Aldehyde dehydrogenase from rat intestinal mucosa: purification and characterization of an isozyme with high affinity for gamma-aminobutyraldehyde.
In rat adrenal gland and gastric mucosa putrescine is efficiently oxidized to GABA via gamma-aminobutyraldehyde (ABAL) by action of diamine oxidase and aldehyde dehydrogenase. Having turned our attention on the rat intestinal mucosa, where putrescine uptake and diamine oxidase are active, we have purified and characterized an aldehyde dehydrogenase optimally active on gamma-aminobutyraldehyde. A dimer with a subunit molecular weight of 52,000, the native enzyme binds ABAL and NAD+ with high affinity: at pH 7.4, Km values are equal to 18 and 14 microM, respectively. Affinity for betaine aldehyde is much lower (Km = 285 microM), but the efficiency is equally good, thanks to a high value of V. Unaffected by disulfiram and Mg2+, the enzyme is activated by high NAD+ concentrations (Vnn = 1.6 x Vn) and is competitively inhibited by NADH. According to the best fitting model, the dimeric enzyme only binds one NADH and the mixed complex enzyme-NAD(+)-NADH is inactive. The increase of activity promoted by NAD+ can therefore be ascribed to an allosteric effect, rather than to the activation of a second reaction center. Highly stable at pH 6.8 in the presence of dithiothreitol and high phosphate concentrations, ABALDH is inactivated by ion-exchange resins and by cationic buffers. Our results show that the enzyme can be effectively involved in the metabolism of biogenic amines and, with a K(m) for ABAL lower than 20 microM, in the synthesis of GABA.
Testore
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G
Dipartimento di Medicina e Oncologia Sperimentale, Università di Torino, Turin, Italy.
Cravanzola
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C
Bedino
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D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism; D000818:Animals; D019281:Dimerization; D004221:Disulfiram / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000201:enzymology*; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity
D000447:Aldehydes; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009243:NAD; C006509:4-aminobutyraldehyde; D000444:Aldehyde Dehydrogenase; D008274:Magnesium; D004221:Disulfiram