Identification of an apo-superoxide dismutase (Cu,Zn) pool in human lymphoblasts.

Copper incorporation (64Cu(II)) into Cu,Zn-superoxide dismutase (SOD) was studied in human lymphoblasts. Rapid incorporation of copper with a proportionate increase in SOD activity was detected. No copper incorporation or SOD activation was detected when 64Cu(II) was added to cell cytosols rather than to intact cells. Thus, incorporation of 64Cu was not due to isotopic exchange. Cycloheximide had no significant effect on copper incorporation and activation of SOD when the data were corrected for total cell copper. Thus, the data were consistent with copper incorporation into a preexisting apoSOD pool rather than newly synthesized SOD, and no new SOD synthesis was detected over a 15-h incubation period. The size of the apoSOD pool was estimated to be ≈35% of the total SOD in lymphoblasts. When cells were preincubated for 15 h with excess copper (15 μM Cu(II)), the size of the apo pool markedly decreased but was not eliminated, suggesting that the apoSOD was not due to copper deficiency. These experiments also indicated that newly arrived copper was preferentially incorporated into the apoSOD pool, while the function(s) of an apoSOD pool remains unknown. Copper binding to apoSOD may provide a rapid protective response against copper toxicity.

Cu,Zn-superoxide dismutase (SOD) 1 is found in the cytosolic fraction of all cells (1,2) and has also been detected in nuclei and perioxisomes (2)(3)(4)(5). However, the cellular site(s) and stage of protein synthesis of copper incorporation into SOD remain unknown. Moreover, an apo form of SOD was detected in a variety of copper-deficient cells or in cells in which the amount of the apoSOD pool appeared to vary with the state of differentiation (6 -15). The total amount of SOD mRNA and protein may also vary with the state of differentiation of some cells types (16). The cellular location of the apo form of SOD is unknown. While some copper incorporation into apoSOD was reported when high (2 mM) concentrations of copper were added to whole cell homogenates (10), no copper incorporation has been reported for copper addition to the cytosolic fractions from cells containing an apoSOD pool. Since copper is incorporated into apoSOD in neutral buffer, this suggests that either there are factors in cell cytosols which inhibit copper incorporation or that the apoSOD pool is not cytosolic.
The time and copper concentration dependencies of copper binding to cytosolic copper binding proteins and copper incorporation into SOD were determined during incubations of lymphoblasts with 64 Cu(II). Since SOD copper does not readily exchange isotopic copper, 64 Cu incorporation exclusively into newly synthesized SOD was anticipated. However, little or no de novo synthesis of SOD protein was detected in lymphoblasts over the time course of these experiments. Instead, an apoSOD pool was detected in lymphoblasts, which were neither copperdeficient nor -differentiating. The results in this and a companion study on copper incorporation into SOD in Menkes (17) lymphoblasts (18) suggest a possible role of apoSOD pools in initial cellular defense mechanisms against copper toxicity.

EXPERIMENTAL PROCEDURES
Materials-The Superose-12 HR 10/30 column, HPLC pump (model 2150) HPLC controller, were from Pharmacia Biotech Inc. HEPES, bicinchoninic acid, cycloheximide, protein A suspension, and a mouse monoclonal antibody to human SOD were from Sigma. 64 Cu(NO 3 ) 2 was from the Buffalo Materials Research Center of the State University of New York at Buffalo. The specific activity of the isotope at the time of shipment was Ϸ14 mCi/mg of copper.
Cell Cultures-Human lymphoblasts developed by transformation of peripheral B lymphocytes with Epstein-Barr virus were obtained from NIGMS Human Genetic Cell Repository (Coriell Institute for Medical Research, Camden, NJ). The cells used (repository no. GM03798) were from a normal, 10-year-old, male Caucasian. Lymphoblasts were grown in 175-cm 2 plastic tissue culture bottles (Falcon) as suspension cultures in 100 ml of RPMI 1640 medium (Sigma) supplemented with 5% fetal calf serum and 5% newborn calf serum (Intergen, Purchase, NY). Cell cultures were maintained at a concentration of Ϸ0.7 ϫ 10 6 /ml in logarithmic growth phase by replacing one-third to one-half of cell suspensions with fresh medium every 2nd day. 64 Cu(NO 3 ) 2 was added to 100 ml of cell cultures at the indicated concentrations for the indicated incubation periods. At the end of the incubation with 64 Cu(II), the cells were washed three times with isotonic, phosphate-buffered saline at 4°C before homogenization.
Preparation of Cytosols-The cells from three 100-ml cultures were combined and homogenized with 150 strokes of a motor driven (1000 rpm) homogenizer (Thomas Teflon pestle) in 0.4 ml of isotonic HEPES sucrose buffer (0.25 M sucrose, 5 mM HEPES, pH 7.4) containing phenylmethylsulfonyl fluoride (40 g/ml) and leupeptin (0.5 g/ml) to inhibit proteolysis. Homogenates were centrifuged for 2 min at 1930 ϫ g. The supernatants were centrifuged for 60 min at 100,000 ϫ g and then filtered through 0.22-m Milex GV syringe filters (Millipore, Bedford, MA) before applying to the Superose column. Protein concentrations of cell and cytosol samples were determined by the bicinchoninic acid assay (19) using bovine serum albumin as a standard. Typical protein concentrations of lymphoblast cytosols were 10 -15 mg/ml. in each pooled 64 Cu-binding protein fraction was determined by adding the radioactivities of each of the tubes constituting that fraction. Data are expressed as nanograms of 64 Cu/mg of cytosolic protein.
SOD Activity-SOD activities were determined by a spectrophotometric method using xanthine oxidase (20). A unit of SOD activity represented the amount of cytosol required to decrease the absorbance of control samples (no cytosol) by 50%. SOD activities were corrected for possible mitochondrial Mn-SOD contamination by subtracting the activity remaining in 1 mM KCN. Quadruplicate assays were performed on each sample, and the data were analyzed for statistical significance by a two-tailed, Student's t test.
Immunoprecipitation of SOD-Protein A (0.5 ml) as an insoluble cell suspension (Staphylococcus aureus) was washed once with phosphatebuffered saline (PBS) and resuspended in 0.5 ml of PBS. 50 l of monoclonal mouse antibodies (IgG) to human recombinant Cu,Zn-SOD were added and incubated with occasional mixing for 1 h at a room temperature. Control protein A suspensions were not incubated with antibodies. The antibody-treated protein A suspensions were pelleted by 12,000 ϫ g centrifugation for 2 min and washed once with PBS. Lymphoblast cytosols were mixed with antibody-treated (or control) protein A suspensions. After 1 h of incubation at room temperature, the protein A suspensions were pelleted by 12,000 ϫ g centrifugation for 2 min, and the supernatants were filtered through 0.22-m Milex GV syringe filters (Millipore, Bedford, MA) before applying the samples to the Superose 12 HPLC column.

Distribution of Copper in Cytosols after Incubating Cytosols or Intact Lymphoblasts with 64 Cu(II)-Lymphoblasts
were incubated with 7 M 64 Cu(II) in culture medium supplemented with 10% fetal calf serum for 2 h, and the 64 Cu-labeled cytosols were fractionated on Superose columns. The amount of 64 Cu in the void volume was variable and most likely reflected variable particulate contamination of the cytosols. Three 64 Cu-labeled protein fractions eluted after the void volume. Fraction I (Fig.  1) contains S-adenosylhomocysteine hydrolase which was recently found to have a high affinity for copper (21,22). This was confirmed by Western blots (data not shown). Copper-binding fraction III contains metallothionein(s) (MT) as indicated by the apparent molecular weight, and the elution of MT in this fraction after transfection of lymphoblasts with a MT-expression vector (18). Since it was known from prior studies that Cu,Zn-SOD elutes near the elution volume where the 64 Cu-binding fraction II was detected, the fractions were assayed for SOD activity. Maximal SOD activity occurred at the peak of 64 Cu-incorporation; and the amount of SOD activity correlated well with the amount of 64 Cu detected in each tube comprising fraction II (Fig. 1). Interestingly, copper incorporation into this fraction required intact cells as no significant 64 Cu-binding in fraction II was detected when 64 Cu(II) was added directly to lymphoblast cytosols (data not shown). 64 Cu incorporation into fraction II with intact cells was unlikely due to isotopic exchange with copper in holo-SOD as none occurred when 64 Cu was added directly to cell cytosols that contain active SOD.
The Effect of Immunoprecipitation of SOD on the Cytosolic Distribution of 64 Cu-SOD was immunoprecipitated from lymphoblast cytosols to determine if 64 Cu incorporation into SOD completely accounted for Superose 64 Cu-binding fraction II. Lymphoblasts were incubated with 64 Cu(II), the cytosols were isolated, and SOD was immunoprecipitated with a monoclonal antibody to human SOD bound to protein A. Control cytosols were treated with protein A without antibody. The effects of immunoprecipitation on the distribution of 64 Cu and SOD ac- Insoluble protein A particles were incubated with monoclonal antibodies to human SOD or buffer. After washing with the PBS buffer, protein A particles were then incubated with cytosols from lymphoblasts which had been incubated with 7 M 64 Cu(II) for 2 h at 37°C. After centrifugation, the supernatants were subjected to Superose 12 HPLC. A, 64 Cu (E) and SOD activity (q) in control cytosols which were incubated with protein A particles that were not preincubated with monoclonal antibodies to human Cu,Zn-SOD. B, 64 Cu (E) and SOD activity (q) in cytosols which were incubated with protein A particles that had been preincubated with monoclonal antibodies to human Cu,Zn-SOD. tivity are shown in Fig. 2, A and B. Both SOD activity and 64 Cu incorporation into copper-binding fraction II decreased proportionally, while the amount of 64 Cu in copper-binding fractions I and MT were not significantly affected. Thus, copper incorporation into fraction II reflects 64 Cu incorporation in SOD.
The Concentration Dependence of 64 Cu Incorporation into SOD in Intact Lymphoblasts-Lymphoblasts were incubated at varying times (2-15 h) and 64 Cu concentrations (2-15 M) to obtain varying amounts of cytosolic 64 Cu. The isolated cytosols were chromatographed on Superose. The amounts of 64 Cu in SOD were determined by calculating the areas under the SOD fraction. Since the plot of 64 Cu incorporated into SOD versus total cytosolic 64 Cu followed a simple saturation curve (data not shown), the maximum amount of 64 Cu that was incorporated into SOD, and the cytosolic 64 Cu concentration needed to reach 50% of maximal incorporation of 64 Cu into SOD (K 0.5 ) could be estimated. Maximal incorporation was Ϸ4.6 ng of 64 Cu/mg of cytosolic protein, and K 0.5 was Ϸ6.3 ng of cytosolic 64 Cu/mg of cytosolic protein.
The Effects of Cycloheximide on the Distribution of 64 Cu in Lymphoblast Cytosols and 64 Cu Incorporation into SOD-Lymphoblasts were preincubated for 3 h with 5 g/ml cycloheximide and then incubated for an additional 15 h with 7 M 64 Cu(II) and cycloheximide to determine whether protein synthesis was required for copper incorporation into SOD. Control cells were incubated without cycloheximide. Cytosols were isolated and fractionated on Superose columns. Increasing the incubation time from 2 h (Fig. 1) to 15 h (Fig. 3) increased the total 64 Cu incorporated in all three copper-binding protein fractions in the control cytosols (Fig. 3). Cycloheximide caused a large decrease in the amount of 64 Cu in the MT fraction and a small increase in 64 Cu in fraction I (Fig. 3). Copper incorporation into SOD decreased significantly. However, cycloheximide also led to decreases in total cytosolic 64 Cu levels. The amounts of copper incorporated into SOD with and without cycloheximide were not significantly different when corrected for the total amount of cytosolic copper under these conditions (Table  I). Moreover, the amount of 64 Cu that was incorporated into SOD at the total cytosolic 64 Cu-level when cycloheximide was present (2.03 ng of 64 Cu/mg of cytosolic protein) was in excellent agreement with the amount predicted from the cytosolic copper concentration dependence (2.10 ng of 64 Cu/mg of cytosolic protein).
Copper incorporation into SOD was associated with proportionate increases in SOD activity with or without cycloheximide (Table I). As expected from the copper incorporation data, the increase in SOD activity with cycloheximide-treated lymphoblasts was significantly less than with the controls (Table  I). However, the observed effect of cycloheximide on SOD activity was accounted for by the decrease in cytosolic 64 Cu with cycloheximide, and the increases in SOD activity per amount of copper incorporated into SOD were equal within experimental error (Table I). The strong correlation between the amount of copper incorporated and the increase in SOD activity with or without cycloheximide further argue against 64 Cu exchange with copper in preexisting holo-SOD. Overall, the copper incorporation and increment in SOD activity data with and without cycloheximide indicate that copper was incorporated exclusively into a presynthesized, apoSOD pool under the conditions of these experiments, and no significant 64 Cu-incorporation into newly synthesized SOD was detected over the time course of these experiments. The size of the apoSOD pool in lymphoblasts was estimated from the maximal copper incorporation into this pool as Ϸ35% of the total SOD in lymphoblasts.
The Effect of Preincubation with Nonradioactive Copper on the Distribution of 64 Cu in the Cytosol and 64 Cu Incorporation into SOD-Lymphoblasts were preincubated with 15 M CuSO 4 for 15 h and then incubated with cycloheximide (5 g/ml) for 3 h followed by 7 M 64 Cu(II) for 15 h in the same medium. Control cells were not preincubated with copper. Although the amount 64 Cu incorporated into the apoSOD pool decreased, preincubation with copper did not eliminate the apoSOD pool (Fig. 4). The amount of 64 Cu in fraction I increased in cells preincubated with copper by about the same amount as the decrease in 64 Cu incorporation into SOD with or without cycloheximide present. This is the expected result if

TABLE I The effect of cycloheximide on copper incorporation into SOD and SOD activity
Human lymphoblasts were incubated with 7 M 64 Cu(II) for 15 h with or without cycloheximide (5 g/ml), and the radiolabeled cytosols were fractionated on Superose. Copper incorporation into SOD was determined by determining the total 64 Cu in the SOD fraction. Total cytosolic copper represents the total 64 Cu in the three major 64 Cu-binding fractions, i.e. fraction I, SOD (fraction II), and MT (Fig. 1B) fraction I contains a copper-binding protein(s) that either supplies copper to apoSOD or equilibrates with a copper pool that is a major source of apoSOD copper. The amount of 64 Cu bound in the MT fraction increased in these experiments (Fig. 4), most likely due to isotopic exchange with copper after induction of MT by copper during the preincubation period.
The results of the preincubation with copper experiments suggested that newly arrived copper is preferentially incorporated into apoSOD without significant mixing with preexisting copper pools. When lymphoblasts were incubated with 15 M 64 Cu(II) for 15 h, 3.3 ng of 64 Cu/mg of protein were incorporated into apoSOD, leaving Ϸ1.3 ng/mg of protein to saturate the apoSOD pool. When cells were preincubated with 15 M stable Cu(II) for 15 h followed by 7 M 64 Cu(II) for 15 h, the amount of 64 Cu incorporated was 1.4 ng. Analogous results were obtained with cycloheximide present. These results indicate that the copper pools developed during the preincubation period, including copper that bound to MT, had no significant effect on subsequent radiolabeled copper incorporation. DISCUSSION The results reported here clearly indicate that lymphoblasts grown under normal culture conditions contain a significant pool of apoSOD which is activated when copper is added to the cells. Copper incorporation into SOD was indicated by the clear correlation between copper incorporation and increase in SOD activity and the effects of immunoprecipitation of SOD on copper incorporation and activity. That copper incorporation was exclusively into a preexisting apo pool was indicated by the minimal effects of cycloheximide on copper incorporation and SOD activity when corrected for the effect of cycloheximide on cytosolic copper levels. No new synthesis of SOD was detected over the time course of these experiments. The effects of preincubation with stable Cu(II) on subsequent, radiolabeled copper incorporation into SOD and SOD activity were also consistent with the presence of an apoSOD pool in lymphoblasts which could be nearly saturated by preincubating with copper. Although copper was not added to the standard incubation medium, the apoSOD pool was probably not due to copper defi-ciency because the apoSOD pool was not eliminated when cells were preincubated for 15 h with 15 M stable-copper, which is about 10-fold higher than the normal level of non-ceruloplasmin copper in serum.
Copper incorporation into apoSOD required intact cells as no copper incorporation was detected when copper was added directly to cytosols isolated from cells. The requirement for intact cells may reflect the cellular location of the apoSOD pool in intact lymphoblasts or a condition or factor that is lost during isolation of the cytosols. The exact function of an apoSOD pool remains unknown. Given the apparent slow rate of new synthesis and turnover of SOD in lymphoblasts as indicated by the lack of a significant effect of cycloheximide during 15 h incubations, a preexisting apo pool may provide a mechanism for providing increased SOD activity when required. However, it is unclear whether a maximal increase in SOD activity of Ϸ35% would be significant unless the increase were localized intracellularly. Alternatively, an apoSOD pool may help protect cells against copper toxicity (18) by providing a high affinity, copper-binding pool which can rapidly sequester copper as it enters cells. Interestingly, lymphoblasts incubated with 1 M 64 Cu(II) for 20 h incorporated 64 Cu(II) exclusively into apoSOD without inducing MT synthesis (data not shown).
The results obtained with human lymphoblasts are consistent with detection of an apoSOD pool in a variety of copperdeficient systems (6 -9, 13, 14). ApoSOD pools were also inferred for differentiating K562 cells (10,11,15) and differentiating HL-60 cells (12). However, the apoSOD pool in lymphoblasts that was detected here is unlikely to be due to copper deficiency, and no cell-differentiating factors were used in the lymphoblast studies. Thus, the results with lymphoblasts represent the first example of a copper-replete, nondifferentiating cell having an apoSOD pool. The possibility of normocupric, nondifferentiating cells having an apoSOD pool had been suggested by Harris (13). Cu incorporation into apoSOD. Lymphoblasts were preincubated with 15 M CuSO 4 for 15 h at 37°C in standard growth medium. The cells were washed with medium, and cycloheximide (5 g/ml) was added. After pretreatment with cycloheximide for 3 h, incubation was continued in the same medium plus 7 M 64 Cu(II) for 15 h. Cytosols were fractionated on Superose collecting 0.22-ml fractions. Data are shown for control cells (E) that were not preincubated with stable Cu(II) and the cells that were preincubated (q) with Cu(II).