Characterization of potential selenium-binding proteins in the selenophosphate synthetase system

Abstract

Selenophosphate, an activated form of selenium that can serve as a selenium donor, is generated by the selD gene product, selenophosphate synthetase (SPS). Selenophosphate is required by several bacteria and by mammals for the specific synthesis of Secys-tRNA, the precursor of selenocysteine in selenoenzymes. Although free selenide can be used in vitro for synthesis of selenophosphate, the physiological system that donates selenium to SPS is incompletely characterized. To detect potential selenium-delivery proteins, two known sulfurtransferases and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) were analyzed for ability to bind and transfer selenium. Rhodanese (EC 2.8.1.1) was shown to bind selenium tightly, with only part of the selenium being available as substrate for SPS in the presence of added reductant. 3-Mercaptopyruvate sulfurtransferase (3-MST; EC 2.8.1.2) and GAPDH also bound selenium supplied as selenodiglutathione formed from SeO3(2-) and glutathione. Selenium bound to 3-MST and GAPDH was released more readily than that from rhodanese and also was more available as a substrate for SPS. Although rhodanese retained tightly bound selenium under aerobic conditions, the protein gradually became insoluble, whereas GAPDH containing bound selenium was stable at neutral pH for a long period. These results indicate that 3-MST and GAPDH have more suitable potentials as a physiological selenium-delivery protein than rhodanese. In the presence of a selenium-binding protein, a low level of selenodiglutathione formed from SeO3(2-) and glutathione could effectively replace the high concentrations of selenide routinely used as substrate in the SPS in vitro assays.

Fig. 1.

Elution profiles of products formed from reaction of selenium-binding proteins with GSH and . Reaction mixtures (100 μl), containing PBS (pH 7.4), 0.1 mM , 0.4 mM GSH, and 10 μM rhodanese E form (A and C), 3-MST (B and E), or GAPDH (C and F) were incubated at 37°C for 30 min under anaerobic conditions. After additional incubation with 2 mM DTT (D–F) or without DTT (A–C) at 37°C, reaction mixtures were applied to a gel filtration column (1.0 × 10 cm) and eluted with PBS (pH 7.4). Aliquots of each fraction were assayed to determine selenium and protein contents as described in Materials and Methods.

Fig. 2.

Effect of physiological concentration of GSH on the selenium-binding state of 3-MST and GAPDH. Reaction mixtures (100 μl) containing PBS (pH 7.4), 4 mM GSH, 0.1 mM , and 10 μM 3-MST (A) or GAPDH (B) were incubated for 10 min at 37°C. Reaction mixtures were applied to a gel filtration column, and aliquots of each fraction were assayed for selenium and protein as described in Materials and Methods.

Fig. 3.

Calibration curve for AMP determination by HPLC. Reaction mixtures (100 μl) contained 50 mM Tricine·KOH (pH 8.0), 2 mM DHLA, 8 mM MgCl₂, 50 mM KCl, 0.1 mM Mg triplex, 2 mM ATP, and varying concentrations of AMP. After the addition of HClO₄ and KOH, a 10-μl aliquot of the mixture was injected into the C₁₈ column. The area of each AMP peak from a given run is plotted versus the total nanomoles of AMP injected. HPLC conditions are described in Materials and Methods.

Fig. 4.

Linearity of AMP formation dependent on SPS concentration. Reaction mixtures containing from 0.73 to 18.3 μg of enzyme (0.2–5.0 μM), 50 mM Tricine·KOH (pH 8.0), 2 mM DHLA, 8 mM MgCl₂, 50 mM KCl, 0.1 mM Mg triplex, 2 mM ATP, 0.1 mM , and 4 mM GSH were incubated at 37°C for 30 min under anaerobic conditions. Reactions were terminated by the addition of 1.2 M HClO₄ followed by neutralization with KOH. The amount of AMP produced was determined by reverse-phase HPLC using a standard curve as described in Materials and Methods.

Fig. 5.

SPS assay with selenium-binding protein. Assays were performed anaerobically at 37°C in 50 mM Tricine·KOH (pH 8.0), 2 mM DHLA, 8 mM MgCl₂, 50 mM KCl, 0.1 mM Mg triplex, 2 mM ATP, 0.1 mM , 4 mM GSH, 5 μM SPS, and selenium-binding protein as indicated. The standard assay mixture contained 1.5 mM selenide and 25 mM DTT in place of GSH, DHLA, and a selenium-binding protein. After a 30-min incubation, reactions were terminated, and the reaction products were separated by reverse-phase HPLC and detected as described in Materials and Methods. The AMP formed in this assay was calculated from a standard curve for AMP prepared as described in Materials and Methods. Measurements were performed in duplicate, and the data are presented as means.