme. The enzyme is highly specific towards NAD+ as the coenzyme and is totally inactive towards NADP+. Maximal enzymatic activity was found at about pH 9-10.
Tang
Wai-Kwan
WK
Department of Biochemistry, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, PR China.
Cheng
Christopher H K
CH
Fong
Wing-Ping
WP
D000444:Aldehyde Dehydrogenase; D000595:Amino Acid Sequence; D000818:Animals; D002847:Chromatography, Agarose; D017124:Conserved Sequence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D021541:Sea Bream / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D011506:Proteins; D009243:NAD; C089507:ALDH7A1 protein, human; D000444:Aldehyde Dehydrogenase