Thionein can serve as a reducing agent for the methionine sulfoxide reductases

Reduced Trx Is Not an Efficient Reducing Agent for hMsrB2 and hMsrB3.

In the course of developing the DABS colorimetric assay for Msr activity (see Materials and Methods), it was confirmed that eMsrA and eMsrB could use either DTT or Trx to supply the reducing power, with similar or more activity observed with Trx in vitro. However, although both hMsrB2 and hMsrB3 could use DTT as the reducing agent, these proteins showed very little activity with Trx. Table 1 compares the activity of several recombinant Msr proteins using either DTT or Trx. It can be seen that eMsrA, bovine MsrA (bMsrA), and eMsrB are active with either DTT or Trx as the reducing system. In fact, eMsrB was much more active with Trx than with DTT. In contrast, hMsrB2 and hMsrB3 work very poorly with Trx, having <10% of the activity seen with DTT. One possibility was that the hMsrB proteins specifically required mammalian Trx and not the bacterial Trx that was used in these experiments. We therefore tested hMsrB3, as well as eMsrA and eMsrB, with mammalian Trx and mammalian Trx reductase. Reduced mammalian Trx, like the bacterial Trx, gave very poor activity with hMsrB3, but both eMsrA and eMsrB efficiently used Trx from either source (data not shown). It should be noted that we were not able to test mammalian MsrB1, which differs from MsrB2 and MsrB3 in being a selenoprotein, because all attempts to obtain sufficient amounts of this recombinant protein were unsuccessful.

The weak activity of hMsrB2 and hMsrB3 with Trx suggested that there may be another reducing system for these proteins in mammalian cells that either functions in place of Trx or is an intermediate hydrogen carrier between Trx and the human MsrB proteins.

Zn-MT in the Presence of EDTA Can Serve as a Reducing Agent for Msr.

In our attempts to search for a biological factor that was more efficient than Trx in supplying the reducing system for hMsrB2 and hMsrB3, we initially tested an S-100 from bovine liver. Using hMsrB3, we were able to detect significant reducing activity in the liver S-100 fraction, but only in the presence of EDTA. The active material was stable to heating at 80°C for 10 min. Fig. 1 shows the effect of protein concentration of the heated S-100 extract and the almost complete dependency on EDTA for hMsrB3 activity. Optimal activity was seen with levels of EDTA >2.5 mM (data not shown). Routinely, 5 mM EDTA has been used in the experiments. EDTA by itself had no significant effect on hMsrB3 activity, although hMsrB3 contains zinc. Other chelating agents were tested in place of EDTA with Zn-MT. 1,10-Phenanthroline (5 mM) gave ≈40% of the activity of EDTA, whereas EGTA (5 or 20 mM), deferoxamine (5 mM), and zincon (500 μM) were inactive. Thus, EDTA was used throughout the present studies. A series of metal salts could not replace EDTA or the heated S-100 in the reaction (data not shown).

The heat stability and EDTA requirement suggested that the active factor might be a metallothionein (MT), and the heat-stable factor was further purified as described in Materials and Methods. Fig. 2A shows the elution profile from a DE-52 cellulose column, the last step in the purification. Two distinct peaks of reducing activity were observed, and the fractions in both peaks were active in the Msr assay using hMsrB3 only in the presence of EDTA. The purification profile suggested that the two peaks correspond to MT-1 and MT-2, based on their elution from the DE-52 column.

Because of the requirement for EDTA for the fraction to be active with hMsrB3, metal analyses were initially performed on purified preparations by using inductively coupled plasma MS. Zinc was found in significant amounts [60,795 parts per billion (ppb)], with trace levels of copper and silver (688 and 739 ppb, respectively). Besides using nanopure water, no special precautions were taken to remove trace metals, so the source of these trace metals in the protein sample is unknown. As shown in Fig. 2A, the active, highly purified fractions from the DE-52 column contained high levels of zinc that coeluted with the fractions active in the Msr assay. The amount of MT could be determined spectrophotometrically (ε₂₂₀ = 48,600 M⁻¹·cm⁻¹ at pH 2.0), and zinc analyses using the 4-(2-pyridylazo)resorcinol (PAR) reagent (see Materials and Methods) showed that there were close to seven zinc atoms per mole of MT in each fraction. Although zinc appears to be the major metal associated with the active factor, we cannot eliminate the presence of lower levels of other metals in the sample. Fig. 2B shows the UV spectrum of a fraction from peak 2 from the DE-52 column (both peaks displayed similar spectral characteristics). It can be seen that the active factor has high absorption in the 200- to 250-nm range but essentially no absorbance at 280 nm, indicating the absence of aromatic amino acids. Upon acidification, the high UV absorption is markedly decreased. On SDS/PAGE, the purified protein, as well as a commercial rabbit liver MT preparation, migrated as a diffuse band in the 13- to 16-kDa range (data not shown), double the size of Zn-MT, which is ≈6 kDa. This gel migration pattern could be due to the unique shape of the protein or the presence of dimers through intermolecular bond formation. The liver MT obtained from a commercial source also supported hMsrB3 activity in the presence of EDTA (data not shown). The presence of zinc as well as the spectral and other characteristics of the active fractions indicated that the two peaks off the DE-52 column were Zn-MT-1 and Zn-MT-2. These peak fractions were further analyzed by electrospray MS, and the molecular weights matched those of bovine MT-1 and MT-2 (5,987 and 6,013, respectively). EDTA removed >90% of the zinc from Zn-MT in <10 min, as measured by the appearance of free SH groups (data not shown). We concluded that the purified factor is a Zn-MT, which, in the presence of EDTA, is converted to the metal-free reduced T, and that T, because of the high content of cysteine residues, is able to supply the reducing system for the Msr reaction. The results shown below used Zn-MT-2, although similar results were obtained with Zn-MT-1.

As seen in Table 2, the purified Zn-MT is not a specific reducing agent for hMsrB3 because it also supports eMsrA, eMsrB, and bMsrA, dependent on EDTA. However, to our surprise, the liver factor showed very little activity with hMsrB2 under the conditions used in Table 2.

T Can Function in the Msr System in the Absence of EDTA.

Although it appeared likely that the requirement for EDTA was to release zinc from Zn-MT to form T, this system was obviously artificial, and it was important to demonstrate directly that T could serve as the reducing agent for the Msr system. T was prepared as described in Materials and Methods and tested with hMsrB3 as shown in Fig. 3. It can be seen that hMsrB3 activity was supported by both T and Zn-MT, although T was active in the absence of EDTA, whereas Zn-MT required EDTA for activity. Shorter incubations were used for these experiments to minimize the oxidation of T that occurred at neutral pH. T also was active with MsrA in the absence of EDTA (data not shown). These results support the view that the requirement for EDTA with Zn-MT is to release the zinc from Zn-MT and form T and that T is able to provide the reducing system for the Msr enzymes.

Trx Can Reduce Oxidized T [T(o)].

The reaction mechanism for both MsrA and MsrB involves the formation of an oxidized enzyme intermediate that must be reduced for the Msr protein to act catalytically (6, 7, 11, 21, 22). If T is capable of reducing oxidized Msr, the T would become partly or fully oxidized to T(o), and, ideally, there should be an enzymatic system that could regenerate T and permit it to recycle. T(o) was prepared as described in Materials and Methods. This material had generally lost ≈50–60% of its free SH groups but still remained mostly soluble (see Materials and Methods). Any insoluble material that was formed was removed by centrifugation. Trx was considered a possible candidate to reduce T(o), which could be shown directly by measuring NADPH oxidation in the presence of the Trx reducing system and T(o). As shown in Fig. 4, the oxidation of NADPH depended on Trx, Trx reductase, and T(o). In addition, as shown in Table 3, T(o) could support hMsrB3 activity in the presence of the complete Trx reducing system (row 1) but not in the absence of Trx, Trx reductase, or NADPH (rows 3–5). As discussed previously, the Trx system alone showed very low activity (row 2). It is also apparent from the results in Table 3 that the free SH groups remaining in the T(o) cannot support the Msr reaction, indicating that the SH groups in T are not all equivalent with respect to their ability to function as a reducing agent for the Msr system. The results in Fig. 4 and Table 3 indicate that disulfide bonds in T(o) can be reduced by the Trx system. Thus, Trx may be one of the cellular agents that can enable T(o) to recycle and function as a metabolic reducing system.

In contrast to the results with hMsrB3, hMsrB2, which had low activity with either Trx or Zn-MT (see Table 2), was also not stimulated when both T(o) and the Trx reducing system were present (data not shown).

Purification of an Active Factor from Bovine Liver.

Fresh bovine liver was homogenized in three volumes of 50 mM Tris (pH 7.4) and centrifuged at 10,000 × g for 30 min and then at 100,000 × g for 16 h (S-100). The S-100 fraction was heated at 80°C for 5 min and centrifuged to remove precipitated proteins (heated S-100). Once the active material was suspected to be a MT, further purification followed an established method for MT (35). Using a Bio-Rad DuoFlow HPLC system, the heated S-100 was placed on a sizing column (Superdex 75 HR 10/30) followed by DE-52 anion-exchange chromatography. The fractions were routinely monitored at 240 and 280 nm. As reported in ref. 35, two distinct peaks of activity were eluted from the DE-52 column that corresponded to Zn-MT-1 and Zn-MT-2, as described in Results.

Preparation of T and T(o) and Assay of T(o) Reduction by Trx.

T and T(o) were prepared from Zn-MT by modification of the procedure described in ref. 36. Briefly, purified Zn-MT was dialyzed against 10 mM HCl (pH 2.0) containing 150 mM NaCl for 12 h at 4°C. The protein after dialysis is reduced, metal-free T and appears stable when left at pH 2.0. To study the activity of T in the Msr system, T was neutralized and added to the reaction mixtures immediately before the incubations were initiated. To oxidize T, 0.75 volumes of 50 mM Tris base were added to the T sample to bring the pH to 8.5. Under these conditions, ≈50% of the sulfhydryls will become oxidized after 4 h at room temperature or 2 h at 37°C. With longer incubations, the T(o) started to precipitate. The assay for free sulfhydryl groups used 5,5′-dithiobis(2-nitrobenzoic acid) as described in ref. 37.

To study the reduction of T(o) by Trx, the reaction mixtures contained (in a total volume of 1 ml) 100 μM NADPH, 26 μg of Trx, 6 μg of TrxB, and 28 μg of partially oxidized T (see above). The oxidation of NADPH was followed at 340 nm at room temperature.

Analysis of Zinc Content.

Zinc was quantitatively determined in the MT preparations by using the PAR reagent (38, 39). The samples (100 μl) were incubated with 10 mM N-ethylmaleimide for 1 h at room temperature. PAR (100 nmol) was then added, and the sample was diluted with water to 1 ml. The Zn–PAR complex was measured at 500 nm. Ten nanomoles of zinc gave a reading of 0.720 at 500 nm. A complete metals analysis of the purified MT preparation was performed by Joseph Caruso (University of Cincinnati, Cincinnati) by using an Agilent 7500 inductively coupled plasma mass spectrometer (Agilent Technologies, Palo Alto, CA). Molecular weight determinations of the purified protein were performed by Peter Yau (University of Illinois, Urbana–Champaign) by using electrospray MS.

Colorimetric Assay for Msr Activity Based on the Reduction of DABS-Met(o).

The reaction mixture (200 μl) to measure Msr activity contained 100 mM Tris·Cl (pH 7.4), 100 nmol of the indicated DABS-Met(o) epimer, either 15 mM DTT or the Trx regenerating system (10 μg of Trx/2.4 μg of Trx reductase/500 μM NADPH), and Msr enzyme as indicated. When the liver fractions [Zn-MT, T, or T(o)] were tested, DTT was omitted, and the Trx reducing system and 5 mM EDTA were added where indicated. Incubations were for 60 min at 37°C unless noted otherwise. In experiments using T(o), the incubations did not contain EDTA. The quantitation of DABS-Met formed used a slight modification of an extraction procedure previously described by Etienne et al. (40) for the reduction of sulindac to sulindac sulfide by MsrA. The reactions were stopped by the addition of 200 μl of 1 M sodium acetate (pH 6.0), followed by the addition of 100 μl of acetonitrile and 1 ml of benzene. After thorough shaking and centrifugation, the optical density of the benzene layer was read at 436 nm. One hundred nanomoles of the product, DABS-L-Met, carried through this procedure gave an optical density reading of 1.7, whereas 100 nmol of the substrate, either the R or S epimer of DABS-Met(o), read <0.04. Under these conditions, the reaction was proportional to Msr concentration until >75% of the substrate was reduced. Unless indicated otherwise, the results are presented as nmol of DABS-Met formed in 60 min. As little as 2 nmol of product could be measured. This assay could be used with purified preparations of MsrA and MsrB as well as crude extracts of mammalian tissues. Bacterial extracts, in the presence of a reducing system, destroyed the substrate, and further studies are needed to adapt the assay to bacterial extracts.