Vol. 137, No. 1
JOURNAL OF BACTERIOLOGY, Jan. 1979, p. 313-320 0021-9193/79/01-0313/08$02.00/0
Biosynthesis and Cellular Distribution of the Two Superoxide Dismutases of Dactylium dendroides ALLAN R. SHATZMAN AND DANIEL J. KOSMAN* Bioinorganic Graduate Research Group, Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York 14214
Received for publication 2 November 1978
The synthesis and subcellular localization of the two superoxide dismutases of Dactylium dendroides were studied in relation to changes in copper and manganese availability. Cultures grew normally at all medium copper concentrations used (10 nM to 1 mM). In the presence of high (10 ,uM) copper, manganese was-. poorly absorbed in comparison to the other metals in the medium. However, cells grown at 10 nM copper exhibited a 3.5-fold increase in manganese content, while the concentration of the other metals remained constant. Cultures grown at 10 nM copper or more had 80% Cu/Zn enzyme and 20% mangani enzyme; the former was entirely in the cytosol, and the latter was mitochondrial. Removal of copper from the medium resulted in decreased Cu/Zn superoxide dismutase synthesis with a concomitant increase in the mangani enzyme such that total cellular superoxide dismutase activity remained constant. The mangani enzyme in excess of the 20% was present in the non-mitochondrial fraction. The mitochondria, therefore, show no variability with respect to superoxide dismutase content, whereas the soluble fraction varies from 100 to 13% Cu/Zn superoxide dismutase. Copper-starved cells that were synthesizing predominantly mangani superoxide dismutase could be switched over to mostly Cu/Zn superoxide dismutase synthesis by supplementing the medium with copper during growth. Immunoprecipitation experiments suggest that the decrease in Cu/Zn activity at low copper concentration is a result of decreased synthesis of that protein rather than the production of an inactive apoprotein. In 1938, Mann and Keilin (21) isolated from ox blood a copper-containing protein of molecular weight 35,000 which had no discernible activity. In the following 30 years similar proteins were isolated from horse liver (25), bovine and human brains (29-31), human erythrocytes (5), and human liver (32) and were named according to their source (hepatocuprein, cerebrocuprein, and erythrocuprein). It was not until 1969 that McCord and Fridovich (20) discovered the superoxide dismutase activity of these proteins. The enzyme supero d i b u (EC 22.214.171.124), trac+ which ca H202 + 09 (20), appears to be an important component of the defenses that have evolved to reduce the potential toxicity of the superoxide radical (8, 12, 13, 15) produced by numerous biological reactions (1, 20, 33). The enzyme, which was also shown to contain zinc (5), is ubiquitous to all oxygen-metabolizing organisms (19). Proteins of identical activity but different genetic origin (18) have been found in aerobic organisms, i.e., the mangani- and iron-containing superoxide dismutases. The mangani- and ironcontaining enzymes have been found in several
procaryotes, whereas the mangani and Cu/Zn enzymes are commonly found in eucaryotes (19). The Cu/Zn protein was believed to be the only superoxide dismutase found in the cytosol of eucaryotic cells, while the mangani enzyme (EC 126.96.36.199) was unique to the mitochondria (27, 34, 39). Recently, however, McCord (19) has shown that the mangani superoxide dismutase is not only present in the cytosol of several species, but is actually the principal cytosolic form of this enzyme in some. The study of copper utilization and copper protein biosynthesis in the fungus Dactylium dendroides has led us to study the effects of copper availability on superoxide dismutase synthesis and distribution. We have already shown that decreasing copper availability leads to increased levels of mangani superoxide dismutase activity in this organism (37). In this report, we have investigated the effects of changes in intracellular metal concentration accompanying copper deprivation in relation to the changing profiles of enzyme synthesis. Our results are in accord with the findings of McCord (19) in that the manganese-containing enzyme may be found 313
SHATZMAN AND KOSMAN
in varying amounts in the cytosol. However, our results here indicate that the cytosolic concentration of this protein appears to be a function of intracellular manganese and copper concentrations and that these two trace metals are taken up in a competitive manner. The mitochondrial superoxide dismutase content appears to be constant and critical to cell survival. These results provide further insight into metal uptake and copper utilization while facilitating the isolation of large quantities of either forn of superoxide dismutase. MATERIALS AND METHODS Growth of organism and media preparation. The fungal isolate (NRRL 2903) and growth conditions are those previously described (22, 36). Cells were cultured in an artificial medium that had been treated with Chelex 100 (Bio-Rad) as described earlier (37). Copper additions were made at varying times by adding CuS04 to concentrations between 1 ,uM and 1 mM. Myceial weight determination. Cells were harvested from shake-flask cultures and filtered through several layers of cheesecloth, washed, refltered, and squeezed dry. Cells were suspended in water, centrifuged at 12,000 x g for 15 min, and again squeezed dry through cheesecloth. These cells were weighed on a Mettler single-pan balance to obtain wet weights. Dry weights were determined as reported earlier (36). Metal analyses. The concentration of metals in all solutions, media, and cells was determined by using a -Perkin-Elmer Atomic Absorption Spectrophotometer wjmodel 360 equipped with a model 2100 graphite furnace. Appropriate standards were obtained from Alfa Chemicals. Drying times varied from 10 s for 5-pl samples to 40 s for 50-pl samples; charring and atomizing times remained constant at 20 and 8 a, respectively. Fungal cells were prepared for analysis as previously described (37). The atomizing time for magnesium analysis was increased to 15 s, followed by use of the high-energy mode for 10 to 15 s to remove any residual metal bound to the graphite tube. Eppendorf tips were used, with acid washing necessary before doing zinc analyses. Cell breakage: production of a cell extract and mitochondrial isolation. One hundred and fifty grams (wet weight) of cells were prepared for breakage by an Eaton cell as described previously (37). This process gave a cell extract with intact mitochondria. These organelles could be isolated by differential centrifugation and were assayed by the succinate-INT reductase assay according to the method of Pennington (28). Cell extracts with and without mitochondria were concentrated to a volume of 6 ml by ultrafiltration (Amicon PM 10). Purification of fungal Cu/Zn superoxide dismutase. The enzyme was purified from D. dendroides using a method similar to that developed by Misra and Fridovich (23) with the following modification. To the 1:1 acetone-protein solution, which yields the precipitate containing superoxide dismutase, was added 50 g of microcrystalline cellulose (Sigma) to facilitate
J. BACTERIOL. recovery of the precipitate. This mixture was stirred and then centrifuged at 10,000 x g for 15 min. The pellet was suspended in 0.1 M potassium phosphate buffer (pH 7.8), followed by a second centrifugation. The supernatant was dialyzed and chromatographed as reported (23). Apo-superoxide dismutase was prepared by incubation with an excess of diethyldithiocarbamic acid. The excess diethyldithiocarbamic acid and the copper chelate were removed by repeated ultrafiltration using a UM2 membrane (Amicon). The protein was diluted with copper-free 0.1 M KCI (pH 4.5) between each ultrafiltration step. Assay of superoxide dismutase activity. The superoxide dismutase activities of the concentrates and mitochondrial preparation were assayed according to literature methods (2, 24). The Cu/Zn cytosolic enzyme was inhibited by addition of 5 mM KCN (39). CN- affects neither the epinephrine assay system nor the mangani superoxide dismutase (19, 24, 37, 39). The activities of both forms of dismutase were visualized on polyacrylamide gels using the activity stain described by Beauchamp and Fridovich (2). Addition of KCN was used to distinguish the two dismutase activities as above. Production of antibodies to fungal Cu/Zn superoxide dismutase. A female New Zealand white rabbit was bled from an artery in the ear to make sure that there were no natural antibodies present in the serum that would cross-react with fungal superoxide dismutase. The rabbit was injected four times, over a period of 5 weeks, with 2 mg of pure fungal Cu/Zn superoxide dismutase per injection. The protein was mixed with Freund complete adjuvant (Difco) to increase the antigenicity of the protein. The mixture was injected subcutaneously, alternately on the right and left side of the rear half of the animal. Detection of apo-Cu/Zn superoxide dismutase. Immunodiffusion (26) and immunoelectrophoresis (35) were performed, using the cell extract concentrates and the rabbit anti-fungal superoxide dismutase antiserum. Intensity of the precipitin arcs obtained with the pure fungal protein were compared with those obtained from the purified apoprotein, horse Cu/Zn superoxide dismutase, and the cytosolic extracts from high- and low-copper cells. The presence of apo-superoxide dismutase was also determined by immunoprecipitation experiments using the antiserum and the high- and low-copper cytosolic extracts. Fifty-microliter samples of these media concentrates were added to 10 tubes containing 100 ,tl of rabbit anti-superoxide dismutase and nine serial dilutions of this antiserum. The diluent was copper-free 0.1 M KCI (pH 7.0). After overnight incubation at room temperature, the samples were spun for 10 min at 2,000 x g and assayed for superoxide dismutase activity as described above.
RESULTS Culture growth. As reported earlier (22, 36, 37), cultures of D. dendroides are easily maintained on a minimal medium in which thiamine pyrophosphate is the only added vitamin (22, 36). Growth remained normal at all copper con-
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centrations used in culturing the fungus (10 nM to 1 mM) except for the changes in fungal cell coloration. Cells grown at 10 nM copper are pinkish, those grown, at 10 and 100 ,uM copper are brownish, and those grown at 1 mM copper are blue-green. Low-copper cells remained somewhat pinkish after incubation with 10 ,uM copper, but turned blue when incubated with 1 mM copper. Metal utilization. D. dendroides is a very efficient scavenger of copper and most other metals. In cultures grown at low copper concentration (0.5 ,uM or less), almost all of the extracellular copper is taken up by the cells during growth. At higher copper concentrations, between 50 and 75% of the available copper is utilized or stored by the cells (37). Iron, zinc, magnesium, and calcium levels were generally unaffected by copper availability, since the metal content per milligram of cells (dry weight) did not vary appreciably (± 10%) (Table 1). Iron, zinc, and calcium, all present in the initial growth medium at a concentration of 10 ,uM, were taken up by the cells such that 50 to 75% of the available metal was utilized or stored. A similar percentage of magnesium, present initially at 25 mM, was also taken up by the cells. The uptake of manganese, however, which was present at 10 ,uM, showed a large dependence on copper availability and utilization. Cultures maintained at 10liM copper contained 20 ng of manganese per mg of cells (dry weight) as compared to 68 ng/mg for cells grown at 10 nM copper. This increased manganese content explains the pink color of low-copper cells. Under low-copper conditions, about 50% of the available manganese was taken up by the cells, as compared to only 15% utilization in the highcopper situation. Cells grown at both 10,uM and 10 nM copper, when incubated with 1 mM copper in vitro, rapidly took up copper, giving bluish cells with no change in intracellular manganese concentration. Superoxide dismutase synthesis: subcelTABLE 1. Intracellular metal concentrations in D. dendroides Medium-copper concn' Element
10 nM 2
10 AM 90
130 130 Fe 115 108 Zn 92 85 Ca a Metal concentrations expressed as nanograms per milligram of cell dry weight.
lular localization. As reported earlier (37), cells harvested from cultures grown at all copper concentrations have approximately the same amount of total superoxide dismutase activity, 2,400 units/50 g of cells (wet weight). However, the fraction of the total activity contributed by the Cu/Zn form versus the mangani form is a function of the initial copper concentration in the medium (Fig. 1). Cells grown at 10 ,uM copper had 87% of their superoxide dismutase activity contributed by the Cu/Zn species, whereas cultures grown at 10 nM copper showed only a 13% contribution from that form. The subcellular localization of the superoxide dismutases in D. dendroides was also studied. The mangani enzyme, often called the mitochondrial superoxide dismutase, is the only form 100 -
4) 0 0,
601 0 =1 0. 0
60. 401 v 0 x
6 5 4 3 - Log [Cu] FIG. 1. Composition of total cellular superoxide dismutase activity as a function of initial extracellular copper concentration. Fifty grams (wet weight) of cells was removed from 6-day-old cultures grown at initial copper concentrations ranging from 10 nM to 1 mM. These cells were fractured, and the extracts were concentrated by ultrafiltration to a volume of 6 ml. The total superoxide dismutase activity (full
length of bar, -2,4() units) was determined by the degree of inhibition of the autoxidation of epineph-
rine (24). The percentages of this activity attributable to the mangani (CN- insensitive; white portion of bar) and Cu/Zn (CN- sensitive; shaded area of bar) enzymes were determined by the addition of 5 mM KCN to the epinephrine assay.
SHATZMAN AND KOSMAN
present in the mitochondria of D. dendroides. The superoxide dismutase in the mitochondria contributes about 20% of the total cellular activity, regardless of the copper availability during growth. The cytosol, however, shows great variability with respect to superoxide dismutase content. All cultures grown at 10,M copper and above had the same concentration of Cu/Zn enzyme in the cytosol, and this enzyme is responsible for essentially all the non-mitochondrial superoxide dismutase activity. Cells cultured at lower concentrations of copper (1 ,uM to 10 nM) showed a decrease in Cu/Zn superoxide dismutase activity accompanied by the appearance of increasing amounts of the mangani enzyme in the cytosol. Whereas the cytosolic fraction of 10 ,uM copper cells had nearly 100% of its superoxide dismutase in the Cu/Zn form, cells grown at 0.01, 0.1, and 1.0 ,uM copper had only 15, 37, and 69%, respectively, in that form. The total cytosolic superoxide dismutase activity was maintained at a constant level, regardless of copper availability, by increasing concentrations of the mangani form, which compensated for the decrease in the level of the Cu/Zn enzyme.
The addition of copper to these copper-depleted cultures during growth drastically changed the ratio of synthesis of these enzyme forms. The magnitude of the effect was dependent upon the time and level of copper supplementation. The addition of 10 ,uM copper to copper-starved cultures after 2 days of growth produced a rapid and permanent reversal in the proportional activities contributed to the whole cell by the Cu/Zn and mangani enzyme forms (Fig. 2A). Within one day of this copper supplementation, the Cu/Zn-to-mangani enzyme ratio changed from 20:80 to 72:28 and eventually to 75:25 (Fig. 2A). This ratio approximates the 80: 0
5 6 2 3 Growth Tlm (days)
FIG. 2. Changes in the total cellular levels of the two superoxide dismutases, measured at 1-day inter-
vals after copper supplementation. (A) Either I (0) copper was added to cultures grown (@) or 10 at 10 nM copper for 2 days. (B) Either EM (S) or 10 (E) copper was added to cultures grown at 10 nM copper for 3 days.
20 ratio seen in cultures maintained at 10 ,iM for all 6 days of growth. The normal 80: 20 ratio could not be reached under these conditions due to the presence of the mangani enzyme, some of which remains in the cytosol from the first 2 days of culture growth. This mangani enzyme was reduced from 90 to 7% of the cytosolic superoxide dismutase after copper supplementation by dilution with Cu/Zn superoxide dismutase, cell division, and (perhaps) proteolysis. When 1 ttM copper was added to the copper-starved cells after 2 days of growth, the reversal in proportional activities of the two superoxide dismutases was much slower than that described above. The Cu/Zn:Mn enzyme ratio only changed from 15:85 to 28:72 within 1 day of supplementation (Fig. 2A), although by day 4 after supplementation the ratio was 53:47, which closely approximates the 55:45 ratio obtained in cultures grown at 1 ,uM copper for all 6 days. Four days were required also for the Cu/Zn-to-Mn enzyme ratio in the soluble fraction to increase from 19:81 to 66:34. Supplementation of cultures after 3 days of low-copper growth with 1 and 10lM copper gave similar results, though somewhat less dramatic than those reported above. When 10lM copper was added to these cells, it took 2 days for the cellular enzyme ratios to change from 10: 90 to 31:69 and eventually to 29:71 (Fig. 2B). This represents a change in mangani enzyme content of the soluble fraction from an initial 89% to a final 11%. The enzyme ratio changed slowly from 10:90 to 40:60 when 1 ,uM copper was added to these cultures (Fig. 2B). This is appreciably different from the 55:45 ratio seen for cells grown at 1 ,iM copper for all 6 days. Under these conditions, the mangani superoxide dismutase was present in amounts equal to the Cu/Zn enzyme in the non-mitochondrial fraction. The decrease in Cu/Zn superoxide dismutase activity in cells grown below 10 ,uM copper is due to the decreased synthesis of this protein rather than to the production of an apo-, inactive form. Copper and zinc added to the cells for a short time (60 min) did not yield an increase in enzyme activity. Free copper as well as copper plus ascorbate added to cell extracts also failed to give any increase in activity. Immunodiffusion and immunoelectrophoresis using rabbit anti-D. dendroides superoxide dismutase antiserum also showed that no apo-Cu/Zn superoxide dismutase was present in the cytosolic fraction of lowcopper cells. A sharp, distinct precipitin arc was obtained with purified apo- and holo-superoxide dismutase and with the high-copper cytosolic extract: The low-copper extract gave a very faint arc, due to the low levels of the enzyme present. copper
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No cross-reactivity was observed between horse superoxide dismutase and the anti-fungal superoxide dismutase antiserum. Immunoprecipitation results indicate that there is about six times more Cu/Zn superoxide dismutase in high-copper cells than in low-copper cells (Fig. 3), which agrees with the activity measurements of the respective extracts and the Ouchterlony results described above.
DISCUSSION The ability of D. dendroides to maintain normal growth rates at nanomolar to millimolar copper concentrations makes this organism uniquely suited for the study of copper protein biosynthesis. In these experiments, changes in protein synthesis and copper content can be directly related to changes in copper availability and utilization without adding the complications that accompany changes in growth, such as variations in cell weights and doubling times. In contrast, studies with this isolate involving deprivation of iron, zinc, magnesium, or manganese would all be affected by changes in growth to different degrees (37). Similar observations concerning variations in trace metal requirements have been observed in gram-positive and gramnegative bacteria (38). The large variations observed in intracellular manganese concentration accompanying changes in copper availability indicate that the uptake of these metals is somehow coordinated.
u~ 60 0 cn
FIG. 3. Determination of total Cu/Zn superoxide dismutase protein by immunoprecipitation. The soluble fractions of cells grown at 10 nM (U) and 10 M (0) copper were concentrated to a volume of 6 ml and assayed for superoxide dismutase activity as described in Fig. 1. Fifty microliters of the respective cell extracts, containing 400 units per ml, was added to serial dilutions of the rabbit anti-fungal.superoxide dismutase antiserum. After overnight incubation and a 10-min centrifugation at 2,000 x g, the supernatant fractions were assayed for superoxide dismutase activity using the epinephrine autoxidation method. Activities are recorded as a percentage of the maximal activity present in the control (no antiserum).
The inability of cells grown at 10 uM copper to effectively concentrate manganese suggests that these two metals may "compete" for the same route of uptake and/or transport. This competitiqn apparently favors copper uptake over manganese, since when copper is available in quantity (10 ,uM) only 15% of the available manganese (also at 10 ttM) is utilized. However, when copper availability is poor (10 nM) and competition is minimized, manganese uptake is increased 3.5-fold. Copper, when taken up in large amounts, may remain bound to the cell wall (3) and thereby inhibit intracellular and extracellular free manganese. from reaching equilibrium, even after 5 to 6 days of growth, at which time extracellular copper has been depleted (37). That the competition is for uptake and not storage is illustrated by the result that low-copper, high-manganese cells, incubated in 1 mM copper, rapidly took up copper while maintaining the elevated intracellular manganese levels. There is no indication that the other metals present in the growth medium compete with copper or manganese for transport into the cell, since their intracellular concentrations do not vary appreciably with those of copper and manganese. The competition for uptake between copper and manganese makes it tempting to suggest that these two metals are competing for the same sites on the cell wall to which they bind or through which they enter the periplasmic space. Copper and manganese have been shown to affect magnesium efflux in Bacillus megaterium and Bacillus subtilis (38). In this case the loss of magnesium which occurs when exponential-phase cells are placed in a magnesium-free medium is prevented completely by copper (5 nM) and reduced by 85% in the presence of manganese (25 nM). This similar action of copper and manganese on the cell wall is further evidence that their binding site(s) on the cell wall may be the same. A metal-binding protein may also be implicated in the uptake and retention of copper and other metals. A copper-binding protein and a copper-binding ligand have been isolated from this organism; they tightly bind 2 and 1 g-atoms of copper per mol, respectively (A. R. Shatzman and D. J. Kosman, Arch. Biochem. Biophys., in press). These species, which do not bind manganese in vivo, may participate in mediating the preferential transport of copper into the fungus. There have been no manganese-binding proteins isolated in microorganisms to date, although manganese-binding proteins, similar to ferritin, have been isolated from rabbit and rat blood plasma (14). On the other hand, the low intracellular manganese concentration could be the
SHATZMAN AND KOSMAN
mechanism which "pumps" manone which blocks its entry. This manganese pump could involve active transport of manganese out of the cell or could be coupled to the influx of another metal, possibly copper. Although the mechanism by which the intracellular manganese content is maintained is not known, the fate of a large percentage of the elevated manganese in low-copper cells has been elucidated. Manganese has been shown to provide a sparing action for magnesium in bacteria (38), and appears to provide a copper-sparing action in D. dendroides. Indeed, a large portion of the increased manganese appears in the elevated levels of the mangani superoxide dismutase found in D. dendroides maintained in lowcopper media (37). Under these conditions the synthesis of the Cu/Zn-containing superoxide dismutase is reduced while total cellular superoxide dismutase activity is maintained at a constant level by the increased synthesis of the mangani enzyme. Hyperbaric oxygen and thus increased levels of the superoxide radical have been shown to induce this enzyme in several species (11, 12, 15). Copper catalysis of many nonenzymatic oxidation reactions (6, 9) can also be a source of superoxide radical. This might lead one to expect cells grown at 0.1 and 1 mM copper to have increased levels of total superoxide dismutase activity. The fact that this is not seen may indicate that this organism is not sensitive to superoxide levels or that copper does not in fact produce increased levels of superoxide. Very likely, however, this level of activity is constitutive and independent of copper or superoxide concentrations. The subcellular distribution of the Cu/Zn and mangani enzyme shows extensive variability with respect to the cytosol, but none at all in the mitochondria. Until recently, it was believed that the Cu/Zn enzyme was the only superoxide dismutase present in the cytosol of eucaryotic cells, while the manganese-containing enzyme appeared solely in the mitochondria (27, 34, 39). However, McCord showed that in chicken liver the mangani superoxide dismutase is three times as prevalent in the cytosol as in mitochondria, whereas in baboon liver the mitochondria-tocytosol ratio of this enzyme is 1:6 (19). In contrast, all of the mangani enzyme present in rat liver is localized in the mitochondria. Furthermore, liver homogenates from chickens, baboons, and humans contain 50 to 75% of their superoxide dismutase in the mangani form, as compared to 7 to 8% in rats. Perhaps the availability of copper and manganese in the liver in some way regulates superoxide dismutase synresult of
out rather than
thesis in these organisms as it does in D. dendroides. Whereas the mangani form, and never the Cu/Zn, of superoxide dismutase is found in mitochondria, the cytosol shows no such rigid requirements. The cytosol contains a constant 80% of the total cellular dismutase activity, but shows little specificity with respect to which form(s) of the enzyme is present. The constant amount of activity in the cytosol may indicate a constitutive level of synthesis or a response to an unchanging level of superoxide radical. In any event, it seems that the predominant cytosolic form of superoxide dismutase is determined by the availability of copper and manganese. When intracellular copper is plentiful, 100% of the cytosolic activity is contributed by the Cu/Zn enzyme, whereas only a 15% contribution is shown for that enzyme form in cells grown at 10 nM copper. A further illustration of the dependence of synthesis on metal availability is the change in synthesis which takes place when copper is added to copper-deficient cultures during growth. As soon as copper is added, the synthesis of Cu/Zn superoxide dismutase is greatly increased while mangani enzyme synthesis is slowed. The mangani enzyme that is now made is probably destined for mitochondria alone. The mechanism(s) by which the synthesis and localization of the two superoxide dismutase forms are controlled is not known. Whatever regulating mechanism is involved must account for the constant level of total cellular superoxide dismutase activity as well as the variations in Cu/Zn and mangani superoxide dismutase synthesis in relation to changing intracellular manganese and copper concentrations. The metals may bind to some site in the fungal genome and act as inducers or repressors in a lac operontype model (16) or could compete for some factor necessary for translation. It has been shown that metals can regulate protein synthesis at the transcriptional level, as with ceruloplasmin (7), and at the translational level, as reported for ferritin (40). It is also possible that there is a constitutive level of total superoxide dismutase synthesis in these cells, such that when synthesis of one enzyme form is decreased, the synthesis of the other form is proportionally increased. This can be compared to the linkage of a a- and ,8-globin chain synthesis in rabbit reticulocytes (10). In these cells, decreased trnslation of a-chain mRNA is coupled to an increase in the translation of ,8-chain mRNA. Consequently, a constant level of total globin chain synthesis is observed. The inflexibility of the mitochondrion with respect to the amount and type of superoxide
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dismutase it contains, as well as the appearance of varying amounts of mangani superoxide dismutase in the cytosol, must also be accounted for by any mechanism proposed for the regulation of the biosynthesis of these two proteins. "Secretory proteins" localized in the plasma membrane, and perhaps also those in subcellular organelles, are probably synthesized on ribosomes that are in contact with the plasma membrane, golgi, or organelle (4, 17). This hypothesis can be used to explain the appearance of the excess mangani superoxide dismutase in the cytosol by postulating that the increased synthesis of this enzyme is due to increased production of message, which saturates these bound ribosomes. As a result of the saturation, the excess message is translated on free ribosomes, which release the protein into the soluble fraction. However, the increase in the mangani enzyme in the non-mitochondrial fraction could also be due to an increased rate of translation of preexisting message. In this case, the rate of protein synthesis exceeds the rate of incorporation into the mitochondria, causing a release of the enzyme into the soluble fraction. Also possible is that message is normally translated in the mitochondria, but the entry of the message into the mitochondria is the rate-limiting step. Excess message would be translated in the soluble fraction, giving rise to cytosolic mangam superoxide dismutase. All of these mechanisms support the findings that organelle localization occurs during synthesis so that once mangani superoxide dismutase appears outside the mitochondria it must remain there. This explains why the "extra" mangani enzyme remains in the cytosolic fraction even after copper supplementation. The mechanism that regulates superoxide dismutase synthesis must also account for the result that, after copper is added to low-copper, high-manganese cells, all new non-mitochondrial superoxide dismutase synthesis is of the Cu/Zn form. This is shown by the similar Cu/Zn-to-mangani enzyme ratios observed in cells grown at 10 uM copper for from 50 to 100% of total culture growth. The fact that Cu/Zn superoxide dismutase synthesis is greatly favored after the addition of copper, even in the presence of elevated manganese, may mean that copper, and not manganese, controls the rate of superoxide dismutase synthesis. If metal availability actually determines which metalloprotein will be synthesized, the lack of copper should decrease synthesis of the Cu/Zn protein rather than result in the synthesis of an apoprotein. This was clearly shown by the Ouchterlony, immunoelectrophoresis, and im-
munoprecipitation experiments. These tests showed that no stable apo-Cu/Zn superoxide dismutase is synthesized, although they do not exclude the possibility that the apoenzyme is turned over very rapidly. The finding that lowcopper cells have about one sixth as much Cu/Zn superoxide dismutase protein as do highcopper cells is in excellent agreement with the difference in the cyanide-sensitive enzyme activity levels. The failure of the horse Cu/Zn superoxide dismutase to cross-react with the rabbit antifungal Cu/Zn superoxide dismutase antiserum indicates that the two proteins have diverged significantly through evolution. This was not surprising, since antibodies to the fungal protein were raised rapidly, whereas great difficulty has been encountered in developing antisera to the Cu/Zn enzyme from higher species such as humans. ACKNOWLEDGMENTS This work is supported by Public Health Service grant AM-19708 from the National Institute of Arthritis, Metabolism and Digestive Diseases. The very capable assistance of Anne Fagundus in obtaining rabbit anti-fungal superoxide dismutase antiserum is greatly appreciated.
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