An NADP/thioredoxin system in leaves: purification and characterization of NADP-thioredoxin reductase and thioredoxin h from spinach

Abstract

An NADP/thioredoxin system, consisting of NADPH, NADP-thioredoxin reductase (NTR), and its thioredoxin, thioredoxin h, has been previously described for heterotrophic plant tissues, i.e., wheat seeds and cultured carrot cells. Until now there was no evidence for this system in green leaves. Here, we report the identification of protein components of the NADP/thioredoxin system in leaves of several species. Thioredoxin h and NTR, which were both recovered in the extrachloroplastic fraction, were purified to apparent homogeneity from spinach leaves. This represents the first time that NTR has been characterized from a plant source. Similar to that from bacterial and mammalian sources, spinach leaf NTR was a flavoprotein (Mr 68,000) composed of two subunits of identical molecular mass (Mr 33,000) that resembled Escherichia coli NTR immunologically. Spinach thioredoxin h existed in two forms (Mr of 13,500 and 12,000) and was highly specific for plant NTR. Thioredoxin h and NTR partially purified from spinach roots showed properties similar to their counterparts from leaves. Spinach cytosolic thioredoxin h differed from chloroplast thioredoxin m or f from the same source but was similar to thioredoxin h from wheat seed in immunological properties.