SP‐22 is a Thioredoxin‐Dependent Peroxide Reductase in Mitochondria

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Abstract
SP‐22 is a mitochondrial antioxidant protein in bovine adrenal cortex. The protein is homologous to thioredoxin peroxidase and other antioxidant proteins. It protects radical‐sensitive enzymes from oxidative damage by a radical‐generating system (Fe2+/dithiothreitol) in the presence of a small amount of serum. In this study we purified a second mitochondrial protein with M, 11 777, which cooperates with SP‐22 to protect glutamine synthetase and other proteins from Fe2+/dithiothreitol‐mediated damage. Without SP22, the protein had no protecting activity. We determined amino acid and nucleotide sequences of the protein and its cDNA, respectively, and found that it was a protein of the thioredoxin family. The protein, designated as mt‐Trx (mitochondrial thioredoxin), had a presequence composed of 59 amino acids that seemed to be a mitochondrial targeting signal. Mitochondrial extract prepared from adrenal cortex contained NADPH‐dependent 5,5’dithiobis(2‐ni‐trobenzoic acid) (Nbs2) reductase activity. The enzyme was thought to have thioredoxin reductase activity, since the Nbs2‐reducing activity was stimulated by mt‐Trx. We partially purified the Nbs2 reductase from bovine adrenocortical mitochondria. In the presence of the partially purified reductase, mt‐Trx, and NADPH, SP‐22 showed the activity to protect oxyhemoglobin against ascorbate‐induced damage. Furthermore, with the three protein components (Nbs2 reductase, mt‐Trx, and SP‐22) NADPH was oxidized in the presence of hydrogen peroxide or tert‐butyl hydroperoxide. The oxidation of NADPH was concomitant with the disappearance of an equimolar amount of hydrogen peroxide. Without any one of the protein components no hemoglobin‐protecting and peroxide‐dependent NADPH‐oxidizing activities were observed. From these results we concluded that SP‐22 is thioredoxin‐dependent peroxide reductase or so‐called thioredoxin peroxidase in mitochondria from the adrenal cortex.

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