Thioredoxin h, the thioredoxin characteristic of heterotrophic plant tissues, was purified to homogeneity from wheat endosperm (flour) and found to resemble its counterpart from carrot cell cultures. In the presence of NADPH, homogeneous thioredoxin h and partially purified wheat endosperm thioredoxin reductase (NADPH), (EC 1.6.4.5), purothionin promoted the activation of chloroplast fructose-1,6-bisphosphatase (EC 3.1.3.11). Under these conditions, NADPH provided the reducing equivalents for a series of thiol reactions in which (a) thioredoxin reductase reduced thioredoxin h thereby converting it from disulfide (S-S) to sulfhydryl (SH) form; (b) the sulfhydryl form of thioredoxin h reduced the disulfide form of purothionin-a 5 kilodalton seed storage protein with 4 S-S bridges; and (c) the sulfhydryl form of purothionin reductively activated fructose-1,6-bisphosphatase. The results show that, since thioredoxin h does not react effectively with fructose-1,6-bisphosphatase, the thioredoxin system can activate an enzyme through purothionin by secondary thiol redox control. In a related type reaction, purothionin, inhibited the activity of either Escherichia coli or calf thymus ribonucleotide reductase with reduced thioredoxin as hydrogen donor. The results suggest that purothionin competes with ribonucleotide reductase for reducing equivalents from thioredoxin. Thus, inhibition of deoxyribonucleotide synthesis should be considered a possible mechanism when examining the toxic effects of purothionin on mammalian cells in S-phase. purothionin is closely related to several proteins identified by various designations-crambin from Crambe abyssinca seeds (9, 24), viscotoxin and phoratoxin from parasitic higher plants (17), and a purothionin-like protein from corn seeds (27). It has been suggested that these proteins are members of a distinct protein group (27).It has been shown that wheat purothionin can substitute for thioredoxin f from spinach chloroplasts in the DTT3 linked activation of chloroplast FBPase, thus providing a biochemical assay for purothionin (26). The results also suggest that purothionin is effectively reduced by thioredoxin f that, in turn, is reduced with either DTT or photoreduced ferredoxin plus ferredoxin-thioredoxin reductase. However, there is no indication as to whether a thioredoxin-linked reduction of purothionin can take place in a system derived from seeds.We now report evidence for such a mechanism in wheat endosperm. A thioredoxin system, consisting of a homogeneous preparation of thioredoxin (thioredoxin h) (14) and partially purified thioredoxin reductase (NADPH) effectively reduced purothionin with NADPH as the hydrogen donor. The reduced purothionin, in turn, activated chloroplast FBPase. In the presence of either NADPH-or DTT-reduced thioredoxin, purothionin inhibited ribonucleotide reductase from bacterial and animal sources apparently by competition for reducing equivalents from thioredoxin. The results suggest that the NADP/ thioredoxin system functions in the reduction...
Dark-grown carrot (Daucus carota L.) tissue cultures were found to contain both protein components of the NADP/thioredoxin system--NADP-thioredoxin reductase and the thioredoxin characteristic of heterotrophic systems, thioredoxin h. Thioredoxin h was purified to apparent homogeneity and, like typical bacterial counterparts, was a 12-kdalton (kDa) acidic protein capable of activating chloroplast NADP-malate dehydrogenase (EC 1.1.1.82) more effectively than fructose-1,6-bisphosphatase (EC 3.1.3.11). NADP-thioredoxin reductase (EC 1.6.4.5) was partially purified and found to be an arsenite-sensitive enzyme composed of two 34-kDa subunits. Carrot NADP-thioredoxin reductase resembled more closely its counterpart from bacteria rather than animal cells in acceptor (thioredoxin) specificity. Upon greening of the cells, the content of NADP-thioredoxin-reductase activity, and, to a lesser extent, thioredoxin h decreased. The results confirm the presence of a heterotrophic-type thioredoxin system in plant cells and raise the question of its physiological function.
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