Increase of Flux Control of Cytochrome c Oxidase in Copper-deficient ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 271, No. 1, Issue of January 5, pp. 283–288, 1996 Printed in U.S.A.

Increase of Flux Control of Cytochrome c Oxidase in Copper-deficient Mottled Brindled Mice* (Received for publication, April 18, 1995, and in revised form, October 6, 1995)

Andrey V. Kuznetsov, Joseph F. Clark‡§, Kirstin Winkler, and Wolfram S. Kunz¶ From the Laboratory of Neurobiochemistry, Klinik fu¨r Neurologie, Universita¨tsklinikum, Leipziger Str. 44, D-39120 Magdeburg, Germany and the ‡Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom

The brindled mottled mouse (Mobr), an animal model of the Menkes’ copper deficiency syndrome, was used for the investigation of changes in respiratory flux control associated with cytochrome c oxidase deficiency in muscle. Enzymatic analysis of cardiac and skeletal muscles showed an approximately 2-fold decrease in cytochrome c oxidase activity of brindled mutants in both types of muscles as compared with controls. The activities of NADH-cytochrome c oxidoreductase (respiratory chain segment I–III) and succinate-cytochrome c oxidoreductase (segment II–III) were normal. Assessment of mitochondrial respiratory function was performed using chemically skinned musculus quadriceps or heart muscle fibers isolated from control and brindled mottled mice. In skeletal muscle, there was no difference found in maximal rates of respiration. In the Mobr hearts, this parameter was slightly lower than control. Alternately, the determination of flux control coefficients of cytochrome c oxidase performed by a step by step inhibition of respiration with increasing concentrations of azide or cyanide revealed significantly sharper inhibition curves for brindled mice than for control, indicating more than 2-fold elevated flux control coefficients of cytochrome c oxidase. This investigation proved essential in characterizing the metabolic effect of a cytochrome c oxidase deficiency. We conclude, therefore, that application of metabolic control analysis can be a valuable approach to study defects of mitochondrial oxidative phosphorylation.

The brindled mouse is a variant of the X-linked mottled mutants (Mobr) with severe copper deficiency and is considered to be an animal model of Menkes’ syndrome (Menkes’ kinky hair disease) (1–5). The copper homeostasis disorders in humans, in mottled mice, or in copper-deficient rats are all associated with distinct mitochondrial alteration in various tissues. The brain and other regions of the central nervous system are particularly affected (6 –7). Among mitochondrial abnormalities are various ultrastructural and biochemical changes, such as the well documented depressed activity of cytochrome c * This work was supported in part by grants from Deutsche Forschungsgemeinschaft, the Land Sachsen-Anhalt (1795A/0084), and the Sandoz Foundation for Therapeutic Research (Germany) (to W. S. K.). Travel grants were supplied by Hansatech (UK) and Zenaca Pharmaceuticals (UK) (to J. F. C. and A. V. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Supported by the Medical Research Council (UK). ¶ To whom correspondence should be addressed: Neurobiochemisches Labor der Klinik fu¨r Neurologie, Universita¨tsklinikum der Otto-vonGuericke Universita¨t Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany. Tel.: 49391-6715219; Fax: 49391-6715228.

oxidase (a mitochondrial cuproenzyme) (7–10). Thus, it was suggested that the lower level of energy metabolism caused by the decrease in both copper concentration and cytochrome c oxidase activity may be responsible for brain degeneration associated with the Menkes’ disease (6, 11). However, there is little information available concerning mitochondrial function or cytochrome c oxidase activity in cardiac and skeletal muscles of animals with copper deficiency. Measurements of maximal rates of mitochondrial respiration are often used for the functional determination of the different mitochondrial defects (12–14). It has been shown that assessment of respiratory activities of saponin-skinned muscle fibers can be especially applicable for the study of small human biopsy specimens (15–17). However, in cases where the defect is located at a non-rate-limiting step of oxidative phosphorylation, simply determining the maximal rate of respiration will not reveal the mitochondrial defect. Alterations in individual respiratory complexes, for example in cytochrome c oxidase activity, can therefore be hard to find or even missed. Recently it was suggested that metabolic control analysis of oxidative phosphorylation can be a successful approach for quantifying the enzymatic defect in certain mitochondrial diseases (18 –20). However, direct evidence is still absent, and acceptable pathological models were not used. In this work we applied metabolic control analysis (21, 22) to study the consequences of cytochrome c oxidase deficiency in Mobr mice. For this we used incremental inhibition of the mitochondrial respiration with increasing concentration of azide and cyanide. Due to the limited amount of tissue, and in order to avoid possible artifacts of the preparation, we used chemically skinned muscle fibers, which allowed us to investigate mitochondrial functions in situ, without isolation of mitochondria (15, 17, 23). EXPERIMENTAL PROCEDURES

Animals—Five mottled brindled mutant male mice (Mobr) obtained from Genetic Division MRC Radiobiology Unit (Chilton, DIDCOT Oxon OX11 ORD, United Kingdom) and five normal controls were used to study mitochondrial and enzymatic properties of cardiac and skeletal (quadriceps) muscles. The mice were not exercised. Animals were euthanized by cervical dislocation, hearts and m. quadriceps1 were rapidly removed, washed, and placed in ice-cold Krebs solution. 50 –100 mg of each tissue were frozen in liquid nitrogen for enzyme assays, and the remaining tissue was used for respiration experiments. The histochemical analysis of the used m. quadriceps portions revealed 40 6 3% slow oxidative fibers (succinate dehydrogenase staining). No difference between Mobr quadriceps and control quadriceps muscles using myosin ATPase and succinate dehydrogenase staining was detected. Isolation of Skinned Fibers—The bundles of muscle fibers were isolated from heart (left ventricle) or skeletal muscle (m. quadriceps) of control and Mobr mice. Saponin-skinned fibers were prepared by incu-

1 The abbreviations used are: m. quadriceps, musculus quadriceps; MES, 4-morpholinopropanesulfonic acid.

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TABLE I Enzyme activities of cardiac homogenates from control and brindled mottled mice Enzyme activities (in units/g, wet weight) and the cytochrome aa3 content (in nmol/g, wet weight) were measured as described under “Experimental Procedures.” LDH, lactate dehydrogenase; AK, adenylate kinase; CPK, creatine kinase; ASAT, aspartate aminotransferase; CS, citrate synthase; COX, cytochrome c oxidase; NADH-cyt c, NADHcytochrome c oxidoreductase, Succ-cyt c, succinate-cytochrome c oxidoreductase; aa3, cytochrome aa3; NS, not significant.

TABLE II Enzyme activities of quadriceps homogenates from control and brindled mottled mice Enzyme activities (in units/g, wet weight) and the cytochrome aa3 content (in nmol/g, wet weight) were measured as described under “Experimental Procedures.” LDH, lactate dehydrogenase; AK, adenylate kinase; CPK, creatine kinase; ASAT, aspartate aminotransferase; CS, citrate synthase; COX, cytochrome c oxidase; NADH-cyt c, NADHcytochrome c oxidoreductase; Succ-cyt c, succinate-cytochrome c oxidoreductase; aa3, cytochrome aa3.

Enzyme

Control mice

Brindled mice

Significance

Enzyme

Control mice

Brindled mice

Significance

LDH AK CPK ASAT CS COX NADH-cyt c Succ-cyt c aa3

106 6 6 318 6 9 802 6 30 302 6 6 216 6 6 38 6 2 17 6 1 10.5 6 1.4 44 6 6

110 6 6 273 6 10 732 6 32 317 6 11 237 6 6 20 6 2 20 6 2 12.2 6 0.6 31 6 1

NS (n 5 11) p , 0.01 (n 5 10) NS (n 5 10) NS (n 5 11) p , 0.05 (n 5 12) p , 0.001 (n 5 11) NS (n 5 10) NS (n 5 10) p , 0.001 (n 5 5)

LDH AK CPK ASAT CS COX NADH-cyt c Succ-cyt c aa3

360 6 11 718 6 22 3553 6 156 58 6 2 32 6 1 10.1 6 0.6 2.3 6 0.2 1.7 6 0.1 6.0 6 0.8

278 6 17 485 6 37 2580 6 200 59 6 5 37 6 3 5.5 6 0.5 2.5 6 0.3 1.6 6 0.2 6.2 6 0.7

p , 0.001 (n 5 12) p , 0.001 (n 5 11) p , 0.01 (n 5 11) NS (n 5 11) NS (n 5 12) p , 0.001 (n 5 12) NS (n 5 10) NS (n 5 10) NS (n 5 6)

bation of bundles of intact fibers in relaxing solution (composition see below) containing 50 mg/ml saponin as described in Ref. 17. Glycerolskinned fibers were prepared by placing the dissected fibers into the same relaxing solution containing 20% glycerol and 10 mg/ml fatty acid-free bovine serum albumin (without saponin). These fibers were then immediately frozen in liquid nitrogen and kept in liquid nitrogen until analysis. Solutions—The relaxing solution contained 10 mM Ca-EGTA buffer, 0.1 mM free concentration of calcium, 20 mM imidazole, 20 mM taurine, 100 mM K-MES, 0.5 mM dithiothreitol, 5 mM MgCl2, 5 mM ATP, 15 mM phosphocreatine, pH 7.2. Respiration—Respiration of skinned fibers was measured at 25 °C using an Oroboros oxygraph (Anton Paar, Graz) in a medium containing 5 mM MgCl2, 60 mM KCl, 110 mM mannitol, 10 mM KH2PO4, 0.5 mM Na2EDTA, 60 mM Tris-HCl (pH 7.4), and 10 mM glutamate 1 5 mM malate as mitochondrial substrates or additionally 10 mM succinate and 0.08 mM cytochrome c. Enzyme Activities—All enzyme activities were determined at 30 °C. Cytochrome c oxidase activity was measured spectrophotometrically in a medium containing 100 mM phosphate buffer, pH 7.4, and 80 mM ferrocytochrome c at 550 nm (24). Activities of NADH:cytochrome c and succinate:cytochrome c oxidoreductases were measured in 100 mM phosphate buffer (pH 7.4) containing 1 mM KCN, 10 mM NADH, or 20 mM succinate and 80 mM ferrocytochrome c at 550 nm. Lactate dehydrogenase, aspartate aminotransferase, citrate synthase activities were measured as described in Ref. 17. Creatine kinase and adenylate kinase activities were measured in a coupled enzyme assay in the respiration medium (see above) supplemented with 20 mM glucose, 0.6 mM NADP, 0.5 mM dithiothreitol, 1.0 mM ADP, 2 IU/ml hexokinase and glucose-6phosphate dehydrogenase. The adenylate kinase reaction was started by the addition of homogenate sample and the creatine kinase reaction by the addition of 10 mM phosphocreatine. Determination of Cytochrome aa3 Content—The content of cytochrome aa3 in muscle was measured spectrophotometrically at 605– 630 nm using e 5 12 mM21 cm21 (25). To completely remove hemoglobin, the cardiac and skeletal muscle homogenates were centrifuged 15 min at 15,000 3 g. The resultant pellet was resuspended in a medium containing 0.5% Triton X-100 and 100 mM Tris-HCl (pH 7.4) and analyzed spectrophotometrically. Materials—Saponin, MES, taurine, creatine phosphate, sodium azide, potassium cyanide, ADP, NADP, and albumin were purchased from Sigma. All enzymes were from Boehringer, and double-distilled glycerol was from Serva. Reduced cytochrome c was prepared from bovine heart cytochrome c (Sigma) by incubation with 20 mM ascorbate and separated from ascorbate using a PD-10 column (Pharmacia Biotech Inc.). Statistical Analysis—The results are presented as mean 6 S.E. Significant changes were assessed by Student’s t test. A value of p , 0.05 was accepted as the level of significance. RESULTS

Tables I and II summarize the data of enzymatic analysis of cardiac and skeletal muscles from control and Mobr mice. It can be seen that cytochrome c oxidase activity of copper-deficient animals was 50% of normal activity in both cardiac and skeletal muscles. On the other hand, the activities of other mito-

chondrial enzymes, NADH-cytochrome c oxidoreductase and succinate-cytochrome c oxidoreductase, were not significantly different from the control values. The activity of the mitochondrial marker enzyme, citrate synthase, was slightly higher in the muscles of brindled mice with significant differences in the heart. This may reflect a possible adaptational increase in mitochondrial content due to depressed oxidative metabolism, since no difference in fiber typing of m. quadriceps was observed. This skeletal muscle of brindled mutants also has decreased activities of lactate dehydrogenase, adenylate kinase, and creatine kinase, while in cardiac muscle only adenylate kinase activity was reduced. The activities of aspartate aminotransferase were normal in both heart and m. quadriceps (Table I). Next, we determined the maximal respiration activities of chemically skinned muscle fibers. Saponin is frequently used as a skinning agent to ensure mitochondrial intactness (15,17). In addition to this we adapted a procedure of plasma membrane permeabilization with glycerol as a chemical skinning agent previously used for mechanical experiments only (26). As shown in Table III and Fig. 1 the mitochondrial function in these fibers is similarly preserved as reported for saponinskinned fibers (15, 17). When comparing the maximal rates of mitochondrial respiration in skinned m. quadriceps fibers isolated from normal and brindled mice (Table III) almost no difference was observed. The same result was obtained using saponin-skinned fibers and glycerol-skinned fibers with two different substrate combinations: glutamate 1 malate, and glutamate 1 malate and succinate. In each case the maximal rate of respiration was achieved by the addition of 1 mM ADP. On the other hand, skinned fibers isolated from the cardiac muscle of copper-deficient animals demonstrated slightly lower respiratory parameters than control (Table III). To determine the flux control coefficients for cytochrome c oxidase we used titrations of the rate of respiration of skinned fibers with the specific inhibitors of this enzyme: sodium azide (28) and potassium cyanide (29). To do this, the maximal rate of respiration (in the presence of 1 mM ADP) was inhibited by incrementally increasing sodium azide (0 – 800 mM) or potassium cyanide (0 – 60 mM) concentrations. The inhibition of respiration of skinned cardiac fibers from control (open circles) and Mobr mice (filled circles) by sodium azide is shown in Fig. 2 (upper part). It can be seen that the shapes of the curves are different for control and brindled mice. The inhibition curve for brindled mutants is characterized by a sharper slope and, therefore, higher sensitivity to azide. A similar effect was found for skinned fibers isolated from m. quadriceps of control and brindled mice (Fig. 2, lower part; compare open and filled

Flux Control of Cytochrome c Oxidase

285

TABLE III Maximal rates of respiration of cardiac and quadriceps skinned fibers isolated from normal and brindled mottled mice The rates of respiration are expressed in ng atoms of oxygen/min/mg, dry weight, at 25 °C: NS, not significant. Tissue

Heart Control mice Brindled mice Significance Quadriceps Control mice Brindled mice Significance

Saponin-skinned fibers

Glycerol-skinned fibers

67.6 6 0.9 39.4 6 3.0 p , 0.05 (n 5 3)

50.5 6 6.6 43.2 6 0.9 NS (n 5 3)

16.8 6 0.6 17.7 6 0.6 NS (n 5 3)

12.1 6 1.0 12.3 6 1.1 NS (n 5 5)

FIG. 1. Representative traces of respiration from glycerolskinned fibers from control mouse heart. Skinned fibers (1.4 mg, dry weight) in the medium for oxygraphic measurement. Additions were as follows: 10 mM glutamate (GLU), 5 mM malate (MAL), 1 mM ADP, 10 mM carboxyatractyloside (CAT). The top line is the direct measure of oxygen concentration, and the bottom line is the traces differentiation.

circles). In terms of metabolic control analysis, this means that the flux control coefficient of cytochrome c oxidase is much higher in copper-deficient brindled mice than in control mice. To determine the flux control coefficient (Ci) using this noncompetitive inhibitor, the following equation can be used. Ci 5 2~dJ/J!/~dI/Kd!

(Eq. 1)

where J is the respiration flux, dJ is the decrement of respiration flux caused by the increment of inhibitor addition dI, and Kd is the dissociation constant for sodium azide. To exclude possible changes of the Kd value under the conditions of copper deficiency it was necessary to determine the inhibition curves of cytochrome c oxidase activity by azide. The dependence of cytochrome c oxidase activity of heart muscle homogenates from control and Mobr mice on azide concentration is shown in Fig. 3. In the inset the data are transformed using the Dickson linearization. Both curves were within experimental error similar with the same Kd value for azide (85.5 6 5.1 mM and 83.9 6 9.2 mM for control and brindled mice, respectively). Interestingly, in the m. quadriceps homogenates we determined Kd values of 73.5 6 6.9 mM and 107.3 6 6.7 mM for control and brindled mice, respectively. Thus, it was possible to calculate the flux control coefficients using Equation 1. As shown in Table IV, both heart and quadriceps muscles of Mobr mice have significantly higher values of flux coefficients of cytochrome c oxidase. To prove the result of these titration experiments, another specific inhibitor of cytochrome c oxidase, potassium cyanide, was applied. The inhibition curves of oxygen consumption of

FIG. 2. Inhibition of mitochondrial respiration of glycerolskinned fibers by azide. Upper part, skinned fibers (1.5–2.0 mg, dry weight) were isolated from heart (left ventricle) of control (open circles) and brindled (filled circles) mice. The rate of respiration was measured in the presence of 10 mM glutamate, 5 mM malate, 10 mM succinate, and 1 mM ADP with subsequent additions of the azide concentrations indicated. The data points are averages of three experiments. Lower part, skinned fibers (3.0 –3.5 mg, dry weight) were isolated from m. quadriceps of control (open circles) and brindled (filled circles) mice. The rate of respiration was measured as described above. The data points are averages of five experiments.

skinned muscle fibers with KCN are shown on Fig. 4. Once again, it can be seen that KCN titrations for control and brindled mice are strikingly different for both types of skinned fibers from heart and m. quadriceps with less pronounced sigmoidal behavior in copper-deficient mutants. The control coefficients obtained from KCN titrations are summarized in Table IV. In this case of irreversible enzyme inhibition the flux control coefficients were calculated using the following equation. Ci 5 2~dJ/J!/~dI/Imax!

(Eq. 2)

where Imax is the maximal amount of inhibitor. To avoid overestimation of flux control coefficients, we performed nonlinear regression analysis of the entire inhibitor titration curves (27). Interestingly, the values determined by titrations with cyanide are substantially smaller than the values with azide. Nevertheless, as seen in Table IV, the values of flux control coefficients obtained from copper-deficient brindled mice are approximately 2-fold elevated, compared with the control values for both cardiac and skeletal muscles. To examine the cause for the reduced cytochrome c oxidase activities in copper-deficient mice, we determined the cytochrome content in Triton X-100 solubilized membrane pellets of homogenates from heart and skeletal muscles from difference spectra in the a-band of cytochromes. Reduced minus oxidized spectra of these membrane pellets are shown in Fig. 5, A and B. It was found that the cytochrome aa3 content is in cardiac

286

Flux Control of Cytochrome c Oxidase

FIG. 3. Inhibition of cytochrome c oxidase activity of cardiac homogenates by azide. The homogenate (50 mg, wet weight/ml) was obtained from hearts of control mice (open circles) and of Mobr mice (filled circles). The cytochrome c oxidase activity was measured as described under “Experimental Procedures.” Inset, Dickson plot of azide inhibition of cytochrome c oxidase activity.

TABLE IV Flux control coefficients of cytochrome c oxidase on maximal mitochondrial respiration of glycerol-skinned fibers from control and brindled mice The values of control flux coefficients for azide titration were calculated from the initial slopes of inhibition curves of respiration of glycerol-skinned fibers (Fig. 2) using the corresponding Kd for azide measured by the titration of cytochrome c oxidase of total tissue homogenates (Fig. 3). The values of control flux coefficients for the KCN titrations were obtained by nonlinear regression analysis of the complete inhibition curves (Fig. 4) as described in Ref. 27. Tissue

Heart Control mice Brindled mice Significance Quadriceps Control mice Brindled mice Significance

Azide titration

Cyanide titration

0.31 6 0.06 0.59 6 0.2 p , 0.05 (n 5 5)

0.13 6 0.03 0.38 6 0.12 p , 0.05 (n 5 4)

0.33 6 0.1 0.71 6 0.29 p , 0.05 (n 5 6)

0.12 6 0.024 0.35 6 0.18 p , 0.05 (n 5 4)

homogenates of Mobr mice about 1.4-fold lower in comparison with control homogenates (cf. quantitative data in Table I), whereas no significant difference was observed for homogenates from skeletal muscle (cf. quantitative data in Table II). DISCUSSION

In the present study mottled brindled copper-deficient mice were used as a model of the more common cytochrome c oxidase deficiency (30 –35). Previous findings provided direct evidence for decreased activities of copper-containing enzymes (particularly of cytochrome c oxidase), being most probably responsible for mitochondrial abnormalities and brain degeneration associated with Menkes’ disease (11). Histochemical investigations of Menkes’ mutants showed elevated copper concentration in organelle-free cytoplasm as compared with nuclei, mitochondria, or lysosomes, suggesting the disturbed copper transport from the cytosol to the organelles in the cell (36). Using 31P NMR spectroscopy, it was shown that in the brain the observed decreased energy metabolism (decline in ATP content, PCr:Cr ratio, and mitochondrial respiration) can be a pathophysiological mechanism of disturbed nervous function in copper deficiency and Menkes’ diseases (37). In our study an approximately 2-fold lower cytochrome c

FIG. 4. Inhibition of mitochondrial respiration of glycerolskinned fibers by cyanide. Upper part, skinned fibers (1.5–2 mg, dry weight) of control (open circles) and brindled (filled circles) mice heart. The respiration was measured in the presence of 10 mM glutamate, 5 mM malate, 10 mM succinate, and 1 mM ADP. Curves with the following parameters (cf. Ref. 27) were fitted to the titration points. Open circles: flux control coefficient (Ci) 5 0.11; maximal amount of inhibitor (Imax) 5 5.8 mM; dissociation constant of the inhibitor (Kd) 5 0.55 mM; initial rate of respiration (J) 5 57.1 ng atoms of oxygen/min/mg, dry weight. Filled circles: Ci 5 0.54; Imax 5 5.6 mM; Kd 5 0.41 mM; J 5 53 ng atoms of oxygen/min/mg, dry weight. Lower part, skinned fibers (3–3.5 mg, dry weight) from m. quadriceps of control (open circles) and brindled (filled circles) mice. The respiration was measured as described above. Curves with the following parameters were fitted to the titration points. Open circles: Ci 5 0.09; Imax 5 7.1 mM; Kd 5 0.35 mM; J 5 16.1 ng atoms of oxygen/min/mg dwt. Filled circles: Ci 5 0.38; Imax 5 8.9 mM; Kd 5 0.9 mM; J 5 15.4 ng atoms of oxygen/min/mg, dry weight.

Flux Control of Cytochrome c Oxidase

FIG. 5. Difference spectra of cytochromes in homogenates of control and Mobr mice. The membrane fractions of homogenates from heart (containing 50 mg, wet weight, of tissue/ml) and m. quadriceps (containing 100 mg, wet weight, tissue/ml) were prepared as described under “Experimental Procedures.” Upper spectrum of each part, dithionite reduced minus oxidized difference spectrum of membrane fraction from controls; lower spectrum of each part, dithionite reduced minus oxidized difference spectrum of membrane fraction from Mobr mice.

oxidase activity is seen in cardiac and skeletal muscles of brindled mice (Tables I and II). As has been pointed out (38), this decline cannot be related exclusively to the role of copper as a metal center of cytochrome c oxidase, but also may be due to a decreased synthesis of nuclear encoded subunits. Reduced expression of cytochrome c oxidase subunits in the cerebellum, spinal cord, and other regions of central nervous system in Menkes’ disease was shown using specific antibodies against subunits II and IV of cytochrome oxidase (10). This reflects also a decrease of synthesis of mtDNA-encoded subunits. The Kd values for azide (70 –100 mM) obtained in this study are close to the value recently reported for isolated cytochrome c oxidase (28). As has been shown previously, the CuB and cytochrome a3 sites are involved in the binding of cytochrome c oxidase inhibitors like azide and cyanide (29). It is therefore noteworthy that the Kd values for azide inhibition obtained in heart homogenates were not significantly different for control and mottled brindled mice. This indicates an absence of changes in cytochrome c oxidase binding sites for azide in this copper deficiency. Therefore, similarly as in brain (10, 38), the decreased activity of this enzyme in cardiac muscle of Mobr mice seems to be related (at least in part) to a decreased expression of the enzyme and is visible from the decreased levels of cytochrome aa3 obtained in heart homogenates. In skeletal muscle, these changes are small (and due to the higher experimental error not significant) even if the elevated amount of mitochondria per mg, wet weight, is taken into consideration (cf. citrate synthase levels in Table II). On the other hand, different Kd values for azide inhibition were observed in quad-

287

riceps homogenates, pointing to possible alterations in enzymatic function of cytochrome c oxidase in this muscle. To date little is known concerning changes in other enzymatic patterns of cardiac and skeletal muscles associated with, or caused by, copper deficiency. As has been suggested recently, copper deficiency may cause compensatory mechanisms to maintain cardiac ATP levels (39). In our study we estimated these possible adaptational alterations in brindled mutants by assaying the activities of certain key enzymes. The most remarkable changes were revealed in m. quadriceps (Table II). We found a significant decrease in the activities of lactate dehydrogenase, adenylate kinase, and creatine kinase in muscle homogenates of brindled mottled mice. We obtained, however, an elevation in the activity of citrate synthase, which was even more pronounced in heart muscle. This is most probably indicative of the mentioned adaptational response. Increasing the mitochondrial volume and/or density may allow the tissue to compensate for the lack of cytochrome c oxidase. This observation is in accordance with increased mitochondrial:myofibrillar ratios and an expanded mitochondrial area reported for copper-deficient rat hearts (40, 41). The apparent conflicting results of mitochondrial respiration and oxidative phosphorylation in various models of copper deficiency (39, 42, 43) may be explained by different procedures of mitochondrial isolation and examination in those studies. To avoid possible artifacts of the mitochondrial preparation the respiratory parameters of cardiac and skeletal muscle mitochondria were determined using chemically skinned fibers. As has been shown in our previous studies, this approach has a number of advantages (15, 17). Selective permeabilization of plasma membrane essentially results in unobstructed access to the mitochondria in skinned fibers for substrates or ADP. In this way, the mitochondrial parameters can be estimated inside thin muscle fibers. It allows one to investigate the total mitochondrial population, in situ, without isolation of these organelles and using very small samples of tissue. The significantly lower values of flux control coefficients for cytochrome oxidase observed with KCN titration as compared with azide titration can be explained by the more specific inhibition of cytochrome c oxidase by cyanide. Due to the fact that in some conditions azide may also inhibit ATP splitting and ATP synthesis activities of mitochondrial ATPase (44), the application of this inhibitor may have resulted in an overestimation of the value of flux control coefficient for cytochrome c oxidase (Table IV). Summarizing, this report demonstrates that determination of flux control coefficients can be a valuable approach to study defects of mitochondrial oxidative phosphorylation. Acknowledgment—The excellent technical assistance of K. Kaiser is gratefully acknowledged. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

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