PURIFICATION AND PROPERTIES OF BETAINE ALDEHYDE DEHYDROGENASE EXTRACTED FROM DETACHED LEAVES OF AMARANTHUS-HYPOCHONDRIACUS L SUBJECTED TO WATER-DEFICIT

The activity of the enzyme betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8) from leaves of Amaranthus 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 homogeneity in only three consecutive steps: fractional precipitation with ammonium sulfate, ion exchange, and affinity chromatography on 5'-AMP Sepharose. A specific activity of 2.6 mol/min kg (protein) was obtained. The enzyme has a native molecular mass of 125 kDa, estimated by gel filtration chromatography, 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 degrees C for NAD(+), NADP(+), and betaine aldehyde were 80 mu M, 2.5 mM, and 69 mu M respectively. The reaction could not be reversed even at very high glycine betaine concentrations. The optimum pH and temperature were 8.0 and 50 degrees 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 NH4+, Na+, and high concentrations of glycine betaine. Mg++ up to 150 mM and Ca++ up to 50 mM did not have any effect on the activity.