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A number of fluorescence studies, both of trp residues and bound NADH, have been reported for porcine malate dehydrogenase (MDH). The large number of trp residues (six) complicates the interpretation of some studies. To circumvent this we have performed studies with a two-tryptophan (per subunit) MDH from Bradyrhizobium japonicum 3I1B-143 bacteroids. We have performed phase/modulation fluorescence lifetime measurements, as a function of temperature and added quencher KI, in order to resolved the 1.2-ns (blue) and 6.5-ns (red) contributions from the two classes of trp residues. Anisotropy decay studies have also been performed. The binding of NADH dynamically quenches the fluorescence of both trp residues, but, unlike mammalian cytoplasmic and mitochondrial MDH, there is not a large enhancement in fluorescence of bound NADH upon forming a ternary complex with either tartronic acid or D-malate.

The effect of drought upon phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31), malate dehydrogenase (MDH; EC 1.1.1.37), alcohol dehydrogenase (ADH; EC 1.1.1.1) and β-hydroxybutyrate dehydrogenase (β-OH-BDH; EC 1.1.130) enzyme activities as well as the leghemoglobin (Lb), malate and ethanol contents of alfalfa nodules (Medicago sativa L. cv Aragón) were examined. Both the leghemoglobin (Lb) content and the Lb/soluble protein ratio were significantly reduced at a nodule water potential (ψmod ) of -1.3 MPa. At lower ψmod,Lb content decreased further, but the ratio remained unchanged. Slight stress (-1.3 MPa) drastically affected acetylene reduction activity (ARA; 60% reduction)' whereas in vitro PEPC activity was maintained at relatively constant values. As stress progressed (-2.0 MPa), a simultaneous reduction in both activities was observed. Severe stress (ψmodlower than: -2.0 MPa) stimulated in vitro PEPC. Bacteroid β-OH-BDFI activity was stimulated by slight (-1.3 MPa) and moderate (-2.0 MPa) drought. MDH activity rose in slightly stressed nodules (ψmod -1.3 MPa). Greater water deficits sharply decreased. MDH activity to! values significant]y lower than those found in control nodules. Nodule malate content followed the same pattern as MDH. The plant fraction of the nodule showed constitutive ADH activity and contained ethanol, ADH was stimulated at slight (-1.3 MPa) and moderate drought levels (-2.0 MPa). Ethanol content showed similar behavior to ADH activity. Inhibition of ARA, reduction of Lb con tent and stimulation of the fermentative metabolism induced by water stress suggest some reduction in O2 availability within the nodule. [ABSTRACT FROM AUTHOR]