©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Selective Oxidative Modification and Affinity Cleavage of Pigeon Liver Malic Enzyme by the Cu-Ascorbate System (*)

(Received for publication, January 23, 1995; and in revised form, July 31, 1995)

Wei-Yuan Chou (1)(§) Wen-Pin Tsai (1)(¶) Ching-Chun Lin (2) Gu-Gang Chang (1)(§)

From the  (1)Department of Biochemistry, National Defense Medical Center, and (2)Institute of Zoology, Academia Sinica, Taipei, Taiwan, 100, Republic of China

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Pigeon liver malic enzyme was rapidly inactivated by micromolar concentration of Fe in the presence of ascorbate at neutral pH. The inactivated enzyme was subsequently cleaved by the Fe-ascorbate system at the chemical bond between Asp and Ile (Wei, C. H., Chou, W. Y., Huang, S. M., Lin, C. C., and Chang, G. G.(1994) Biochemistry, 33, 7931-7936), which was confirmed by site-specific mutagenesis (Wei, C. H., Chou, W. Y., and Chang, G. G.(1995) Biochemistry 34, 7949-7954). In the present study, at neutral pH, Cu was found to be more reactive in the oxidative modification of malic enzyme and the enzyme was cleaved in a similar manner as Fe did. At acidic pH, however, Fe was found to be ineffective in oxidative modification of the enzyme. Nevertheless, Cu still caused enzyme inactivation and cleaved the enzyme at Asp-Gly, Asp-Pro, or Asp-Asp. Mn and L-malate synergistically protect the enzyme from Cu inactivation at acidic pH. Cu is also a competitive inhibitor versus Mn in the malic enzyme-catalyzed reaction with K value 70.3 ± 5.8 µM. The above results indicated that, in addition to the previously determined Asp at neutral pH, Asp, Asp, and Asp are also the coordination sites for the metal binding of malic enzyme. We suggest that the mechanism of affinity modification and cleavage of malic enzyme by the Cu-ascorbate system proceed in the following sequence. First, Cu binds with the enzyme at the Mn binding site and reduces to Cu by ascorbate. Next, the local oxygen molecules are reduced by Cu, thereby generating superoxide or other reactive free radicals. These radicals interact with the susceptible essential amino acid residues at the metal-binding site, ultimately causing enzyme inactivation. Finally, the modified enzyme is cleaved into several peptide fragments, allowing the identification of metal site of the enzyme. The pH-dependent different specificities of metal-catalyzed oxidation system may be generally applicable for other enzymes or proteins.


INTRODUCTION

Cytosolic malic enzyme ((S)-malate:NADP oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) catalyzes the divalent metal ion-dependent reversible oxidative decarboxylation of L-malate to give CO(2) and pyruvate, with concomitant reduction of NADP to NADPH ().

The role of metal ion in the enzymatic reaction entails providing a bridge between malate and the enzyme and functions as a second sphere complex with the substrate (Hsu et al., 1976). Therefore, identification of the ligands for metal binding is ultimately important in understanding the structure-function relationship of this enzyme. In most metalloproteins, the amino acid residues involved in metal binding are dispersed along the complete sequence (Regan, 1993). Without a three-dimensional crystal structure available, affinity cleavage at the putative metal-binding site by the metal-catalyzed oxidation system (MCO) (^1)may be the optimal approach of reaching the above goal. Using this technique with the Fe-ascorbate system, Asp is successfully identified in our previous study as one of the metal-binding sites (Wei et al., 1994), as confirmed by site-directed mutagenesis (Wei et al., 1995). In that study, some divalent metal ions were found to be capable of providing protection of the enzyme against Fe-induced inactivation. Among the divalent metal ions, only Cu was found to accelerate the Fe-ascorbate-induced enzyme inactivation rate.

In this work, the inactivation of malic enzyme by Cu-ascorbate system is investigated. We demonstrate, for the first time, that at different pH values Cu-ascorbate system shows different specificities in protein modification and peptide bond cleavage. Taking the advantage of this selectivity, three more metal binding ligands of pigeon liver malic enzyme are successfully identified. Novel sequence motifs for the metal-binding site of malic enzyme are also deduced.


EXPERIMENTAL PROCEDURES

Enzyme Purification and Assay

Malic enzyme from pigeon liver was purified to apparent homogeneity according to published procedure (Chang and Chang, 1982). The enzyme activity was assayed by monitoring the formation of NADPH at 30 °C as described previously (Chang et al., 1992).

Enzyme Modification and Cleavage

The inactivation experiments were performed at 0 °C by adding freshly prepared solutions of cupric nitrate (6 µM) and ascorbate (20 mM) into the enzyme solution (0.97 µM) in sodium acetate buffer (66.5 mM, pH 5.0). The progress of enzyme inactivation was monitored by assaying the enzyme activity in small aliquots withdrawn at the designated time intervals. For modification of the enzyme at different pH values, Bis-Tris (pH 4.0-7.0) or Bis-Tris-propane (pH 6.3-9.0) was used as buffer. Other experimental conditions are provided in the figure legends.

For detecting the peptide bond cleavage, the samples withdrawn from the reaction mixture were added to EDTA solution (4 mM) to prevent further reaction from occurring. Next, the protein samples were subjected to SDS-PAGE for separating the peptide fragments. The resulting gel pattern was quantified by densitometric analysis and the isolated peptide fragments were then transblotted to an Immobilon-P membrane and subjected to automatic amino acid sequence analysis as described previously (Wei et al., 1994).

Inhibition Study

Inhibition study was performed at several Cu concentrations, and the concentration of Mn was varied from 1 to 10 µM. Concentrations of other components were held constant. The results were fitted to for a competitive inhibition by using the EZ-FIT computer program (Perrella, 1988).

where v is the observed enzyme activity, V(m) is the maximum enzyme activity, K(m) denotes the Michaelis constants for Mn, and K(i) denotes the inhibition constant for Cu.


RESULTS

Selective Inactivation of Pigeon Liver Malic Enzyme

Pigeon liver malic enzyme was highly sensitive to metal-catalyzed oxidation (Wei et al., 1994). The inactivation rate of the enzyme by the MCO system was highly pH-dependent. At pH 7.0 and 0 °C, 20 µM Fe-20 mM ascorbate caused 98% enzyme activity loss in 1 h (Fig. 1), i.e. in correlation with our previous observation (Wei et al., 1994). On the other hand, under otherwise identical conditions, the enzyme lost only 10% activity in 1 h at pH 5.0. Experimental results indicated that Cu caused a faster inactivation at a smaller concentration than Fe, especially at pH 5.0, in which Cu caused substantial inactivation (Fig. 1).


Figure 1: Inactivation of pigeon liver malic enzyme by Cu- or Fe-ascorbate systems at different pH values. The reaction mixture contained malic enzyme (0.97 µM) in 66.7 mM sodium acetate (pH 5.0, bullet, ) or triethanolamine-HCl (pH 7.4, circle, box) buffer, 6 µM cupric nitrate (bullet, circle) or 20 µM ferrous sulfate (, box), and 20 mM ascorbate. Incubation was at 0 °C.



Inactivation of malic enzyme by Cu required ascorbate in the system; Cu or ascorbate alone did not cause any inactivation. Cu was much less effective; Cu (6 µM)-ascorbate (20 mM) caused only 10% inactivation in 1 h under conditions that caused >95% inactivation by the Cu-ascorbate system.

Effect of pH on the Cu-catalyzed Inactivation of Malic Enzyme

The above results indicate that malic enzyme has a different sensitivity toward the metal-catalyzed oxidation system at two different pH values. Next, the inactivation of malic enzyme was investigated by the Cu-ascorbate system between pH 4.0-9.0 in which the enzyme was stable. There are two optima for the inactivation rate: one at pH 6.0-7.0, and the other at approximately pH 4.0 (Fig. 2). These results would suggest that different modification mechanisms involved in acidic or neutral pH. For obtaining manageable inactivation rates, the inactivation of malic enzyme by Cu at pH 5.0 is explored in the following experiments.


Figure 2: Effect of pH on the Cu-induced inactivation of pigeon liver malic enzyme. Experimental conditions were the same as in Fig. 1with 6 µM Cu, except that different buffers were used (Bis-Tris for pH 4.0-7.0, circle; Bis-Tris-propane for pH 6.3-9.0, box). The enzyme activity lost after a 1-h incubation was measured.



Dependence of Cu-catalyzed Inactivation of Malic Enzyme on CuConcentration

At pH 5.0, the inactivation of malic enzyme activity does not follow a pseudo-first-order kinetics as the natural logarithmic of residual activity versus time does not result in a straight line. The inactivation rate is clearly dependent on Cu concentration (Fig. 3), i.e. much smaller in a concentration than Fe requiring to cause the same extent inactivation.


Figure 3: Effect of Cu concentration on the inactivation of pigeon liver malic enzyme. Experimental conditions were the same as in Fig. 1at pH 5.0, except that the Cu concentrations used were: up triangle, 2 µM; , 3 µM; box, 4 µM; bullet, 5 µM; circle, 6 µM.



Similar to Fe-induced inactivation, Cu-induced inactivation could be stopped by EDTA (4 mM), which, however, did not reverse the already inactivated enzyme activity.

Protection of Malic Enzyme against Cu-induced Inactivation by Substrates

For demonstrating that the inactivation of enzyme activity was due to modification of essential amino acid residues in or near the active site, the inactivation process was examined in the presence of various combinations of substrates. In contrast to Fe-ascorbate inactivation of malic enzyme, which was completely protected by some divalent cations, Mn (4 mM) alone only protected 56% enzyme activity against the Cu-ascorbate induced inactivation at pH 5.0 (Fig. 4). L-Malate, which did not give any protective effect in the Fe system, provided 15% protection in the inactivation induced by the Cu system. L-Malate plus Mn yielded synergistic protection (90%). Nucleotide NADP, on the other hand, did not provide any protection by itself or in combination with Mn and L-malate in both Fe and Cu systems.


Figure 4: Protection of pigeon liver malic enzyme from Cu-catalyzed inactivation by substrates. Experimental conditions were the same as in Fig. 1with 6 µM Cu and pH 5.0, but with the following additions: circle, none; , 5 mML-malate; , 4 mM Mn; bullet, 5 mML-malate + 4 mM Mn; box, 5 mML-malate + 4 mM Mn + 0.23 mM NADP.



Inhibition of Pigeon Liver Malic Enzyme by Cu

The above results indicate that at pH 5.0 the binding mode between divalent cation and the enzyme is different from that at pH 7.0. Direct kinetic evidence for the binding of Cu at the Mn binding site of malic enzyme was provided by inhibition studies shown in Fig. 5, where Cu was demonstrated to be a competitive inhibitor with respect to Mn with K(i) value of 70.3 ± 5.8 µM. This result indicates that Cu and Mn compete for the same binding site. The fitted value of K(m) was 2.7 ± 0.23 µM, which is within the range (1.8-9 µM) as determined previously (Hsu et al., 1976).


Figure 5: Competitive inhibition of pigeon liver malic enzyme by Cu with respect to Mn. In the routine assay mixture, the Mn concentration was varied from 1 µM to 10 µM with all other components held at fixed concentration. Malic enzyme activity was assay in the presence of various concentrations of Cu: circle, none; bullet, 100 µM; box, 200 µM. Lines are computer fitted to .



For comparison, Cu was also tested for inhibition of malic enzyme. Cu as high as 160 µM showed no inhibition on the enzyme. At 169 µM, the inhibition was only 7%. These results strongly suggest that only divalent Cu has a high affinity with malic enzyme.

Peptide Bond Cleavage Pattern of the Cu-inactivated Malic Enzyme

We have demonstrated previously that, at pH 7.0, the Fe-ascorbate system cleaved malic enzyme at Asp-Ile (Wei et al., 1994). In this study, the cleavage pattern of the enzyme inactivated by Cu-ascorbate system is also examined. The results shown in Fig. 6clearly indicate that Cu induced the same cleavage as Fe at pH 7.0. However, different specificities were observed at pH 5.0. Fe showed minimum cleavage at pH 5.0, which is in correlation with the minimum enzyme inactivation observed (Fig. 1). Cu produced entirely different cleavage patterns at pH 5.0 (Fig. 6, lane6). The two fragments IV and V, which are those seen at pH 7.0, were relatively small in quantity at pH 5.0. Instead, six major fragments are detected (E`(I), E`, E`, E`, E`, and E`). On the basis of M(r) estimation, these fragments seem to be due to three cleavages. Site A cleavage produces E` (48,000) and E` (M(r) 17,000), site B cleavage produces E` (M(r) 38,000) and E` (M(r) 27,000), and cleavage at site C produces E`(I) (M(r) 55,000) and E` (M(r) 10,000). Probably because of a low molecular mass, the diffusion problem renders E` and E` less defined visually. However, we successfully obtained a discrete amino acid sequence that is the fourth coordination site of Mn of malic enzyme as described in the following sections.


Figure 6: SDS-PAGE pattern of the metal-catalyzed oxidized malic enzyme. A, lane1 is M(r) standards (phosphorylase b, M(r) 94,000; bovine serum albumin, M(r) 67,000; ovalbumin, M(r) 43,000; carbonic anhydrase, M(r) 30,000; trypsin inhibitor, M(r) 20,000; alpha-lactalbumin, M(r) 14,400). Lane2 is the unmodified native enzyme. Lanes3 and 4 are enzymes modified at pH 7.0. Lanes5 and 6 are enzymes modified at pH 5.0. Lanes3 and 5 are enzymes modified with Fe-ascorbate system. Lanes4 and 6 are enzymes modified with Cu-ascorbate system. B, cleavage positions of malic enzyme by the MCO systems identified by amino acid sequence analysis of the peptide fragments.



Correlation between Enzyme Activity Inactivation and Peptide Bond Cleavage

To correlate the Cu-induced enzyme inactivation and peptide bond cleavage, the modification was performed in various stages, the reaction was stopped with EDTA, and the protein samples were subjected to SDS-PAGE to examine the peptide bond cleavage. Results shown in Table 1clearly indicate that with the increasing of incubation time, a rapid loss in enzyme activity occurred and the peptide bond cleavage increased. However, the enzyme activity lost proceeded much faster than the peptide bond cleavage. When the enzyme activity was down to 27%, 91% of the enzyme molecules were still intact. We can conclude that peptide bond cleavage follows the enzyme inactivation, as for other MCO system-induced enzyme inactivation. Site C is a minor cleavage site as compared to sites A and B. Since E` + E` and E` + E` appear to have an equal amount (Table 1), site A and site B seem to have a similar probability of cleavage. However, these sites do not cleave simultaneously in the same enzyme molecule. Similar mutually exclusive cleavages were also observed for pig heart isocitrate dehydrogenase with Fe-ascorbate system (Soundar and Colman, 1993).



The switch of specificity was examined by monitoring the protein cleavage pattern at various pH values. As the pH decreased from neutral to acidic, fragments I, II, III, VI, VII, and VIII gradually increased, while fragments IV and V decreased (data not shown).

Identification of the Metal-binding Site of Pigeon Liver Malic Enzyme

From Fig. 6, we see that, at pH 5.0, Cu cleaved malic enzyme at three sites that are different from the Asp-Ile observed at pH 7.0. The SDS-PAGE-separated peptide fragments were electrophoretically transblotted onto an Immobilon-P membrane and each peptide was analyzed via an automatic protein sequencer. The N terminus of fragment II was found to have the following sequence: Gly-Glu-Arg-Ile-Leu-Gly-Leu-Gly-Asp-Leu-X-X-X-Gly-Met-Gly-Ile-X-X-Gly, which is identified as the known sequence between Gly-Gly of the cDNA sequence of pigeon liver malic enzyme (Chou et al., 1994). Peptide E` has the N-terminal sequence Pro-Leu-Tyr-Ile-Gly-Leu-Arg-His-Lys-Arg-Ile-Arg-Gly-Gln-Ala-Tyr-Asp-Asp, which is identified as Pro-Asp of the cDNA sequence. Peptide E` has the N-terminal sequence Asp-Val-Phe-Leu-Thr-Thr-Ala-Glu-Val-Ile-Ala-Gln-Glu-Val-Ser-Glu-Glu-Asn-Leu-Gln, which is identified as Asp-Asn of the cDNA sequence.

Corresponding to peptide E` and E` are peptides E` and E`, which contain the N-terminal peptides Met^1-Asp and Met^1-Asp, respectively, and should not detect any sequence since the N terminus of malic enzyme is blocked (Wei et al., 1994). When peptides E` and E` were subjected to amino acid sequence analysis, multiple small peaks were detected and identifying any reliable sequence was relatively difficult. When consulting the gel pattern shown in Fig. 6, we can conclude that while peptides E`-E` represent the major cleavage products, minor cleavage at other sites also occurred. Peptide E` contains sequence Met^1-Asp and some other minor peptides. This is also true for peptide E`. The failure in identifying some amino acid residues in E` may be due to the interference of other minor peptides. A sufficient quantity of fragment I for sequence analysis was not collected in this study to confirm that it is blocked at the N terminus.

Although the peptide cleavage caused by Cu system is complicated at pH 5.0, three more metal ligand sites are unequivocally identified, i.e. Asp, Asp, and Asp. A comparison with the known amino acid sequences for malic enzyme from various sources (Bagchi et al., 1987; Börsch and Westhoff, 1990; Chou et al., 1994; Hsu et al., 1992; Loeber et al., 1991, 1994; Magnuson et al., 1986; Kobayashi et al., 1989; Kulkarni et al., 1993; Rothermel and Nelson, 1989) (^2)reveals that these aspartate residues are highly conserved (Fig. 7Fig. 8Fig. 9).


Figure 7: Comparison of the putative Mn binding site Asp of malic enzymes. Figure shows amino acid sequences around Asp of pigeon and duck, Asp of human, rat and mice, Asp of human liver mitochondria, Asp of Ascaris suum mitochondria, Asp of maize chloroplast, Asp of dicotyledonous C4 plant Flaveria trinervia, and Asp of Bacillus stearothermophilus malic enzymes were aligned to show the high conservation in this region. The cleavage site of pigeon liver malic enzyme by the Cu-ascorbate was indicated by an arrow. The dashedunderlined sequence was confirmed by amino acid sequence analysis. The gray area showed identical amino acid residues between different malic enzymes. The putative Mn-binding ligand Asp was highlighted.




Figure 8: Comparison of the putative Mn binding site Asp of malic enzymes. Figure is same as Fig. 7but with amino acid sequences around Asp of pigeon and duck, Asp of human, rat and mice, Asp of human liver mitochondria, Asp of Ascaris suum mitochondria, Asp of maize chloroplast, Asp of Flaveria trinervia, and Asp of Bacillus stearothermophilus malic enzymes.




Figure 9: Comparison of the putative Mn binding site Asp of malic enzymes. Figure is same as Fig. 7but with amino acid sequences around Asp of pigeon, Glu (may be Asp) of duck, Asp of human, rat, and mice, Asp of human liver mitochondria, Asn (may be Asp or Asp) of Ascaris suum mitochondria, Glu (may be Asp) of maize chloroplast, Asp of Flaveria trinervia, and Val (may be Glu) of Bacillus stearothermophilus malic enzymes.




DISCUSSION

Pigeon liver malic enzyme is among the most sensitive enzymes toward MCO system. We have demonstrated previously that this enzyme was rapidly inactivated by the Fe-ascorbate system at neutral pH. The inactivation was rapid that incubation at 0 °C was necessary to slow down the reaction rate. In this study, the Cu-ascorbate system was found to be more effective than the Fe system. Experimental results indicated that the Cu system produced an entirely different cleavage pattern at pH 5.0 as compared to the pattern at pH 7.0. The following criteria indicate that Cu-ascorbate caused affinity modification and cleavage of pigeon liver malic enzyme at the putative metal-binding site. (a) Cu is essential for the process; Cu is much less effective, suggesting that Cu must bind with the enzyme at the divalent metal ion binding site before inactivation takes place. (b) Cu is a competitive inhibitor versus Mn for the enzyme, indicating that Cu and Mn compete for the same binding site. (c) Inactivation of the enzyme is prevented by Mn plus L-malate, indicating that modification is at the active site.

Based on the above discussion, the reaction sequence of oxidative modification and peptide bond cleavage of pigeon liver malic enzyme by the Cu-ascorbate system can be summarized in Fig. SI. First, Cu binds with the enzyme at the Mn binding site (step 1). Second, ascorbate reduces Cu to Cu (step 2), which, in the presence of dissolved O(2), generates reactive free radicals (e.g. O(2), OH) that in turn modify the essential amino acid residue(s) nearby and forming the inactivated enzyme (E`) (step 3). Finally, depending on pH of the solution, the enzyme molecule is cleaved at four possible sites giving peptide fragments E`(I) + E`, E` + E`, or E` + E` at pH 5.0, or E`+ E`(V) at pH 7.0 (step 4). In this manner, different specificities are achieved by manipulating the reaction conditions. We suggest that this strategy can be generally applied to other enzymes or proteins in elucidating the metal-binding sites. We propose that the catalytically essential carboxyl group of Asp has a pK(a) value of 6.7, as determined by chemical modification experiments (Chang et al., 1985). At pH 5.0, this carboxyl group is protonated and loss its metal-binding ability and is not reactive toward oxidative modification. Under this circumstance, Fe is inactive but other metal ligands Asp, Asp, or Asp, which might have pK(a) values near 4.7, are modified by the more reactive Cu.


Figure SI: Scheme IProposed reaction mechanism for the oxidative modification and affinity cleavage of pigeon liver malic enzyme by the Cu-ascorbate system. The N and C under each peptide fragment denote the N- or C-terminal half before cleavage.



Interestingly, the metal ligands Asp, Asp, Asp, and Asp of malic enzyme were all aspartate residue, which has been indicated to be the major metal-binding ligand for many metal-proteins (Higaki et al., 1992; Vallee and Auld, 1993; Traut, 1994). Furthermore, isocitrate dehydrogenase, which catalyzes a similar oxidative decarboxylation reaction as malic enzyme, was demonstrated to have Asp, Asp from the same subunit, and Asp` from the other subunit as the metal coordinates (Hurley et al., 1990); this enzyme from pig heart was also sensitive to MCO system (Soundar and Coleman, 1993). Examination of the sequences shown in Fig. 7Fig. 8Fig. 9reveals that Asp and Asp, the major cleavage sites by the Cu system, are strictly conserved in all malic enzyme with known amino acid sequences. Asp is also highly conserved; however, this region has higher variations among malic enzyme of different origins. This site is a minor cleavage site. Maximum alignment of other malic enzyme sequences with Asp of pigeon enzyme reveals that Glu of duck, Asn of ascaris, and Glu of maize enzymes may be the metal coordinates. Although Asn and Glu are found as metal ligand in many metal-proteins (Villafranca and Nowak, 1992; Higaki et al., 1992; Vallee and Auld, 1993; Traut, 1994), an observation of the nearby amino acid residues reveals that Asp of duck, Asp or Asp of ascaris, and Asp of maize enzyme may be the authentic metal ligands. These results enforce the critical value of aspartate residue as the metal coordinate in proteins. Only bacillus malic enzyme was nonconservatively substituted this Asp with Val. The actual metal ligand, however, may be Glu, which is also a conservative substitution.

Malic enzyme is a bifunctional enzyme. It catalyzes both the oxidoreduction and decarboxylation reactions. The reaction mechanism of the enzyme proceeds in two steps with hydride transfer preceding decarboxylation (Hermes et al., 1982). These two functions can be assessed separately by assaying the partial reactions with appropriate substrate (Hsu, 1982). One of the distinguishing features of the decarboxylase activity of malic enzyme is its pH optimum being at 4.5 (Salles and Ochoa, 1950). We propose that the proton released during dehydrogenase reaction provides a favorable local active site environment for the decarboxylation reaction, which involves metal ion-stabilized enolate anion transition state (Hsu et al., 1976; O'Leary, 1992). During the catalytic cycle, the enzyme might undergo an isomerization that favors the decarboxylation reaction. Results presented in this study support the hypothesis that the enzyme exists as different conformational isoforms at neutral or acidic environment. However, another possibility that the observed pH effects were due to changes in the catalytic cleavage rate of different sites rather than to global conformational changes was not ruled out (Kufel and Kirsebom, 1994).

Metal-catalyzed oxidation of proteins has been suggested to be the marker for protein turnover in vivo (Stadtman and Oliver, 1991; Stadtman, 1992). According to this theory, the oxidized protein molecules are unstable and prone to degradation by multicatalytic proteases (proteasomes) in cells (Rivett, 1993). The normal copper concentration in serum is 16-31 µM (Murray et al., 1990); both iron and copper ions are normally presented in the cells, which also contain various reducing compounds. If malic enzyme is sensitive to only a few micromolar concentration of Cu in the cytoplasm, an intriguing question is that how could malic enzyme survive in vivo for a reasonable period of time to perform its metabolic roles? Protection of the enzyme by Mg or other divalent cations and substrate is one of the answers. Furthermore, cells contain other defense mechanisms against oxidative damage. The endogenous antioxidant enzymes (catalase, dismutase) are active in normal young tissues; a rapid physiological response of the translational or transcriptional control of the detoxification genes might play an important role when metal ion concentrations exceed a dangerous threshold (O'Halloran, 1993). These detoxification gene products may play important roles in cell protection, e.g. the high Fe content and oxygen carrier function of red blood cells indicate the high oxidation stress experience by red blood cells, which contains an abundant natural killer enhancing factor that protects red blood cells from oxidation injuries (Shau and Kim, 1994). These protecting proteins are highly homologous with yeast thiol-specific antioxidant, which protects yeast cells from oxidation insults (Chae et al., 1993). It is these natural protecting mechanisms that prevent cell proteins and other cellular components from experiencing oxidative damage.


FOOTNOTES

*
This work was supported by National Science Council (Republic of China) Grants 84-2331-B016-063 (to W.-Y. C.) and 82-0412-B016-032 (to G.-G. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, National Defense Medical Center, P. O. Box 90048, Taipei, Taiwan, 100, Republic of China. Fax: 886-2-365-5746.

Portions of this work were submitted in partial fulfillment of a M. S. degree (Biochemistry), National Defense Medical Center, Taipei.

(^1)
The abbreviations used are: MCO, metal-catalyzed oxidation system; PAGE, polyacrylamide gel electrophoresis.

(^2)
Chou, W.-Y., Huang, S.-M., and Chang, G.-G., unpublished results.


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