Properties of a Large Complex with NADH Dehydrogenase Activity from Barley Thylakoids

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Abstract
When assays for NAD(P)H-ferricyanide oxidoreductases were performed, activities specific for NADH (0.23 unit (mg protein)−1) and NADPH (0.68 unit (mg protein)−1) were detected in chloroplasts isolated from leaves of barley (Hordeum vulgare L.). Activities of chloroplast NADH- and NADPH-ferricyanide oxidoreductase were 5-fold and 25-fold higher, respectively, than the maximum activity that could be attributed to mitochondrial contamination. Moreover, most of the chloroplast NADH-ferricyanide oxidoreductase (60 to 80%) was solubilized by deoxycholate (DOC) from thylakoids as a single, high-molecular-mass complex that was distinguishable from the mitochondrial complex by its lower electrophoretic mobility in 3% polyacrylamide, as revealed by reduction of nitro blue tetrazolium (NBT) in the presence of NADH or NADPH on gels after electrophoresis. The stroma yielded a single band of a dehydrogenase (66 kDa) that used NADH as its electron donor. Several NADPH-dependent activities were detected after electrophoresis of the stromal fraction. Moreover, chloroplast-specific activities could be distinguished from mitochondrial activities on the basis of the specificity of the donor and the acceptor of electrons, the dependence of the activities on pH, and the sensitivity to various inhibitors. Km values for NADH (26 μM) and NADPH (75 μM) were in the same range as those of mitochondrial activities. Most of the NADPH-dependent activity probably corresponds to the chloroplast ferredoxin-NADP+ oxidoreductase. The possibility is discussed that thylakoid NADH dehydrogenase(s) might be the product of chloroplast ndh genes and that this activity is involved in chlororespiration.

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