Enzymatic properties of mitochondria isolated from normal and vitamin B12-deficient rats

ARCHIVES OF BIOCHEMISTRY Vol. 197, No. 2, October 15,

Enzymatic M. ABDUL Pre-Clinical Texas

AND BIOPHYSICS

pp. 388-395, 1979

Properties of Mitochondria Vitamin B,,-Deficient MATLIB,2 JERRY

EUGENE HENSLEE,

Isolated Rats1

P. FRENKEL, AND PAUL

from

Normal

and

AMAL MUKHERJEE, A. SRERE

Sciences Unit and Nuclear Medicine Seruice, Veterans Administration Medical Center, and the Department of Biochemistry and the Evelyn L. Overton Hematology-Oncology Research Laboratory, Department of Internal Medicine, The University of Texas Health Science Center, Dallas, Texas 75235

Dallas,

75216,

Received April 9, 1979; revised June 7, 1979 The mitochondria from livers of normal and vitamin B,,-deprived animals were compared. The rate of oxygen uptake (states 3 and 4) of mitochondria from vitamin B,,-deprived livers was higher than that of normal mitochondria while the respiratory control index of B,,-deprived mitochondria was lower than normal. Most Krebs cycle enzyme activities were increased in the hepatic mitochondria from B,,-deprived animals. Two-dimensional gel electrophoresis of mitochondrial matrix proteins also indicated that an increase in Krebs cycle enzyme occurred but the increase was not general for all mito_ quantities _ chondrial proteins.

Previous studies have demonstrated that vitamin B12 deficiency leads to increased fatty acid synthesis in animals and man (l-5). This increased fatty acid synthesis has been shown to be related to an increased rate of synthesis of the enzyme fatty acid synthetase (4, 6). Recently, it was also observed that hepatic citrate synthase, one of the Krebs cycle enzymes located in the mitochondrial matrix was increased twofold due to its increased rate of synthesis in vitamin B,,-deficient rats (7,s). The present study was to determine whether the increased citrate synthase activity was correlated with increases in the activity of other enzymes of the Krebs cycle located in the mitochondrial matrix from hepatic cells. In addition, the changes in the activity of enzymes located in the outer membrane

and the intermembrane space were measured. Since the work of Pette et al. (9) indicated that the enzymes of the Krebs cycle constitute a constant proportion group of enzymes, the present study also appeared to be an excellent model to test this proposal in a single tissue. MATERIALS

1 Supported by the Veterans Administration, NSF GB 41851, and National Institutes of Health Grants AM11313 and CA23115. 2 Present address: Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267. 3 Present address: Department of Biochemistry and Nutrition, University of North Carolina, Chapel Hill, N. C. 27514. 0003-9861/79/120388-08$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

AND METHODS

The following materials were obtained from indicated sources: ADP, ATP, NAD, NADP, NADH, 5,5dithiobis(2-nitrobenzoic acid) (DTNB),” L-malic acid, a-ketoglutaric acid, L-glutamic acid, succinic acid, /3-hydroxybutyric acid, bovine serum albumin (BSA), cytochrome c, rotenone, oligomycin, and L-aspartic acid from Sigma Chemical Company, St. Louis, Missouri; oxalacetic acid from Calbiochem, LaJolla, California; and, CoA from P-L. Biochemicals, Milwaukee, Wisconsin. Acetyl-CoA was prepared according to Simon and Shemin (10). All other chemicals were of the purest commercially available grades. Animals and their maintenance. Animal maintenance, characterization of the diets, and manner of achieving vitamin B,, deprivation were previously described (4, 5). The animals were decapitated, at 4 Abbreviations used: DTNB, 5,5-dithiobis(2-nitrobenzoic acid); BSA, bovine serum albumin; RC, respiratory control; Hepes, N-2-hydroxyethylpiperazine-N’2-ethanesulfonic acid.

388

MITOCHONDRIAL

ENZYMES

which time serum and liver were obtained. Serum vitamin B,, was determined as previously described (11, 12). Isolation of mitochondria. Mitochondria were isolated from livers of fed rats according to a procedure described earlier (13). Equal amounts of livers from normal and B,,-deficient animals were used during isolation of mitochondria. Total protein of isolated mitochondria was determined and used as an estimate of the yield of mitochondria obtained by each isolation. On this basis, the yield of mitochondria from livers of normal and deficient animals was similar. Protein was determined by the method of Lowry et al. (14) using crystalline BSA as standard. Measurement of respiration of phosphoqlation of isolated mitochondria Respiration was measured with a Clark oxygen electrode at 25°C according to a procedure described previously (15). Calculations of respiratory control (RC), ADP/O ratios and the terminology of the “state” of mitochondria were according to Chance and Williams (16). The assay solution (3.3 ml) contained 220 mM mannitol, 70 mM sucrose, 10 mM Hepes (pH 7.4), 2-5 mM potassium phosphate buffer (pH 7.4), 2.5 mM MgCl,, 0.05% BSA, and 5 mg mitochondrial protein. Enzyme assays. All enzymatic assays were carried out at 25°C in 1 ml (lo-mm light path) in a Beckman DU spectrophotometer fitted with a Gilford recording assembly. Isolated mitochondria were sonicated in a Branson sonifier at 50W output in 5-s intervals with cooling at 0°C until the suspension was fairly clear and no further increase in citrate synthase activity was observed. The following enzymes were assayed according to the procedures described previously with one unit of enzyme defined as 1 pmol substrate utilizedimin: citrate synthase according to Srere et al. (17); aconitase according to Fansler and Lowenstein (18); NAD-isocitrate dehydrogenase according to Plaut (19); NADP-isocitrate dehydrogenase according to Cleland et al. (20); cY-ketoglutarate dehydrogenase acTABLE

389

IN B,, DEFICIENCY

cording to Sanadi (21); fumarase according to Hill and Bradshaw (22); malate dehydrogenase according to Kitto (23); sulfite-cytochrome c reductase according to Cohen and Fridovich (24); glutamate dehydrogenase according to Schmidt (25); glutamate-oxalacetate transaminase according to Bergmeyer and Brant (26); ATPase was measured by assaying liberated Pi according to Ames and Dubin (27). Incubation was carried out at 25°C in 0.02 M MgCl,, 0.1 M Tris-malate (pH 7.4), and 0.25 M sucrose. The reaction was terminated with an equal volume of cold 12% HCIO,. The following enzyme activities were assayed in mitochondria swollen in presence of 10 mM potassium phosphate buffer (pH 7.4), for 15 min at 25°C: Succinate dehydrogenase was determined spectrophotometrically using dichlorphenol indophenol according to Veeger et al. (28); succinate cytochrome c reductase was assayed by the method of Wojtczak and Zaluska (29); cytochrome c oxidase was done by following the oxidation of reduced cytochrome c at 550 nm in 0.05 M potassium phosphate buffer (pH 7.4), and NADH oxidase was measured by following the oxidation of NADH at 340 nm in a buffer containing 0.05 M potassium phosphate buffer (pH 7.4). Rotenone-sensitive NADH-cytochrome c reductase activity was measured according to Sottocasa et al. (30). P-Hydroxybutyrate dehydrogenase was measured spectrophotometrically according to Bergmeyer et al. (31). Rotenone-insensitive NADH-cytochrome c reductase was determined in intact mitochondria according to Sottocasa et al. (30). Gel electrophoresis. Two-dimensional gel electrophoresis was run using the method of O’Farrell(32) as modified by Henslee and Srere (33). RESULTS

Characterization

of Study Animals

The status of the animals in each dietary study group at the time of sacrifice is shown in Table I. Methylmalonic aciduria was I

CHARACTERISTICS OF STUDY ANIMALS~ Vitamin B,, Study animals

Body weight (g)

Liver weight

Serum W/ml)

Liver Wg)

Normal (4) Vitamin B,, deprived (4)

303 * 34*

821

1103 k 14

142 k 16

224 t 21

11 k 1

84 t 15

22 e 1

a Data characterizing the study animals at the time of sacrifice. Each group refers to the dietary maintenance program as described under Materials and Methods. Figures in parentheses refer to the number of animals in each group. * tl SD.

390

MATLIB

identified in the B,,-deprived animals prior to sacrifice to help characterize their cobalamine deficiency (34). The liver weights in the B,,-deprived animals were about 37% greater than those of the control animals although body weights were about 26% less. Serum and liver vitamin Blz levels were decreased to 8 and 15%, respectively, of the control values in the vitamin B,,-deprived animals. Respiration and Phosphorylation Isolated Mitochondria

of

Mitochondria were isolated from livers of normal and B,,-deprived animals under identical conditions. Homogenization of equal amounts of tissue was performed in equal volumes of solution and the specimens were simultaneously centrifuged. The yield of mitochondrial protein after isolation from the livers of the two groups was within 10% of each other. Isolated mitochondria from normal rats were found to be tightly coupled with ADP/O ratios very close to theoretical values and very high respiratory control values approaching infinity with NAD-linked sub-

ET AL.

strates. Respiratory and phosphorylative activity of mitochondria isolated from normal rats with cY-ketoglutarate as the substrate is presented in Fig. 1N. The rate of oxidation of hepatic mitochondria from vitamin &,-deprived rats was higher with lower respiratory ~control values compared to hepatic mitochondria from normal rats (Fig. 1D). However, the difference in oxygen uptake in “state 4” rate was much higher than the difference in “state 3” rate. This indicated that the mitochondria isolated from vitamin B,,-deprived rats were slightly less tightly coupled compared to mitochondria from normal rats. Although only results with (Yketoglutarate as substrate are presented here, similar results were obtained with other substrates. Enzymatic Activities Mitochondria

of Isolated

All Krebs cycle enzyme activities, except NADP-isocitrate dehydrogenase and (Yketoglutarate dehydrogenase, were found to be increased in activity (Table II). Almost a twofold increase in activity of citrate

FIG. 1. Respiration and phosphorylation of mitochondria isolated from the livers of normal and vitamin B,,-deficient rats. The oxygraph trace on the left (labeled N) was obtained from normal animals; the trace on the right (D) was obtained from cobalamin-deficient animals. The measurements were carried out as described under Materials and Methods. The normal study (N) used 4.96 mg of mitochondria obtained from the livers of normal rats and the deficient study (D) used 4.48 mg of mitochondria obtained from the livers of cobalamin-deficient rats. The substrate was 10 mM a-ketoglutarate (aKg) and ADP (600 nmol) was added as indicated by the arrows. The numbers along the trace are n atoms of oxygen consumed per minute per milligram of mitochondrial protein. The respiratory control (RC) and the ADP/O values are also indicated on the chart.

MITOCHONDRIAL TABLE

ENZYMES

II

ENZYMATIC ACTIVITIES OF MITOCHONDRIA ISOLATED FROM NORMAL AND VITAMIN B12-D~~~~~~~ RATS Activity

Enzymes Citrate synthase Aconitase NAD-Isocitrate dehydrogenase NADP-Isocitrate dehydrogenase a-Ketoglutarate dehydrogenase Succinate dehydrogenase Fumarase Malate dehydrogenase Glutamate dehydrogenase Glutamate-oxaloacetate transaminase P-Hydroxybutyrate dehydrogenase Succinate-cytochrome c reductase Cytochrome c oxidase NADH oxidase Rotenone-sensitive NADH-cytochrome c reductase Rotenone-insensitive NADH-cytochrome c reductase Sulfite-cytochrome c reductase Oligomycin-sensitive ATPase (intact mitochondria) Oligomycin-sensitive ATPase (sonicated mitochondria)

(mU/mg

Vitamin B,, deprived

Normal 116 k 28 94 c 18 5kl

260 + 19 155 2 14 11 + 2

58 k 14 11 97 577 2807 74

protein)

+ iz + k +

2 13 46 307 5

51 2 18 8+3 123 I 984 2 4062 zi 136 ”

4 43 342 2

930 + 10

705 + 9

46 k 3

38 + 4

86 k 10 970 k 20 78 h 16

108 2 18 870 k 31 76 + 12

45 2 6

40 + 4

243 + 9

202 k 11

93 + 9

55 zk 8

3+1

14 f 4

530 -r- 42

717 2 43

synthase, NAD-isocitrate dehydrogenase, and fumarase was observed in hepatic mitochondria from vitamin B,,-deprived rats compared to hepatic mitochondria from normal rats. About 50% increased activity of malate dehydrogenase and aconitase was observed in mitochondria of B,,-deprived rats compared to normal rats. Only a 25% increase in succinate dehydrogenase and succinate-cytochrome c reductase activities was observed. Glutamate dehydrogenase,

IN B,, DEFICIENCY

391

an enzyme not directly involved in Krebs cycle, was also increased in activity due to B,, deprivation. Total oligomycin-sensitive ATPase activity was also increased. Inner membrane-bound rotenone-sensitive NADHcytochrome c reductase, cytochrome c oxidase, NADH oxidase and /3-hydroxybutyrate dehydrogenase activity remained unaffected. The outer membrane-bound enzyme rotenone-insensitive NADH-cytochrome c reductase activity was also unaffected. The only enzyme found to be decreased in activity was sulfite-cytochrome c reductase located in the intermembrane space. The measured activities of NAD-isocitrate dehydrogenase and a-ketoglutarate dehydrogenase were low. These are labile enzymes and the values (Table II) may not reflect their true mitochondrial activities. Nonetheless the values were those determined under the conditions of the study and are recorded for future comparison. Furthermore, it is of interest that the value for NAD-isocitrate dehydrogenase was higher in the mitochondria from B,,-deficient animals. By contrast, the reverse was true for a-ketoglutarate dehydrogenase, thereby suggesting that the measured values for the enzymes were not simply due to the labile status. Gel Electrophoresis

Two-dimensional polyacrylamide gel electrophoresis (32) has been applied to resolving the polypeptides of the matrix subfraction prepared from rat liver mitochondria (33). Since enzymatic differences were observed between liver mitochondria of normal and vitamin B,,-deprived rats, it was of interest to determine whether differences could also be detected by two-dimensional electrophoresis. Equal amounts of mitochondrial matrix protein from normal and vitamin B,,-deprived rats were analyzed (Figs. 2 and 3). Protein components 1, 2, 3, and 4 appeared as larger “spots” in the normal pattern than in the vitamin B,,deprived pattern. Components 1 and 2 have previously been identified as carbamyl phosphate synthase and ornithine transcarbamylase respectively (33). The patterns therefore suggest that the enzymes repre-

392

MATLIB

ET AL.

FIG. 2. Two-dimensional gel electrophoretic pattern of the liver mitochondrial matrix subfraction (100 pg protein) obtained from normal rats. Identified components include: Component 1 is carbamyl phosphate synthase; 2 is ornithine transcarbamylase; 5 is aconitase; 6 is glutamate dehydrogenase; and 7 is citrate synthase.

sented by these “spots” were present at higher concentrations in normal rat liver than in B,,-deprived liver. Protein components observed to be at higher concentrations in the vitamin B,,-deprived liver mitochondria were 5,6,7,8, and 9; 5,6, and ‘7 have been identified as aconitase, glutamate dehydrogenase, and citrate synthase, respectively (33). Protein components 10, 11, and 12 were observed to be at similar concentrations in normal and vitamin Blzdeprived mitochondria; these protein components have not yet been identified, but studies to characterize them are currently in progress. DISCUSSION

Mitochondria isolated from livers of B,,deprived rats appear to have retained the normal oxidative and phosphorylative properties although the rate of oxygen uptake was increased. The increase in state 3 rate

probably was due to an increase in the enzymatic activities of the Krebs cycle enzymes. On the other hand, the state 4 rate increase may be due to a slightly altered inner membrane; for instance, one consideration being an increased proton permeability of the inner mitochondrial membrane. This would activate the normally latent ATPase activity which would cause a recycling of ADP. Since fatty acids accumulate in the B,,-deficient condition (2,4) it is possible that some residual fatty acids in intact mitochondria isolated from B,,deprived rats would stimulate latent ATPase activity. During isolation of mitochondria and in the studies of their oxidative and phosphorylative properties, BSA, which was known to remove uncoupling agents by binding, (35,36) was included in the media. Good respiratory control observed in mitochondria from B,,-deprived rats may be due to the protective method of isolation employed in the present study. It is uncertain

MITOCHONDRIAL

ENZYMES

FIG. 3. Two-dimensional gel electrophoretic pattern of the liver mitochondrial (100 PI; protein) obtained from vitamin B,,-deficient rats.

whether these mitochondria would be tightly coupled in vivo where there may be an abnormal accumulation of fatty acids. The increase in vitamin B,,-deprived rat liver mitochondria of citrate synthase, aconitase, and glutamate dehydrogenase activities and the increased “spot” size of their two-dimensional components in the deprived pattern suggest a higher concentration of these enzymes in vitamin B,,-deprived liver mitochondria than in the normal. The two-dimensional patterns also suggest that the effect of vitamin B,, deficiency upon enzyme concentrations in the liver mitochondrial matrix was specific since some protein components increased in “spot” size while others decreased or remained unchanged relative to the normal pattern. It appears from our studies that most of the enzymes of the Krebs cycle were increased in activity except NADP-isocitrate dehydrogenase and a-ketoglutarate dehydrogenase. Direct involvement of NADPisocitrate dehydrogenase in the Krebs cycle

393

IN B,, DEFICIENCY

matrix subfr *action

has not yet been established. Oxygen uptake studies of intact mitochondria with a-ketoglutarate as substrate indicated that a-ketoglutarate dehydrogenase activity was increased in vitamin B,,-deprived rats compared to normal rats (Fig. 1). A similar increase in activity was not observed in the direct assay system for a-ketoglutarate dehydrogenase in sonicated mitochondria. This may be due to the instability of the liver enzyme (37). Nevertheless, another similarly labile enzyme (NAD-isocitrate dehydrogenase) was increased in activity, suggesting that the changes seen were not simply the result of instability, but may reflect a true physiologic status. An increase in the cristae of the inner membrane of mitochondria in livers of vitamin B,,-deficient rats and humans has been demonstrated in this laboratory (7,8). The present study indicates that there is a correlation between the amount of cristae membrane and Krebs cycle enzymes in mitochondria as noted earlier by Srere (38).

394

MATLIB

Although increased citrate synthase activity in B12-deprived rats was demonstrated to be due to the increased rate of synthesis of the enzyme, it remains to be determined if a similar mechanism applies to other enzymes of the Krebs cycle that are increased due to vitamin B,, deprivation. It is of some interest to consider the potential mechanism that underlies the increase in Krebs cycle enzymes which occurs in the presence of cobalamin (vitamin B,,) deficiency. One possibility is that as a result of B,2 deficiency some crucial Krebs cycle intermediate(s) is decreased in the liver and in an attempt to compensate for the decreased energy production additional enzyme synthesis occurs. Alternatively, it is possible that the B,, deficiency results in an increase in some negative effector with the same consequences. The fact that most enzymes of the Krebs cycle are seen to increase in concert seems to indicate, as suggested also by other studies (9) on this enzyme system, that no rate-limiting enzyme exists in this metabolic sequence. Final testing of this hypothesis will require further measurements of all of the Krebs cycle intermediates and potential effecters in the two conditions. REFERENCES 1. CARDINALE, G. J., CARTY, T. J., AND ABELES, R. H. (1970) J. Biol. Chem. 245, 3’771-3’775. 2. FRENKEL, E. P. (1973) J. Clin. Invest. 52, 1237-1245. 3. KISHIMOTO, Y., WILLIAMS, M., MOSER, H. W., HIGNITE, C., AND BIEMANN, K. (1973) J. Lipid. Res. 14, 69-77. 4. FRENKEL, E. P., KITCHENS, R. L., AND JOHNSTON, J. M. (1973) J. Biol. Chem. 248, 75407546. 5. FRENKEL, E. P., AND WHITE, J. E. (1973) Lab. Invest. 29, 614-619. 6. FRENKEL, E. P., KITCHENS, R. L., JOHNSTON, J. M., AND FRENKEL, R. (1974) Arch. Biochem. Biophys. 162, 607-613. 7. FRENKEL, E. P., MUKHERJEE, A., HACKENBROCK, C. R., AND SRERE, P. A. (1976) J. Biol. Chem. 251,2147-2154. 8. MUKHERJEE, A., SRERE, P. A., AND FRENKEL, E. P. (1976) J. Biol. Chem. 251, 2155-2160. 9. PETTE, D., KLINGENBERG, M., AND BUCHER, T. (1962) Biochem. Biophys. Res. Commun. 7, 425-429.

ET AL. 10. SIMON, E. J., AND SHEMIN, D. (1953) J. Amer. Chem. Sot. 75, 2520. 11. FRENKEL, E. P., KELLER, S., AND MCCALL, M. S. (1966) J. Lab. Clin. Med. 85, 487-496. 12. FRENXEL, E. P., MCCALL, M. S., AND WHITE, J. D. (1970)Ame-r. J. Clin. Pathol. 53,891-903. 13. MATLIB, M. A., AND SRERE, P. A. (1976) Arch. Biochem. Biophys. 174, 705-712. 14. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 15. ESTABROOK, R. W. (1967) in Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. 10, pp. 41-47, Academic Press, New York. 16. CHANCE, B., AND WILLIAMS, G. R. (1956) in Advances in Enzymology (Nord, F. F., ed.), Vol. 17, pp. 65-134, Interscience, New York. 17. SRERE, P. A., BRAZIL, H., AND GONEN, L. (1963) Acta Chem. &and. 17, S129-S134. 18. FANSLER, B., AND LOWENSTEIN, J. M. (1969) in Methods in Enzymology (Lowenstein, J., ed.), Vol. 13, pp, 26-30, Academic Press, New York. 19. PLAUT, G. W. E. (1969) in Methods in Enzymology (Lowenstein, J., ed.), Vol. 13, pp. 34-42, Academic Press, New York. 20. CLELAND, W. W., THOMSON, V. W., AND BEARDEN, R. E. (1969) in Methods in Enzymology (Lowenstein, J., ed.), Vol. 13, pp. 30-33, Academic Press, New York. 21. SANADI, D. R. (1969) in Methods in Enzymology (Lowenstein, J., ed.), Vol. 13, pp. 52-55, Academic Press, New York. 22. HILL, R. L., AND BRADSHAW, R. A. (1969) in Methods in Enzymology (Lowenstein, J., ed.), Vol. 13, pp. 91-99, Academic Press, New York. 23. KITTO, G. B. (1969) in Methods in Enzymology (Lowenstein, J., ed.), Vol. 13, pp. 106-116, Academic Press, New York. 24. COHEN, H. J., AND FRIDOVICH, I. (1971) J. Biol. Chem. 246, 359-366. 25. SCHMIDT, E. (1965) in Methods in Enzymatic Analysis (Bergmeyer, H. U., ed.), p. 752, Academic Press, New York. 26. BERGMEYER, H. U. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), p. 976, Academic Press, New York. 27. AMES, B. N., AND DUBIN, D. T. (1960) J. Biol. Chem. 235, 769. 28. VEEGER, C., DER~ARTANIAN, D. V., AND ZEYLEMAKER, W. P. (1969) in Methods in Enzymology(Lowenstein, J., ed.), Vol. 13, pp. 81-90, Academic Press, New York. 29. WOJTCZAK, L., AND ZALLJSKA, H. (1969) Biochim. Biophys. Acta 193, 64-72. 30. SOTTOCASA, G. L., KUYLENSTIERNA, B., ERNSTER, L., AND BERGSTAND, A. (1967) J. Cell. Biol. 32, 415.

MITOCHONDRIAL

ENZYMES

31. BERGMEYER, H. U., GAWEHN, K., KLOTZSCH, H., KREBS, H. A., AND WILLIAMSON, D. H. (1967) J. Biol. Chem. 102, 423-431. 32. O’FARRELL, 4021. 33. HENSLEE,

P. (1975)

J.

Biol. Chem. 250, 4007-

J. G., AND SRERE,

P. A. (1979) J.

Biol.

Chem. 254, 5488-5497. 34. FRENKEL,

E. P.,

Blood 49, 125-138.

AND

KITCHENS,

R. L. (1977)

395

IN B,* DEFICIENCY 35. WEINBACH,

E. C., AND GARBUS,

J. (1965) J.

Biol.

J. (1966)J.

Biol.

Chem. 241, 169-175. 36. WEINBACH,

E. C., ANDGARBUS,

Chem. 241, 3708-3713. 37. LINN, T. (1974) Arch. Biochem. Biophys.

161, 505-574. 38. SRERE, P. A. (1972) in Energy Metabolism and Regulation of Metabolic Processes in Mitochondria (Mehlman, M. A., and Hanson, R. W., eds.), pp. 79-91, Academic Press, New York.