Reduction of Purothionin by the Wheat Seed Thioredoxin System

Abstract

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.