Copper accumulation and phosphatase activities of Aspergillus and Rhizopus

Copper accumulation and phosphatase activities of three Aspergillus species resistant to copper were compared to three copper-sensitive Rhizopus species. High level of acid phosphatases and decreased Cu2+-uptake were found with resistant in contrast to sensitive strains. The presence of copper(II) ions in the medium increased the production of acid phosphatases in the resistant A. niger and decreased their activity in the sensitive R. delemar. Copper ions inhibited the activity of A. niger cellular acid phosphatase with a Ki of 8.9x10(-4) M and slightly activated the R. delemar enzyme.     

Cultivation of Methylosinus trichosporium OB3b: III. production of particulate methane monooxygenase in continuous culture

Continous culture experiments with the obligatory methanotroph, Methylosinus trichosporium OB3b, were conducted to study the whole-cell methane monooxygenase (MMO) and nitrogenase activities in a nitrate minimal salts medium under oxygen-limited conditions with methane as the carbone source. The important variables investigated were the feed medium concentrations of copper and nitrate, CO(2) addition, the agitation speed, and the dilution rate. M. trichosporium OB3b required quantitative amounts of copper (2.6 x 10(-4) g Cu/g dry cell Wt) for the exclusive production of particulate MMo during continous culture growth. When the feed medium nitrate concentration was varied in the range of 5-50 mM, the whole-cell specific pMMO activity exhibited a maximum at 40 mM. The elimination of external CO(2) gassing decreased pMMO activity by more than 30%. The steady-state cell density increased continuously over a 300-700 rpm range of agitation speed, whereas, the pMMO activity became maximal at 400 rpm. Also, the pMMO activity increased with the dilution rate up to 0.06 h(-1) and remained constant thereafter. Maximal continuous pMMO productivity was, thus, achieved in Higgin's medium containing 10 muM Cu, 80 muM Fe, and 40 mM nitrate with an agitation speed of 500 rpm and a dilution rate of 0.06 h(-1). Nitrogenase activity, on the other hand, increased over a feed medium copper concentration of 2-15 muM, falling sharply at 20 muM, and it exhibited a minimum at 20 mM when the feed medium nitrate concentration was varied. (c) 1992 John Wiley & Sons, Inc.     

Batch cultivation of Methylosinus trichosporium OB3b: II. Production of particulate methane monooxygenase

The obligatory methanotroph, Methylosinus trichosporium OB3b, was studied to optimize the batch culture conditions for the formation of particulate methane monooxygenase (pMMO) in a nitrate minimal salts medium. The important medium components investigated were copper, carbon dioxide, and nitrate. The whole-cell specific pMMO activity decreased sharply with increasing copper concentrations in the range of 10-40 muM and remained constant upon further increases of the copper concentration to 120 muM. The cell growth rate (mu), on the other hand, decreased over the entire range (10-120 muM) of copper concentrations tested. When pMMO was produced in a bioreactor with an optimal initial copper concentration of 10 muM, M. trichosporium OB3b exhibited a much faster overall growth rate and a higher whole-cell propene epoxidation activity compared to our earlier study, in which soluble methane monooxygenase (sMMO) was produced with copper-deficient medium. The addition of external carbon dioxide to the bioreactor culture eliminated an initial lag period in the cell growth. When the standard culture medium nitrate concentration (10 mM) was depleted, the pMMO activity, but not the growth rate, decreased rapidly. The whole-cell specific pMMO activity could be maintained by subsequent supplementation of nitrate. A 4-fold higher initial culture medium nitrate concentration of 40 mM, however, resulted in slower cell growth and lower pMMO activity. These observations demonstrate that, in addition to affecting the exclusive production of pMMO, copper also has an important previously unrecognized role in enhancing the growth rate of M. trichosporium OB3b. They also indicate that for the optimal batch production of pMMO with the minimal medium under study, nitrate should be supplied intermittently during the course of cultivation until other culture medium components become growth-limiting.     

Biosynthesis and Cellular Distribution of the Two Superoxide Dismutases of Dactylium dendroides

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 μM) 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.     

Differential effects of urea on yeast and bovine copper, zinc superoxide dismutases, in relation to the extent of analogy of primary structure.

Incubation in 8M urea (pH 7.4) inactivated yeast Cu, Zn superoxide dismutase with biphasic first order kinetics (k for the decrease from 100% to 16% activity = 6.5 × 10^?3 min^?1; k for the decrease from 16% to 0.1% activity = 2.5 × 10^?3 min^?1). The inactivation was fully reversible on dilution with or dialysis against urea-free buffer. No inactivation was shown to occur in similar experiments with the bovine Cu, Zn enzyme. EPR spectra recorded immediately after addition of 8M urea showed a more axial line shape and a higher A_″ of the copper signal typical of the native enzyme. In the case of the yeast enzyme, this change was more pronounced and further incubation led to a new type of copper signal, typical of the inactivated enzyme. All EPR changes were reversible. Comparative analysis of the amino acid sequence of the two enzymes showed substantial identity of the protein regions contributing the ligands to the metals and the disulfide bridge. Differential destabilization of active sites by urea should be due to replacements in other protein segments, such as the three C-terminal and some N-terminal residues.     

Response of shape in vitro cultures of shape Nicotiana tabacum L. to copper stress and selection of plants from Cu-tolerant callus

In Nicotiana tabacum L. var. BEL W3 copper (Cu) at concentrations higher than 50 μM significantly inhibited callus growth and shoot regeneration. After 5–6 months of culture only a few morphogenic callus lines survived in the presence of 100 μM Cu. These calluses showed the capacity to grow and regenerate shoots through successive subcultures on medium containing 100 μM Cu. The 100 μM Cu-tolerant shoots, in comparison to regenerated control shoots, formed roots only when cultured in the presence of 100 μM Cu. From five independent Cu-tolerant callus lines in a culture period of 4–5 months more than 50 plants (defined ‘tolerant’) able to grow in presence of 100 μM Cu were obtained. These plants showed normal xylem tissue formation while control regenerated plants growing in normal Cu MS content (0.1 μM) had few xylem elements in the central cylinder. No difference as far chlorophyll content and chloroplast structure was found among Cu-tolerant and control plants. In Cu-tolerant plants, dry matter production was higher than in controls, particularly in roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Purification and some properties of rat liver cysteine oxidase (cysteine dioxygenase).

Cysteine oxidase (cysteine dioxygenase, EC 1.13.11.20) was purified approximately 1000-fold from rat liver. The purified enzyme (protein-B) was obtained as an inactive form, which was activated by anaerobic preincubation with L-cysteine. The active form of protein-B was inactivated during aerobic incubation to produce cysteine sulfinate. This inactivation of protein-B was protected by a distinct protein in rat liver cytoplasm, namely stabilizing protein (protein-A). The Ka and Km values for L-cysteine were 0.8-10(-3) M and 1.3-10(-3) M respectively. The enzyme was strongly inhibited by Cu+ and/or Fe2+ chelating agents but not by Cu2+ chelating agent. The optimum pH of enzyme reaction was 8.5-9.5 while that of enzyme activation was 6.8-9.5, with a broad peak.     

Leukotriene B4 synthesis and metabolism by neutrophils and granule-free cytoplasts.

Copper-deprived sycamore (Acer pseudoplatanus) cells do not excrete molecules of active laccase in their culture medium. In the range of 2-100 micrograms of copper initially present per litre of nutrient solution, the total laccase activity measured in the cell suspensions at the end of the exponential phase of growth was closely proportional to the amount of added copper. However, copper-deprived cells excreted the laccase apoprotein (laccase without copper) at the same rate as copper-supplied cells excreted the active, copper-containing, laccase. When the culture medium was initially supplied with limiting amounts of copper, the active laccase was excreted until all copper molecules were metabolized. Thereafter, the laccase apoprotein was excreted. Consequently, at the end of the exponential phase of growth, the cell supernatants contained a mixture of apoprotein and copper-containing laccase. After purification and concentration, this mixture of copper-containing laccase (blue) and laccase apoprotein (slightly yellow) showed a yellow-green colour. Under copper-limiting culture conditions an equivalent decrease of Type 1, Type 2 and Type 3 Cu2+ was observed. Addition of copper to copper-deficient enzyme solutions does not result in a recovery of the enzyme activity. However, when added to copper-deficient sycamore-cell suspensions, copper induced a recovery of the excretion of active enzyme, at a normal rate, within about 10 h. The first molecules of active laccase were excreted after 3-4 h.     

Acclimation of Daphnia magna to environmentally realistic copper concentrations

It may be hypothesised that as the bioavailable background concentration of an essential metal increases (within natural limits), the natural tolerance (to the metal) of the acclimated/adapted organisms and communities will increase. In this study the influence of acclimation to different copper concentrations on the sensitivity of the freshwater cladoceran Daphnia magna Straus was investigated. D. magna was acclimated over three generations to environmentally relevant copper concentrations ranging from 0.5 to 100 microg Cu/l (copper activity: 7.18 x 10(-15) to 3700 x 10(-12) M Cu2+). A modified standard test medium was used as culture and test medium. Medium modifications were: reduced hardness (lowered to 180 mg CaCO3/l) and addition of Aldrich humic acid at a concentration of 5 mg DOC/l (instead of EDTA). The effects of acclimation on these organisms were monitored using acute mortality assays and long-term assays in which life table parameters, copper body concentrations and energy reserves were used as test endpoints. Our results showed a two-fold increase in acute copper tolerance with increasing acclimation concentration for second and third generation organisms. Copper acclimation concentrations up to 35 microg Cu/l (80 pM Cu2+) did not affect the net reproduction and the intrinsic growth rate. The energy reserves of the acclimated daphnids revealed an Optimal Concentration range (OCEE) and concentrations between 5 and 12 microg Cu/l (0.5-4.1 pM Cu2+) and 1 and 35 microg Cu/l (0.023-80 pM Cu2+) seemed to be optimal for first and third generation daphnids, respectively. Lower and higher copper concentrations resulted in deficiency and toxicity responses. It was also demonstrated that up to 35 microg Cu/l, third generation daphnids were able to regulate their total copper body concentration. These results clearly indicate that bioavailable background copper concentrations present in culture media have to be considered in the evaluation of toxicity test results, especially when the toxicity data are used for water quality guideline derivation and/or ecological risk assessment for metals.     

Copper modulation of macrophage cyclooxygenase metabolite synthesis

The effect of copper on the release of cyclooxygenase metabolites from starch elicited, rat, peritoneal macrophages was investigated. Copper sulphate, in the range 10(-6)-10(-5) M, inhibited the formation of prostaglandin (PG) E2 and thromboxane (Tx) B2, the stable metabolite of TxA2, in a dose dependent manner but had no effect on the production of 6-keto-PGF1 alpha, the stable product of prostacyclin. At higher concentrations (5 x 10(-5) and 10(-4) M) the synthesis of all three metabolites of arachidonic acid (AA) was stimulated as was the release of radioactivity from macrophages prelabelled with 14C AA. Copper had no effect on the metabolism of exogenous AA however. At 10(-4) M copper also stimulated secretion of the lysosomal enzyme, beta-glucuronidase (GUR). Copper nitrate (10(-4) M), but not zinc sulphate, also stimulated eicosanoid formation and lysosomal enzyme release. Our results are consistent with the idea that copper stimulates eicosanoid formation via an effect on PL activity.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactytium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, an extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (haloenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (< 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 μM, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 μM medium copper, holoenzyme secretion is maintained throughout cell growth.The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN^?-insensitive, manganese form of this enzyme. Cells grown at 10 μM copper shown 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

Glucocorticosteroid regulation of prostaglandin biosynthesis in human myometrial smooth muscle cells in monolayer culture

In the present investigation, we evaluated the production of prostaglandins by human myometrial smooth muscle cells maintained in monolayer culture in the absence or presence of glucocorticosteroids. In the presence of cortisol (10(-7) M) or dexamethasone (10(-8) M), the rate of production of prostacyclin (PGI2) by these cells was decreased significantly. The glucocorticosteroid-mediated inhibition of prostaglandin production was attenuated when cortisol-21-mesylate (10(-6) M), a glucocorticosteroid antagonist, was present in the culture medium. The rate of conversion of radiolabeled arachidonic acid to radiolabeled prostaglandins as determined by use of sonicates of myometrial cells and optimal assay conditions, however, was not affected significantly by treatment with cortisol or dexamethasone in concentrations sufficient to inhibit prostaglandin formation by more than 80%. These findings are suggestive that glucocorticosteroids act in human myometrial smooth muscle cells in culture to inhibit prostaglandin formation by way of a receptor-mediated process that does not involve inhibition of enzyme activities that are involved in the biosynthesis of prostaglandins, i.e. the conversion of arachidonic acid to prostaglandin.     

Stimulation of bovine endothelial cell angiotensin-I-converting enzyme activity by cyclic AMP-related agents

Bovine pulmonary artery endothelial cells in culture were used to assess the influence of cyclic nucleotides, isoproterenol (beta adrenergic agonist), and theophylline (phosphodiesterase inhibitor) on angiotensin-I-converting enzyme (ACE) activity of the cells and culture medium. Dibutyryl cAMP (10(-3) M) but not cAMP or dibutyryl cGMP stimulated angiotensin-I-converting enzyme (ACE) activity of cells in culture approximately 50-100% but had little influence on ACE activity of the medium. Theophylline at 10(-3) M concentration produced a three- to fourfold stimulation of both cellular and medium ACE activity. Isoproterenol by itself had no effect on cellular ACE activity but produced a stimulatory effect at 10(-7)-10(-5) M concentration after pretreatment of cells for 24 hr with 10(-4) M theophylline. The results support the concept that ACE activity of endothelial cells is influenced by the cyclic AMP system. ACE activity in cells and that released into medium may be under different regulatory controls.     

Dietary copper deficiency and endothelium-dependent relaxation of rat aorta.

Endothelium-dependent relaxation of aortas was studied in dietary copper (Cu) deficiency. Male, weanling Sprague-Dawley rats were fed diets deficient (CuD, less than 0.5 ppm) or adequate (CuA, 5.0-5.5 ppm) in Cu for 4 weeks. Aortic rings from paired Cu-deficient and Cu-adequate rats were isolated from the descending thoracic aorta, placed in tandem tissue baths, and attached to force transducers. Aortas were contracted with phenylephrine (3 x 10(-7) M) and the degree of force reduction was measured after successively increasing the dose of acetylcholine (10(-8)-10(-5) M), histamine 10(-6)-10(-3) M), or sodium nitroprusside (10(-9)-10(-6) M). Cu deficiency was found to significantly reduce the relaxation responses of each relaxing agent at the highest three of the four doses tested. The ability of Cu-adequate and Cu-deficient aortas to relax was not different, as indicated by their complete relaxation in response to 10(-4) or 10(-5) M papaverine. Because the relaxation responses to both acetylcholine and histamine in rat aorta are dependent on the presence of endothelium, the reduction of these responses suggests that endothelium, or its interaction with smooth muscle, was disrupted in dietary Cu deficiency. The reduction in response to sodium nitroprusside, an endothelium-independent analog of endothelium-derived relaxing factor, indicates that the interaction of endothelium-derived relaxing factor with smooth muscle was disrupted. These findings have implications regarding blood pressure regulation in Cu deficiency.     

Organ culture of suckling rat intestine: comparative study of various hormones on brush border enzymes

Jejunal mucosa of 6 d-old rats were cultured for 24 and 48 h in the presence of thyroxine, insulin, pentagastrin, glucagon, epidermal growth factor (EGF) or dibutyryl-A-3:5-MP cyclic with or without dexamethasone (DX). The enzymes were assayed on the purified brush borders. The various agents added alone to the basic culture medium had no effect with the exception of DX on the levels of enzyme activities. Dexamethasone alone induced sucrase, stimulated maltase, and protected other brush border enzyme activities (aminopeptidase, lactase, and alkaline phosphatase). When added to DX-supplemented medium, only the following factors modified the levels of enzymatic activities observed with DX alone. Insulin (10(-6) M) increased maltase, alkaline phosphatase, and lactase activity to a greater extent than DX at 24 h culture, the effect being maintained at 48 h on alkaline phosphatase only. At 48 h culture, both EGF (10(-8) M) and dbcAMP (10(-3) M) decreased DX-induced sucrase activity. The latter agent also depressed DX-stimulated aminopeptidase activity.     

Agaricus bisporus metapotyrosinase: preparation, characterization, and conversion to mixed-metal derivatives of the binuclear site

The alpha, beta, and gamma isozymes of Agaricus bisporus tyrosinase undergo inactivation in the presence of oxalate. The inactivation rate law is first order in enzyme and second order in oxalate. On a more rapid time scale than inactivation, oxalate acts as a competitive inhibitor of the catecholase reaction of tyrosinase. After removal of oxalate by dialysis, the inactivated enzyme is found to contain 50% of the original copper, all of which is present as paramagnetic, mononuclear copper sites. The ESR parameters of this copper indicate a tetragonal environment with nitrogen and oxygen ligands. The product of oxalate inactivation has lost one copper from each binuclear site and is thus a metapo derivative. Addition of Cu(II) to metapotyrosinase results in complete recovery of copper and catalytic activity. Prolonged storage of metapotyrosinase, in the absence of any additional Cu(II), results in copper migration, producing a 50% recovery of the original specific activity, expressed on a protein basis. Copper migration converts metapo sites into equal numbers of reconstituted, holo sites and fully apo sites. Both copper migration and copper reconstitution follow apparent first-order kinetics and are pH dependent. The involvement of two ionizable groups accounts for the observed pH dependence of each process. For copper migration pKa values of 6.0 and 8.8 were found, while for copper reconstitution the pKa values were 5.4 and 6.9. Addition of either Co(II) or Zn(II) to metapotyrosinase results in the formation of enzymatically inactive, mixed-metal derivatives of the binuclear copper site having one Cu(II) and one Co(II) or Zn(II) ion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional activity of rat testicular cells in culture

Testicular cells from adult hypophysectomized rats were cultured for 10 or 12 days, and the effect of treatment with hCG (10 ng/ml) on testosterone and progesterone production and the activity of the Leydig cell enzyme, 3 beta-hydroxysteroid dehydrogenase, were studied. Regardless of hormone treatment, on 4th day in culture a decline in the steroidogenic activity of cultured cells could be observed. Treatment with hCG resulted in stimulation of steroidogenesis on days 6 to 10 in culture, as measured by testosterone and progesterone production. Hormone treatment stimulated or inhibited the enzyme activity depending on the presence or absence in the culture medium of 10(-6) M spironolactone, an inhibitor of 17 alpha-hydroxylase, or an anti-androgen, cyproterone acetate.     

Cu(I) analysis of blue copper proteins.

A simple colorimetric test for the Cu(I) content in blue copper proteins is described. The procedure is based on the formation of a complex between Cu(I) and 2,2'-biquinoline in an acetic acid medium. Analyses of spinach plastocyanin, Pseudomonas aeruginosa azurin and Rhus vernicifera stellacyanin show that the cysteine residue in the type 1 site does not induce Cu(II) reduction under our conditions. There is evidence in laccase samples for the presence of an endogenous reductant that can reduce 0.14 +/- 0.04 mol of Cu(II)/mol of protein; however, the addition of EDTA eliminates the interference. The analysis shows that 25 +/- 2% of the type 3 copper ions are in the reduced form in the resting enzyme and that 80 +/- 15% of the type 3 copper ions are reduced in preparations of type-2-depleted laccase. There is growing interest in the development of chemically modified forms of laccase, and our method should be very useful for establishing the valence state of the metal centres in the various derivatives.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactylium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, and extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (holoenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (less than 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 micrometer, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 micrometer medium copper, holoenzyme secretion is maintained throughout cell growth. The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN(-)-insensitive, manganese form of this enzyme. Cells grown at 10 micrometer copper show 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.