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Effects of succinylacetone on growth and respiration of L1210 leukemia cells
253
Cancer Letten, 26 (1985) 253-259 Elsevier Scientific Publishers Ireland Ltd.
EFFECTS OF SUCCINYLACETONE OF L1210 LEUKEMIA CELLS
ON GROWTH
AND RESPIRATION
EUGENE C. WEINBACH’ and PAUL S. EBERTb ‘Laboratory of Pan&tic Dieeaeee, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20205 and bLaboratory of Molecular Oncology, National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD 21701 (U.S.A.) (Received 20 December 1984) (Accepted 25 January 1985)
SUMMARY
4,6-Dioxoheptanoic acid (succinylacetone, SA), a potent inhibitor of heme biosynthesis, suppressed growth and decreased respiration of L1210 leukemia cells in vitro. Growth of cells incubated in the presence of 2--4 mM SA for the first 2 days declined, and after 3 days virtually ceased. L1210 cells in the logarithmic growth phase exhibited active respiration (40 + 9.3 nanoatoms oxygen/min 10’ cells at 37°C) which was inhibited by oligomycin and released by uncouplers of oxidative phosphorylation. These and other inhibitors of mitochondrial function clearly demonstrate a mitochondrial basis for the cellular respiration in both control and SA-treated cells. L1210 cells in the stationary phase exhibited a marked decrease in oxygen consumption compared to cells in logarithmic growth. At the concentrations used in this study, SA was not immediately toxic to L1210 cells, but inhibited growth at 2 days without lowering levels of cellular heme. Thus, it appears unlikely that inhibition of growth of L1210 cells by SA can be ascribed either to heme depletion or to impairment of respiration. l
INTRODUCTION
The L1210 murine leukemia cell has been used in numerous pharmacological, immunological and ultrastructural studies as a model of transformed cells. Relatively few investigations, however, have focused directly on the respiratory and associated bioenergetic processes in this cell [ 2,3,5]. Previous work in our laboratory has shown that 4,6dioxoheptanoic acid (succinylacetone, SA) inhibits heme biosynthesis and growth of several types of tumor cells both in vitro and in vivo [ 6-81. The inhibition of heme synthesis results from the irreversible binding of SA to 6 aminolevulinic acid (ALA) dehydratase, the second enzyme of heme biosynthetic pathway [ 81. Similarly,growth 0304-3835/85/$03.30 o 1985 Elaevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
254
of L1210 cells and Walker 256 carcinosarcoma cells was suppressed by SA in vitro, but no depletion of heme biosynthesis was observed [ 81. The present study was undertaken to investigate certain characteristics of respiration in L1210 cells, and to assess the effect of SA on this metabolism in conjunction with its influence on the growth of these cells. Compounds known to have specific effects on mitochondrial metabolism also were evaluated in intact L1210 cells in an attempt to locate the site of action of SA in the malignant cells. MATERIALS AND METHODS
Chemicals SA was purchased medium was obtained
from Omega Organics (Longmont, CO). Culture from Grand Island Biological Co., (Grand Island, NY).
Cells and culture procedures L1210 leukemia cells were aspirated sterilely from DBA/2 mice and routinely maintained in Liebovitz (L-15) medium: McCoy’s 5A medium (5A) (L-15/5A, 1: 1) containing penicillin/streptomycin/neomycin (10 : 10 : 20 pg/ ml), 2 mM glutamine and 2% heat-inactivated fetal bovine serum (FBS). L1210 cells incubated at 37°C with and without SA were collected by centrifugation on the days indicated, washed once in Earle’s balanced salt solution (EBSS), and suspended in Roswell Park Memorial Institute Medium (RPMI) 1640 without FBS. Viable cells were determined by trypan blue exclusion in a hemacytometer.
Measurement of respiration Oxygen consumption was determined polarographically using the Clark oxygen electrode (Yellow Springs Instrument Co., Inc., Yellow Springs, OH). One milliliter of the cell suspensions containing the indicated numbers of cells was stirred magnetically, and equilibrated with air at 37°C in a glass cuvette mounted in a thermostated chamber. After the pre-incubation period, the electrode was inserted into the cuvette, and recording started. Additions to the respiring cell suspension were made with microliter syringes through a capillary port in the cuvette [ 121. RESULTS
Effect of SA on growth of L1210 leukemia cells Figure 1 shows the effect of various concentrations of SA on the in vitro growth of L1210 cells. The control cells grew logarithmically for 4 days, whereas by day 2, cells grown in the presence of all 3 concentrations of SA exhibited variable decreases in their rate of replication. By the 3rd day, growth of cells in the presence of 2 mM SA declined, and the growth of cells
255
Fig. 1. Growth of L1210 leukemia cells in the presence and absence of SA. The growth of untreated cells reflects cumulative growth. All cultures were divided when the cell concentration reached 1 x lo6 cells/ml.
incubated with 3 and 4 mM SA virtually ceased. By the 4th day, unambiguous growth stasis was observed with all concentrations of SA tested. In some experiments, growth of the L1210 cells ceased after 2 days incubation with 2and3mMSA. Effect of SA on respiration of L1210 cells The leukemia cells growing logarithmically displayed a high rate of respiration (Fig. 2). They responded to mitochondrial reagents in a manner qualitatively similar to that observed with intact mitochondria [lo]. For example, their respiration was inhibited by oligomycin (Olig), and then released by carbonylcyanide 3chlorophenylhydrazone (CCCP), a classical uncoupler of oxidative phosphorylation. Pentachlorophenol(lO0 PM), and 4,5,6,7tetrachlorotrifluoromethyl-benzimidazole (25 PM) also were effective uncouplers. The uncoupled respiration was completely blocked by 500 PM cyanide, and by antimycin, 5 pg per ml (data not shown). Rotenone impeded the endogenous respiration’of the L1210 cells, and, as shown in Fig. 2, also inhibited the uncoupled respiration.
256 Ll210 CELLS 2.2 x 107/ml I
Fig. 2. Respiration of L1210 leukemia cells: effects of inhibitors and uncoupling agents. The numbers on the tracing represent oxygen uptake in nanoatoms/min* 10’ cells. The reaction mixture consisted of cells in the logarithmic phase, plus additions as indicated, in 1.0 ml of RPM1 1640 without FBS. Other details are given in Materials and Methods.
L1210 CELLS: SA-TREATED 4.9 x 107/ml
Fig. 3. Respiration of L1210 leukemia cells in the presence of SA. Conditions were the same as those given in the legend to Fig. 2, except that the cells were grown in the presence of 3 mM SA for 3 days.
257 TABLE 1 EFFECT
OF SA ON RESPIRATION
OF L1210
LEUKEMIA
CELLS
Conditions
Respiration (nanoatoms oxygen/min - 10’ cells)a
Control cells, logarithmic Control cells, stationary t 2mMSA + 3mMSA
40.0 9.0 6.7 6.6
f 9.3 * 2.9 k4.5 * 2.8
(14) (15) (15) (12)
Cells were grown in the absence (control) and presence of the indicated amounts of SA for 3 days prior to harvest. *Values are the means f S.E.M. for the number of experiments shown in parentheses.
Respiration of cells incubated in the presence of 3 mM SA for 3 days was strongly impaired (Fig. 3). Their response to inhibitors and uncouplers was similar to that observed with control cells. These cells, however, were in the stationary phase as a result of incubation with SA (Fig. 1). L1210 control cells in the stationary phase (i.e. cells maintained at a high concentration which did not change in 24 h) also exhibited diminished respiration when compared to control cells growing logarithmically. Statistical evaluation of repetitive polarographic assays are presented in Table 1. Addition of SA in a final concentration of 3 mM to respiring control cells during polarographic assays had no effect (data not shown). These experiments eliminated the possibility of a rapid, direct but transient effect of SA on the respiration of the cells. Experiments with isolated rat liver mitochondria showed that 3 mM SA did not inhibit respiration supported by tricarboxylic acid cycle substrates, or had any effect on electron transport and oxidative phosphorylation. DISCUSSION
The mechanism of inhibition of L1210 cell growth by SA is not clear. Although the compound is a specific inhibitor of ALA dehydratase and thereby inhibits heme biosynthesis in MEL cells in vitro [ 61, and Walker 256 carcinosarcoma and Novikoff hepatoma cells in vivo [ 71, SA did not decrease the level of cellular heme used in L1210 cells maintained in vitro [7]. At the concentrations used in these experiments (2-4 mM), inhibition of cell growth was observed after a-days exposure to the compound. Apparently, the inhibition of cell growth by SA is a specific toxic effect that occurs only after several days of exposure to the inhibitor and after several cell divisions. This effect is similar to that of the slow reduction in heme biosynthesis in MEL cells exposed to SA [6]. Higher concentrations of SA (30 mM) incubated with cultured normal liver cells caused little toxicity [ 43. Furthermore,
258
rats given much larger doses than those used in this study showed no signs of toxicity either grossly or upon histological examination of their vital organs 131. Additionally, these results provide evidence that the respiration of cultured L1210 murine leukemia cells is of mitochondrial origin. The organelles of L1210 cells in situ could be perturbed by reagents known to have highly specific effects on isolated mitochondria [ 111. Particularly impressive was the inhibition of cellular respiration by oligomycin and its release by uncouplers of oxidative phosphorylation (Figs. 2,3). Respiration is mediated by electron transport through the respiratory chain as shown by the marked inhibition obtained with rotenone, cyanide and antimycin. Studies in other laboratories also have shown respiratory inhibition of L1210 cells by rotenone [ 51, oligomycin and antimycin [ 31, but under experimental conditions that differed markedly from those used in the present study. The collective data support the premise that respiration of the L1210 leukemia cell results from electron transport and phosphorylation at the mitochondrial locus. It cannot be assumed a priori, however, that respiration of all eukaryotic cells is of mitochondrial origin. For example, the enteric parasitic protozoa En&moeba histolytica and Giardia lamblia exhibit active endogenous respiration; but do not contain mitochondria [ 111. The endogenous respiration of L1210 cells incubated with 2-3 mM SA was markedly inhibited when compared to control cells growing logarithmicaIly (Figs. 2,3). Respiration of control cells that had ceased growth also was strongly diminished, similar to the effect of SA. Apparently, SA exerts a general effect on respiration analogous to that observed when cells are cultivated at high cell concentration or in low amounts of serum. No specific effects on mitochondrial metabolism were found. The mode of action of SA to inhibit cell growth does not appear to be on a locus directly affecting respiration, but the compound may affect the production of a highly sensitive pool of heme-containing enzymes or cytochromes at levels not presently detectable. It appears unlikely in view of our present data that the inhibition of growth of L1210 leukemia cells in vitro by SA can be ascribed to either heme depletion or respiratory impairment. ACKNOWLEDGEMENTS
We thank C. Elwood Claggett and Richard A. Hess for their technical assistance. We also thank Mrs. Wilma J. Davis and Mrs. Brenda B. Martin for their typing and editorial assistance. REFERENCES 1 Ebert, P.S., Hess, R.A., Frykholm, B.C. and Tschudy, D.P. (1979) Biochem. Biophys. Res. Commun., 88,1382-1390. 2 Granger, D.L., Taintor, R.R., Cook, J.L. and Hibbs, Jr., J.B; (1980) J. Clin. Invest., 65,351--370.
259
3 Pedersen, P.L. (1978) Prog. Exp. Tumor Res., 22, 190-240. 4 Sassa, S. and Kappas, A. (1983) J. Clin. Invest., 71, 625-634. 5 Tucker, A.N. and Friedman, M.A. (1977) Res. Commun. Chem. Pathol. Pharmacol., 23,617-632. 6 Tschudy, D.P., Ebert, P.S., Hess, R.A., Frykhohn, B.C. and Weinbach, E.C. (1980) Biochem. Phsrmacol., 29, 1825-1831. 7 Tschudy, D.P., Ebert, P.S., Hess, R.A., Frykholm, B.C. and Atsmon, A. (1983) Oncology, 40,148-154. 8 Tschudy, D.P., Hess, R.A. and Frykholm, B.C. (1981) J. Biol. Chem., 256,99159923. 9 Tschudy, D.P., Hess, R.A., Frykholm, B.C. and Blaese, R.M. (1982) J. Lab. Clin. Med., 99,526-532. 10 Tzagoloff, A. (1983) Mitochondria, pp. 132-134. Plenum, New York and London. 11 Weinbach, E.C. (1981) Trends Biochem. Sci., 2, 254-257. 12 Weinbach, E.C. and Garbus, J. (1966) J. Biol. Chem., 241, 3708-3713.
Cancer Letten, 26 (1985) 253-259 Elsevier Scientific Publishers Ireland Ltd.
EFFECTS OF SUCCINYLACETONE OF L1210 LEUKEMIA CELLS
ON GROWTH
AND RESPIRATION
EUGENE C. WEINBACH’ and PAUL S. EBERTb ‘Laboratory of Pan&tic Dieeaeee, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20205 and bLaboratory of Molecular Oncology, National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD 21701 (U.S.A.) (Received 20 December 1984) (Accepted 25 January 1985)
SUMMARY
4,6-Dioxoheptanoic acid (succinylacetone, SA), a potent inhibitor of heme biosynthesis, suppressed growth and decreased respiration of L1210 leukemia cells in vitro. Growth of cells incubated in the presence of 2--4 mM SA for the first 2 days declined, and after 3 days virtually ceased. L1210 cells in the logarithmic growth phase exhibited active respiration (40 + 9.3 nanoatoms oxygen/min 10’ cells at 37°C) which was inhibited by oligomycin and released by uncouplers of oxidative phosphorylation. These and other inhibitors of mitochondrial function clearly demonstrate a mitochondrial basis for the cellular respiration in both control and SA-treated cells. L1210 cells in the stationary phase exhibited a marked decrease in oxygen consumption compared to cells in logarithmic growth. At the concentrations used in this study, SA was not immediately toxic to L1210 cells, but inhibited growth at 2 days without lowering levels of cellular heme. Thus, it appears unlikely that inhibition of growth of L1210 cells by SA can be ascribed either to heme depletion or to impairment of respiration. l
INTRODUCTION
The L1210 murine leukemia cell has been used in numerous pharmacological, immunological and ultrastructural studies as a model of transformed cells. Relatively few investigations, however, have focused directly on the respiratory and associated bioenergetic processes in this cell [ 2,3,5]. Previous work in our laboratory has shown that 4,6dioxoheptanoic acid (succinylacetone, SA) inhibits heme biosynthesis and growth of several types of tumor cells both in vitro and in vivo [ 6-81. The inhibition of heme synthesis results from the irreversible binding of SA to 6 aminolevulinic acid (ALA) dehydratase, the second enzyme of heme biosynthetic pathway [ 81. Similarly,growth 0304-3835/85/$03.30 o 1985 Elaevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
254
of L1210 cells and Walker 256 carcinosarcoma cells was suppressed by SA in vitro, but no depletion of heme biosynthesis was observed [ 81. The present study was undertaken to investigate certain characteristics of respiration in L1210 cells, and to assess the effect of SA on this metabolism in conjunction with its influence on the growth of these cells. Compounds known to have specific effects on mitochondrial metabolism also were evaluated in intact L1210 cells in an attempt to locate the site of action of SA in the malignant cells. MATERIALS AND METHODS
Chemicals SA was purchased medium was obtained
from Omega Organics (Longmont, CO). Culture from Grand Island Biological Co., (Grand Island, NY).
Cells and culture procedures L1210 leukemia cells were aspirated sterilely from DBA/2 mice and routinely maintained in Liebovitz (L-15) medium: McCoy’s 5A medium (5A) (L-15/5A, 1: 1) containing penicillin/streptomycin/neomycin (10 : 10 : 20 pg/ ml), 2 mM glutamine and 2% heat-inactivated fetal bovine serum (FBS). L1210 cells incubated at 37°C with and without SA were collected by centrifugation on the days indicated, washed once in Earle’s balanced salt solution (EBSS), and suspended in Roswell Park Memorial Institute Medium (RPMI) 1640 without FBS. Viable cells were determined by trypan blue exclusion in a hemacytometer.
Measurement of respiration Oxygen consumption was determined polarographically using the Clark oxygen electrode (Yellow Springs Instrument Co., Inc., Yellow Springs, OH). One milliliter of the cell suspensions containing the indicated numbers of cells was stirred magnetically, and equilibrated with air at 37°C in a glass cuvette mounted in a thermostated chamber. After the pre-incubation period, the electrode was inserted into the cuvette, and recording started. Additions to the respiring cell suspension were made with microliter syringes through a capillary port in the cuvette [ 121. RESULTS
Effect of SA on growth of L1210 leukemia cells Figure 1 shows the effect of various concentrations of SA on the in vitro growth of L1210 cells. The control cells grew logarithmically for 4 days, whereas by day 2, cells grown in the presence of all 3 concentrations of SA exhibited variable decreases in their rate of replication. By the 3rd day, growth of cells in the presence of 2 mM SA declined, and the growth of cells
255
Fig. 1. Growth of L1210 leukemia cells in the presence and absence of SA. The growth of untreated cells reflects cumulative growth. All cultures were divided when the cell concentration reached 1 x lo6 cells/ml.
incubated with 3 and 4 mM SA virtually ceased. By the 4th day, unambiguous growth stasis was observed with all concentrations of SA tested. In some experiments, growth of the L1210 cells ceased after 2 days incubation with 2and3mMSA. Effect of SA on respiration of L1210 cells The leukemia cells growing logarithmically displayed a high rate of respiration (Fig. 2). They responded to mitochondrial reagents in a manner qualitatively similar to that observed with intact mitochondria [lo]. For example, their respiration was inhibited by oligomycin (Olig), and then released by carbonylcyanide 3chlorophenylhydrazone (CCCP), a classical uncoupler of oxidative phosphorylation. Pentachlorophenol(lO0 PM), and 4,5,6,7tetrachlorotrifluoromethyl-benzimidazole (25 PM) also were effective uncouplers. The uncoupled respiration was completely blocked by 500 PM cyanide, and by antimycin, 5 pg per ml (data not shown). Rotenone impeded the endogenous respiration’of the L1210 cells, and, as shown in Fig. 2, also inhibited the uncoupled respiration.
256 Ll210 CELLS 2.2 x 107/ml I
Fig. 2. Respiration of L1210 leukemia cells: effects of inhibitors and uncoupling agents. The numbers on the tracing represent oxygen uptake in nanoatoms/min* 10’ cells. The reaction mixture consisted of cells in the logarithmic phase, plus additions as indicated, in 1.0 ml of RPM1 1640 without FBS. Other details are given in Materials and Methods.
L1210 CELLS: SA-TREATED 4.9 x 107/ml
Fig. 3. Respiration of L1210 leukemia cells in the presence of SA. Conditions were the same as those given in the legend to Fig. 2, except that the cells were grown in the presence of 3 mM SA for 3 days.
257 TABLE 1 EFFECT
OF SA ON RESPIRATION
OF L1210
LEUKEMIA
CELLS
Conditions
Respiration (nanoatoms oxygen/min - 10’ cells)a
Control cells, logarithmic Control cells, stationary t 2mMSA + 3mMSA
40.0 9.0 6.7 6.6
f 9.3 * 2.9 k4.5 * 2.8
(14) (15) (15) (12)
Cells were grown in the absence (control) and presence of the indicated amounts of SA for 3 days prior to harvest. *Values are the means f S.E.M. for the number of experiments shown in parentheses.
Respiration of cells incubated in the presence of 3 mM SA for 3 days was strongly impaired (Fig. 3). Their response to inhibitors and uncouplers was similar to that observed with control cells. These cells, however, were in the stationary phase as a result of incubation with SA (Fig. 1). L1210 control cells in the stationary phase (i.e. cells maintained at a high concentration which did not change in 24 h) also exhibited diminished respiration when compared to control cells growing logarithmically. Statistical evaluation of repetitive polarographic assays are presented in Table 1. Addition of SA in a final concentration of 3 mM to respiring control cells during polarographic assays had no effect (data not shown). These experiments eliminated the possibility of a rapid, direct but transient effect of SA on the respiration of the cells. Experiments with isolated rat liver mitochondria showed that 3 mM SA did not inhibit respiration supported by tricarboxylic acid cycle substrates, or had any effect on electron transport and oxidative phosphorylation. DISCUSSION
The mechanism of inhibition of L1210 cell growth by SA is not clear. Although the compound is a specific inhibitor of ALA dehydratase and thereby inhibits heme biosynthesis in MEL cells in vitro [ 61, and Walker 256 carcinosarcoma and Novikoff hepatoma cells in vivo [ 71, SA did not decrease the level of cellular heme used in L1210 cells maintained in vitro [7]. At the concentrations used in these experiments (2-4 mM), inhibition of cell growth was observed after a-days exposure to the compound. Apparently, the inhibition of cell growth by SA is a specific toxic effect that occurs only after several days of exposure to the inhibitor and after several cell divisions. This effect is similar to that of the slow reduction in heme biosynthesis in MEL cells exposed to SA [6]. Higher concentrations of SA (30 mM) incubated with cultured normal liver cells caused little toxicity [ 43. Furthermore,
258
rats given much larger doses than those used in this study showed no signs of toxicity either grossly or upon histological examination of their vital organs 131. Additionally, these results provide evidence that the respiration of cultured L1210 murine leukemia cells is of mitochondrial origin. The organelles of L1210 cells in situ could be perturbed by reagents known to have highly specific effects on isolated mitochondria [ 111. Particularly impressive was the inhibition of cellular respiration by oligomycin and its release by uncouplers of oxidative phosphorylation (Figs. 2,3). Respiration is mediated by electron transport through the respiratory chain as shown by the marked inhibition obtained with rotenone, cyanide and antimycin. Studies in other laboratories also have shown respiratory inhibition of L1210 cells by rotenone [ 51, oligomycin and antimycin [ 31, but under experimental conditions that differed markedly from those used in the present study. The collective data support the premise that respiration of the L1210 leukemia cell results from electron transport and phosphorylation at the mitochondrial locus. It cannot be assumed a priori, however, that respiration of all eukaryotic cells is of mitochondrial origin. For example, the enteric parasitic protozoa En&moeba histolytica and Giardia lamblia exhibit active endogenous respiration; but do not contain mitochondria [ 111. The endogenous respiration of L1210 cells incubated with 2-3 mM SA was markedly inhibited when compared to control cells growing logarithmicaIly (Figs. 2,3). Respiration of control cells that had ceased growth also was strongly diminished, similar to the effect of SA. Apparently, SA exerts a general effect on respiration analogous to that observed when cells are cultivated at high cell concentration or in low amounts of serum. No specific effects on mitochondrial metabolism were found. The mode of action of SA to inhibit cell growth does not appear to be on a locus directly affecting respiration, but the compound may affect the production of a highly sensitive pool of heme-containing enzymes or cytochromes at levels not presently detectable. It appears unlikely in view of our present data that the inhibition of growth of L1210 leukemia cells in vitro by SA can be ascribed to either heme depletion or respiratory impairment. ACKNOWLEDGEMENTS
We thank C. Elwood Claggett and Richard A. Hess for their technical assistance. We also thank Mrs. Wilma J. Davis and Mrs. Brenda B. Martin for their typing and editorial assistance. REFERENCES 1 Ebert, P.S., Hess, R.A., Frykholm, B.C. and Tschudy, D.P. (1979) Biochem. Biophys. Res. Commun., 88,1382-1390. 2 Granger, D.L., Taintor, R.R., Cook, J.L. and Hibbs, Jr., J.B; (1980) J. Clin. Invest., 65,351--370.
259
3 Pedersen, P.L. (1978) Prog. Exp. Tumor Res., 22, 190-240. 4 Sassa, S. and Kappas, A. (1983) J. Clin. Invest., 71, 625-634. 5 Tucker, A.N. and Friedman, M.A. (1977) Res. Commun. Chem. Pathol. Pharmacol., 23,617-632. 6 Tschudy, D.P., Ebert, P.S., Hess, R.A., Frykhohn, B.C. and Weinbach, E.C. (1980) Biochem. Phsrmacol., 29, 1825-1831. 7 Tschudy, D.P., Ebert, P.S., Hess, R.A., Frykholm, B.C. and Atsmon, A. (1983) Oncology, 40,148-154. 8 Tschudy, D.P., Hess, R.A. and Frykholm, B.C. (1981) J. Biol. Chem., 256,99159923. 9 Tschudy, D.P., Hess, R.A., Frykholm, B.C. and Blaese, R.M. (1982) J. Lab. Clin. Med., 99,526-532. 10 Tzagoloff, A. (1983) Mitochondria, pp. 132-134. Plenum, New York and London. 11 Weinbach, E.C. (1981) Trends Biochem. Sci., 2, 254-257. 12 Weinbach, E.C. and Garbus, J. (1966) J. Biol. Chem., 241, 3708-3713.