Monitoring of fatty aldehyde dehydrogenase by formation of pyrenedecanoic acid from pyrenedecanal.
Fatty aldehyde dehydrogenase (EC 1.2.1.48) converts long-chain fatty aldehydes to the corresponding acids. Deficiency in this enzyme causes the Sjogren Larsson Syndrome, a rare inherited disorder characterized by ichthyosis, spasticity, and mental retardation. Using a fluorescent aldehyde, pyrenedecanal, and HPLC with fluorescence detection, we developed a novel method to monitor fatty aldehyde dehydrogenase activity by quantification of the product pyrenedecanoic acid together with the substrate pyrenedecanal and possible side products, such as aldehyde adducts. As shown with recombinant enzymes, pyrenedecanal showed a high preference for fatty aldehyde dehydrogenase compared with other aldehyde dehydrogenases. The method allowed detection of fatty aldehyde dehydrogenase activity in nanogram amounts of microsomal or tissue protein and microgram amounts of Sjogren Larsson syndrome patients' skin fibroblast protein. It could successfully be adapted for the analysis of fatty aldehyde dehydrogenase activity in gel slices derived from low-temperature SDS-PAGE, showing that fatty aldehyde dehydrogenase activity from solubilized rat liver microsomes migrates as a dimer. Thus, monitoring of pyrenedecanoic acid formation from pyrenedecanal by HPLC with fluorescence detection provides a robust and sensitive method for determination of fatty aldehyde dehydrogenase activity.
Keller
Markus A
MA
Division of Biological Chemistry, Biocenter, Innsbruck Medical University, A-6020 Innsbruck, Austria.
Watschinger
Katrin
K
Golderer
Georg
G
Maglione
Manuel
M
Sarg
Bettina
B
Lindner
Herbert H
HH
Werner-Felmayer
Gabriele
G
Terrinoni
Alessandro
A
Wanders
Ronald J A
RJ
Werner
Ernst R
ER
D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D003652:Decanoic Acids / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays / Q000379:methods*; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D009038:Motion; D011721:Pyrenes / Q000378:metabolism*; D051381:Rats; D013696:Temperature
D000447:Aldehydes; D003652:Decanoic Acids; D005456:Fluorescent Dyes; D011721:Pyrenes; C021247:pyrenedecanoic acid; D000445:Aldehyde Oxidoreductases; C038828:long-chain-aldehyde dehydrogenase