Human selenium-dependent thioredoxin reductase from HeLa cells: properties of forms with differing heparin affinities.

The TrxRl form of thioredoxin reductase (TrxR) was the major form of the enzyme isolated from HeLa cells grown in a fermentor at 35 degrees C under controlled aeration conditions favorable to growth, nominally 30% of saturation of dissolved oxygen or 8 ml of oxygen in a liter of medium. This TrxR1 form was not retained on a heparin affinity matrix, it contained one selenium per subunit, was highly active catalytically, and showed strong cross-reactivity with anti-rat liver TrxR1 polyclonal antibodies. At higher aeration, 50% of saturation of dissolved oxygen or 12 ml of oxygen in a liter of medium, HeLa cell growth was slower and additional TrxR forms that bound to heparin were present in purified enzyme preparations. A minor component, TrxR2, the mitochondrial form of TrxR, was detected in the heparin-bound enzyme fraction. One enzyme form that contained less selenium (ca. 0.5 Se per TrxR subunit) was only about 50% as active with thioredoxin or 5,5'dithiobis(2-nitrobenzoic acid) as substrate. Cross-reactivity of this form with anti-rat liver TrxR1 polyclonal antibodies was very weak. The isoelectric point of the low Se enzyme, 5.85, was higher than that, 5.2-5.4, of normal Se content enzyme. Affinity of purified fully active TrxR1 to heparin could be induced by reduction with NADPH or tris-(2-carboxyethyl)phosphine (TCEP). Under anaerobic conditions there was complete retention of Se indicating that an enzyme conformation change effected by reduction was involved. The TCEP-reduced enzyme form was very oxygen labile and upon exposure to air both the Se content and catalytic activity decreased by about 50%. Addition of millimolar concentrations of NADPH or NADP(+) to the TCEP-reduced enzyme gave full protection from oxygen inactivation. TrxR1 exhibited weak peroxidase activity with H(2)O(2) as substrate in the presence of an NADPH-generating system but this activity was unstable. Specific alkylation of the selenocysteine residue of TrxR1 which completely inhibits the NADPH-dependent reduction of disulfides also destroyed peroxidase activity. Copyright 1999 Academic Press.