].PlantPbysiol. Vol.143.pp.145-152(1994) Introduction Purification and Properties of Betaine Aldehyde Dehydrogenase Extracted from Detached Leaves of Amaranthus hypochondriacus L. Subjected to Water Deficit EusA M. V ALENZUELA-Soro and RosARIO A. Mu:Noz-CLARES Departamento de Bioqufmica, Facultad de Qufmica, UNAM, Mexico, D.F. 04510 Received February 23, 1993 ·Accepted July 24, 1993 Summary The activity of the enzyme betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8) from leaves of Ama· ranthus hypochondriacus L. rises from undetectable levels to 10- 3 units/ mg protein after 4 h of treatment with 17% (w/v) polyethyleneglycol to produce a water deficit. This enzyme was purified to apparent ho- mogeneity in only three consecutive steps: fractional precipitation with ammonium sulfate, ion ex- change, and affinity chromatography on 5' -AMP Sepharose. A specific activity of 2.6 moll min kg (pro- tein) was obtained. The enzyme has a native molecular mass of 125 kDa, estimated by gel filtration chro- matography, and a subunit molecular mass of 63 kDa, determined by SDS-PAGE. The reaction is highly specific for betaine aldehyde, which is an inhibitor at high concentrations, but can use NAD+ or NADP+ as nucleotide. The estimated Km values at pH 8.0 and 30 °C for NAD+, NADP+, and betaine aldehyde were 80 f.lM, 2.5 mM, and 69 f.1M respectively. The reaction could not be reversed even at very high glyc- ine betaine concentrations. The optimum pH and temperature were 8.0 and 50 °C, respectively. The pH dependence of the velocity indicated the existence of two ionizable groups of macroscopic pK values of 6.78 ± 0.02 and 9.38 ± 0.01 involved in catalysis and/or binding of the substrates. Chemical modification studies suggested the presence of essential cisteine(s), histidine(s), and arginine(s) residues. The enzyme was activated by relatively low concentrations of K+, sucrose, and proline, while it was inhibited by NHt, Na+, and high concentrations of glycine betaine. Mg++ up to 150mM and Ca++ up to SOmM did not have any effect on the activity. Key words: Amaranthus hypochondriacus L., betaine aldehyde dehydrogenase, purification, water deficit, compatible solutes, monovalent cations. Abbreviations: BA = betaine aldehyde; BADH = betaine aldehyde dehydrogenase; DEPC = diethyl pyrocarbonate; DTNB = 5-5' -dithio-bis(2-nitrobenzoic) acid; EDTA = ethylenediaminetetra-acetic acid; Hepes N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid; MES 2-(N-morpho- lino)ethanesulfonic acid; PAGE = polyacrylamide gel electrophoresis; PEG = polyethyleneglycol; RWC = relative water content; SDS = sodium dodecyl sulfate; Tris = Tris(hydroxymethyl) amino methane. Betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8 be- taine aldehyde: NAD+ oxidoreductase) catalyzes the final step in the synthesis of glycine betaine from choline by bac- teria (Nagasawa et al., 1976; Falkenberg and Strom, 1990) and plants (Pan et al., 1981; Weretilnyk and Hanson, 1989; Arakawa et al., 1990). This enzyme has been purified to ho- © 1994 by Gustav Fischer Verlag, Stuttgart