Purification and kinetic characterization of gamma-aminobutyraldehyde dehydrogenase from rat liver.
Oxidative deamination of putrescine, the precursor of polyamines, gives rise to gamma-aminobutyraldehyde (ABAL). In this study an aldehyde dehydrogenase, active on ABAL, has been purified to electrophoretic homogeneity from rat liver cytoplasm and its kinetic behaviour investigated. The enzyme is a dimer with a subunit molecular weight of 51,000. It is NAD(+)-dependent, active only in the presence of sulphhydryl compounds and has a pH optimum in the range 7.3-8.4. Temperatures higher than 28 degrees C promote slow activation and the process is favoured by the presence of at least one substrate. Km for aliphatic aldehydes decreases from 110 microM for ABAL and acetaldehyde to 2-3 microM for capronaldehyde. The highest relative V-values have been observed with ABAL (100) and isobutyraldehyde (64), and the lowest with acetaldehyde (14). Affinity for NAD+ is affected by the aldehyde present at the active site: Km for NAD+ is approximately 70 microM with ABAL, approximately 200 microM with isobutyraldehyde and capronaldehyde, and > 800 microM with acetaldehyde. The kinetic behaviour at 37 degrees C is quite complex; according to enzymatic models, NAD+ activates the enzyme (Kact approximately 500 microM) while NADH competes for the regulatory site (Kin approximately 70 microM). In the presence of high NAD+ concentrations (4 mM), ABAL promotes further activation by binding to a low-affinity regulatory site (Kact approximately 10 mM). The data show that the enzyme is probably an E3 aldehyde dehydrogenase, and suggest that it can effectively metabolize aldehydes arising from biogenic amines.
Testore
G
G
Dipartimento di Medicina e Oncologia Sperimentale, Università di Torino, Italy.
Colombatto
S
S
Silvagno
F
F
Bedino
S
S
D000227:Adenine Nucleotides / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000145:classification / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D000447:Aldehydes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D003593:Cytoplasm / Q000201:enzymology; D004229:Dithiothreitol / Q000494:pharmacology; D004791:Enzyme Inhibitors; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008956:Models, Chemical; D008970:Molecular Weight; D051381:Rats; D017208:Rats, Wistar; D013095:Spermidine / Q000494:pharmacology; D013379:Substrate Specificity; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D000227:Adenine Nucleotides; D000447:Aldehydes; D004791:Enzyme Inhibitors; C006509:4-aminobutyraldehyde; D005680:gamma-Aminobutyric Acid; D000445:Aldehyde Oxidoreductases; C019679:aminobutyraldehyde dehydrogenase; D008274:Magnesium; D004229:Dithiothreitol; D013095:Spermidine