Regulation of Corepressor Function by Nuclear NADH Qinghong Zhang, David W. Piston, Richard H. Goodman

Determination of free NAD/NADH ratio.
Because there should be no barrier to diffusion of NAD/NADH across the nuclear membrane, we presume that the ratios of free NAD/NADH in the cytoplasm and nucleus are the same. We have therefore determined the ratios of free nucleotides in the former compartment to approximate what is going on in the latter. These determinations were based upon methodology described by Krebs and colleagues [see, for example, Williamson et al., Biochem. J. 103, 514 (1967)], in which the concentrations of the oxidized and reduced substrates of an NAD-linked dehydrogenase are used to calculate the NAD/NADH ratio according to the equation:

[pyruvate][NADH] / [lactate] [NAD] = K,

where K is the equilibrium constant for the cytoplasmic lactate dehydrogenase. Cells were de-proteinized and the concentration of lactate and pyruvate were determined as described in Methods of Enzymatic Analysis (Bergmeyer, Academic Press, New York, ed. 2, 1974). As indicated in Fig. 4A, the values for free NAD/NADH exactly recapitulate what was reported by Williamson et al. in liver in and by Katz and Sahlin in skeletal muscle [Acta J. Physiol. Scand., 131, 119 (1987)].

Supplemental Figure 1. Limited proteolytic digestion of CtBP. (A) Dose response of NAD⁺ or NADH on protection of CtBP from partial tryptic digestion. (B) Digestion of G183A mutation of CtBP without (control) or with 100 M NAD⁺ or NADH using various doses of trypsin. Western blots were developed using a His-specific antibody (which detects a carboxyl-terminal epitope).

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Supplemental Figure 2. CoCl₂, azide, or hypoxia treatments increase the interaction of E1A and CtBP in a mammalian two-hybrid assay. For each pair of interacting proteins, the first component is fused to the Gal DNA binding domain and the second to VP16. CREB-D:KIX represents constitutively active CREB:CBP [Cardinaux et al., Mol. Cell Biol. 20, 1546 (2000)]; E1A represents a carboxyl-terminal fragment of E1A. G183A mutation ablates NAD⁺/NADH binding site in CtBP. Cells were treated with 200 M CoCl₂, 10 mM azide, or exposed to hypoxia for 16 hours.

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