Fish Physiology and Biochemistry vol. 5 no. 1 pp 1-8 (1988)
Kugler Publications, Amsterdam/Berkeley
NAD +-linked isocitrate dehydrogenase in fish tissues Kenneth B. Storey I and Jeremy H . A . Fields 2
l lnstitute o f Biochemistry and Department o f Biology, Carleton University, Ottawa, Ontario, Canada KIS 5B6 2Department of Biology, University of San Diego, San Diego, California 92110, U.S.A.
Abstract
N A D + - l i n k e d isocitrate dehydrogenase was found in the brain, heart, gills, kidney, liver and muscle of trout, and in the liver and muscle of eel. A complex homogenization buffer containing 1 mM A D P , 5 mM MgSO 4, 5 mM citrate and 40~ glycerol is required for retrieval of significant amounts of stable enzyme. The highest activities were found in brain of trout and the lowest in white muscle of trout and eel. The enzyme was partially purified from frozen trout heart to a final activity of 0.04 ~ . M / m i n / m g protein, and the kinetic properties of this partially purified enzyme were studied. The enzyme requires either Mn 2+ or Mg 2+ for activity, higher activities being observed with Mn 2§ . Saturation kinetics for DL-isocitrate were sigmoidal, apparent S0. 5 = 8.2 + 0.6 mM and n H = 1.8 _+ 0.2, in the absence of A D P , changing to hyperbolic, apparent So. 5 = 1.4 _+ 0.3 mM and n H = 1.0, with 1 mM A D P added. Citrate and Ca 2+ were found to activate the enzyme to a small extent. N A D H strongly inhibited the enzyme, 150 = 3.7 + 0.5 ~M. A T P was also found to be an inhibitor, 150 = 7.2 +_ 1.4 mM. These properties are consistent with the role of the enzyme as a m a j o r control site of the tricarboxylic acid cycle.
Introduction
The tricarboxylic acid cycle is the m a j o r aerobic pathway involved in the catabolism of organic compounds to provide metabolic energy to living organisms. Because of its central role in energy metabolism, this cycle is tightly coupled to an organism's metabolic needs and shows appropriate biological control. The control of the tricarboxylic acid cycle during various metabolic transitions is complex, but one enzyme, N A D § isocitrate dehydrogenase (EC 1.1.1.41) ( N A D + - I D H ) , has been shown to be a m a j o r control site for flux
through the cycle. This has been established by studies on oxidative metabolism in isolated mitochondria (LaNoue et al. 1970; Hansford 1975; Hansford and Johnson 1975a, b; Johnson and Hansford 1975). The effects of various metabolites on the flux through the tricarboxylic acid cycle are well correlated with their effects on N A D + - I D H (LaNoue et al. 1970; Hansford 1975; Hansford and Johnson 1975a, b; Johnson and Hansford 1975 ; Gabriel and Ptaut 1984b). Several studies have established that A D P is a potent activator of this enzyme (Chen and Plaut 1963; Goebel and Kilingenberg 1964; Plaut and Aogaichi 1968; Cohen and
Correspondence and reprint requeststo: Dr. K.B. Storey, Department of Biology, Carleton University, Ottawa, Ontario, Canada KIS 5B6.
Colman 1972; Zammit and Newsholme 1976), while citrate and Ca 2+ activate to a lesser extent, and N A D H , N A D P H and A T P inhibit the enzyme (Chen and Plaut 1963; Plaut and Aogaichi 1968; Cohen and Colman 1972; Gabriel and Plaut 1984a, b; Gabriel et al. 1985). The tricarboxylic acid cycle is presumed to be ubiquitous in free living organisms that spend most of their lives in an aerobic environment. It is surprising, therefore, to find occasional claims that this key enzyme is missing. Several workers have a t t e m p t e d to measure the activity of this enzyme in tissues of fish with little success (Moon and Hochachka 1971; Moon and Ouellet 1979). Moon and Ouellet (1979) found that isolated mitochondria from liver of the eel Anguilla rostrata could oxidize added isocitrate and possessed N A D P + linked isocitrate dehydrogenase (EC 1.1.1.42) ( N A D P + - I D H ) as well as an active N A D P H : N A D transhydrogenase (EC 1.6.1.1). These authors concluded, therefore, that the oxidation of isocitrate in mitochondria from eel liver probably proceeded via the N A D P + - I D H and then electrons were transferred to N A D + by the transhydrogenase for entry into the normal electron transfer system. A complete lack of N A D + - I D H in selected species of fish would be exceptional in the animal kingdom, particularly since other vertebrates possess the enzyme, and it has been reported from heart and muscle of trout and dogfish (Alp et al. 1976; McCormack and Denton 1981). Rather than accept the view that fish have made adaptive changes to utilize N A D P + - I D H rather than N A D + - I D H in mitochondrial oxidations, we chose to re-examine the situation with the premise that N A D +IDH is probably present but highly unstable. For this purpose we employed several different homogenization media and assay conditions to search for N A D + - I D H in tissues of trout and eel. Fish N A D + - I D H proved to be a highly unstable enzyme requiring a complex homogenization buffer. However, using a buffer which contained several stabilizers of the enzyme, we found low activities of the enzyme in all tissues tested. We then partially purified N A D + - I D H from trout heart, because this tissue was a m o n g those with the highest activity of the enzyme, and studied its kinetic and regula-
tory properties in order to further understand its role in the energy metabolism of fish tissue.
Materials and methods Live rainbow trout (Sahno gairdneri) and American eels (Anguilla rostrata) were obtained from the Department of Biology, University of Ottawa. Fish were killed by a blow to the head and tissues were rapidly dissected out and stored on ice for a few minutes if used fresh, or frozen and stored at -20~ for future use. Additional trout tissues were obtained frozen from C o m m a n d a n t Properties Ltd., Montebello, P.Q. and kept frozen at - 2 0 ~ for future use in enzyme purification. Biochemicals were obtained from Sigma Chemical Co., St. Louis; all other chemicals were reagent grade.
Buffers used Buffer A: 20 mM imidazole-HCl, pH 7.2, 20 mM 2-mercaptoethanol, 1 mM A D P , 40~ (v/v) glycerol, Buffer B: 20 mM imidazole-HCl, pH 7.2, 20 mM 2-mercaptoethanol, 1 mM A D P , 5 mM citrate, 5 mM MgSO4, Buffer C: 20 mM imidazole-HCl, pH 7.2, 20 mM 2-mercaptoethanol, 1 mM A D P , 5 mM citrate, 5 mM MgSO 4, 4007o (v/v) glycerol, Buffer D: 100 mM Tris-HCl, pH 7.5, 5 mM citrate, 5 mM MgSO4, 2 mM E G T A , 2.8 mM 2-mercaptoethanol, 2 mM E G T A , 1 mM ADP, Buffer E: 20 mM imidazole-HCl, pH 7.5, 5 mM citrate, 5 mM MgSO 4, 2 mM E G T A , 2.8 mM 2-mercaptoethanol, 20O7o (v/v) glycerol.
Preparation of tissue extracts Trout tissues were homogenized in buffer in a ratio of 1:3 w/v and eel tissues 1:5 w / v using a Polytron
3 P T I 0 homogenizer. Three homogenizing buffers (A, B, and C) were tested. Phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor, was added immediately before homogenization (at a final concentration of 2 raM) to all eel tissues and previously frozen tissues, and to fresh trout liver and kidney. H o m o g e n a t e s were centrifuged at 30,000 x g for 20 min at 4~
and used for kinetic analyses without further purification.
Protein assay The concentration of protein in the extracts was measured by Coomassie blue binding (Bradford 1976) using the BioRad Laboratories prepared reagent and bovine g a m m a globulin as the standard.
Assa.v o f en~vme activity Standard assay conditions were 50 mM imidazoleHCI, pH 7.0, 5 mM DL-isocitrate, 2 mM MnCI 2, 1 mM N A D + and 1 mM A D P . The enzyme was assayed at 23~ by measuring the increase in A340 with a Pye Unicam SP 8 - 100 or SP 1800 recording spectrophotometer. Assays were started by the addition of enzyme. One unit of enzyme activity is defined as the amount of enzyme producing 1 micromole N A D H per minute. Affinity constants, S0. 5, were determined from Hill plots. 150 values were determined by the method of Job et al. (1978).
Partial purification o f the enzyme from trout heart Frozen trout hearts were thawed and homogenized (1:5 w/v) in Buffer D with the addition of 2 m m PMSF just before homogenization. The homogenate was centrifuged at 30,000 x g for 20 min at 2~ The supernatant was treated with 0.08 g / m l of polyethylene glycol 8000. The suspension was then stirred on ice for 30 min, then centrifuged at 30,000 x g for 20 rain. The supernatant was then treated with the further addition of polyethylene glycol 8000 to a final level of 0.12 g / m l , stirred on ice and centrifuged as before. The pellet, containing N A D + - I D H , was dissolved in a small volume (about 4 ml) of Buffer E. It was applied to a 4 • 1 cm column of DEAE-Sephadex A-50 that had been equilibrated with Buffer E. N A D + - I D H was then eluted from the column with a 0 - 1 M gradient of KCI in a total volume of 50 ml Buffer E; the elution peak was between about 350 and 500 mM KCI. Fractions containing enzyme activity were pooled
Preparation o f mitochondria Cytosolic and mitochondrial fractions were prepared from trout liver, gill and heart using the method of Ballantyne and Storey (1985).
Results
Activities o f N A D +-IDH in various tissues N A D + - I D H activity was found in all trout tissues examined, although in most instances the activity was very low. No activity was found in eel liver or muscle when the tissue was homogenized in Buffer A or Buffer B, but when Buffer C was used the standard assay conditions produced activities of 0.6 unit/g wet weight in liver and 0.15 unit/g wet weight in white muscle. The data for trout tissues are given in Tabl~ 1. Invariably, when the tissues were homogenized in Buffer A or Buffer B no activity or very low activity was found. When homogenization was in Buffer C, however, all tissues showed substantial NAD + - I D H activity. Previously frozen tissues had lower enzyme activities than fresh tissues in most cases. The standard assay conditions appeared to give optimal activity in most cases. In all tissues, the addition of I mM A D P activated the enzyme; the degree of activation ranged from 80% (trout brain) to 260% (gill and heart). The presence of aconitase in crude preparations could potentially reduce the observed activity of N A D + - I D H through competition for the c o m m o n substrate, isocitrate. Therefore, activities were also measured in the presence
4 Table I. Activities (unit/g wet wt) of N A D §
Tissue
dehydrogenase in fresh and previously frozen trout tissues Fresh
Frozen
t
Gill Liver Kidney Heart j Brain White muscle
Buffer A
Buffer B
Buffer C
Buffer C
0.19 0 n.m, 0.47 n,m, 0.01
0.11 0 n.m. 0.39 n.m. 0
0.25 0.24 0.30 0.99 1.35 0.02
0.17 0.14 0.46 n.m. 0.85 0.01
iAssayed with 35 mM m a g n e s i u m citrate added to the standard assay medium; n.m. = not measured.
Table 2. Purification of N A D " -isocitrate dehydrogenase from trout heart
30,000 x g Supernatant Polyethylene glycol DEAE-Sephadex
Total activity (#M/min)
Yield (%)
Specific activity ( # M / m i n / m g protein)
Fold purification
1.68 1.44 0.41
100 86 24
0.007 0.039 0.041
1 5.6 5.9
of 35 mM Mg.citrate (a 7:1 ratio citrate:isocitrate). Trout heart showed a small increase in N A D § 1DH activity under these conditions, but was the only tissue to do so.
Subcellular distribution o f N A D +- I D H
Crude mitochondrial and cytosolic fractions were prepared from trout heart, liver, and gill. In all three tissues, N A D § was found only in the mitochondrial fraction; activity in the cytosolic fraction was below the sensitivity of the assay. Polytron homogenization in Buffer C released all N A D + - I D H activity to the supernatant fraction; no activity was found in cell debris when centrifuged pellets were re-homogenized in Buffer D, which contains no glycerol.
Kinetic properties o f N A D +-IDH f r o m trout heart
Trout heart N A D + - I D H was purified about 6 fold to a final specific activity of approximately 0.04 u n i t / m g protein. Purification steps are summarized in Table 2. Activities of both aconitase and
N A D P + - I D H were absent from the partially purified preparation. The N A D § preparation was quite unstable in the absence of A D P , losing about 50~ of the total activity in 24 hours. Addition of A D P to a final concentration of 1 mM stabilized the enzyme somewhat, but activity was still diminished by 25~ after 24 hours. The data for substrate and co-factor saturation studies are summarized in Table 3. The enzyme required Mn 2§ or Mg 2§ for activity. In the absence of A D P the maximal activity with either co-factor was approximately equal, although the affinity for Mn 2§ was higher than that for Mg 2§ In the presence of 1 mM A D P the enzyme showed a 5 fold greater affinity for Mn z§ than for Mg 2+ and a higher velocity. A D P activation was more pronounced if Mn 2+ was the co-factor (apparent K a = 0.17 _ 0.025 mM under conditions of 0.5 mM N A D § 5 mM DL-isocitrate, 2 mM MnCI2) than if Mg 2§ was the co-factor (apparent K a = 0.8 + 0.12 mM under conditions of 0.5 mM N A D § 5 mM DLisocitrate, 5 mM MgCI2). In the absence of A D P the saturation kinetics for DL-isocitrate were sigmoidal (n H= 1.8 + 0.2) (Fig. 1), but 1 mM A D P changed this to hyperbolic form and also reduced the apparent S0. 5 for DL-isocitrate approximately
Table 3. Apparent S0. 5 and Hill coefficient, n H, values for substrates and co-factors of trout heart N A D §
isocitrate dehydro-
genase no A D P
plus I mM A D P nH
S0. 5 (mM)
nH
So. 5 (mM)
Substrate NAD +
0.52 + 0.04
DL-isocitrate
8.2
1.0
___ 0.6
0.25 + 0.05
1.0
1.8 • 0.2
1.4
1.0
2.3 _+ 0.5 2.3 _ 0.3
0.23 • 0.07 1.3 • 0.4
_+ 0.3
Co-factor MnCI 2 MgCI 2
0.56 + 0.06 2.1 +_ 0.4
1.0 1.0
Values are means • S.D. for determinations on 3 individual preparations of the partially purified enzyme; conditions: 50 mM imidazole-HCl buffer pH 7.5, 23~ plus: for N A D § saturation, 10 mM DL-isocitrate, 2 mM MnCI2; for isocitrate saturation, 1.0 mM N A D § , 2 mM MnCI2; for MnC12 and MgCI 2 saturation, 1.0 mM N A D §
10 mM DL-isocitrate.
12 1.0
4 %
0,9
x
"T 3 0.6 04
~2
log 02 0 t 210 10 [ DL-isocitrate] rnM
o12
0'4
0'6
0 ' 8 ) i 10 1.2 14 1'6 9/ log [DL-isocitrote]
-02
i
3O -04 -05 -04
9 ./
,b
Fig. 1. a. Enzyme velocity versus concentration of DL-isocitrate for N A D § -IDH partially purified from trout heart. Assay conditions are as in Table 3 with no A D P added; b. Data replotted as a Hill plot.
6 fold (Table 3). The apparent S0. 5 for N A D + was also reduced by about one half by 1 mM ADP. The following metabolites were tested for their effect on N A D + - I D H : citrate, malate, succinate, 2-oxoglutarate, ATP and A M P (all at 5 mM), 0.1 mM N A D H , 0.5 mM CoA and 0.05 mM acetyl CoA. Of these, citrate activated slightly, ATP and N A D H were fairly good inhibitors (about 20~ inhibition by CoA and about 10~ inhibition by 2-oxoglutarate at 1 mM N A D , 10 mM DLisocitrate, 2 mM MnCI 2) in the absence of ADP.
The I50 for ATP was 7.2 + 1.4 mM (conditions I mM N A D +, 10 mM DL-isocitrate, 1 mM ADP, 2 mM MnCI 2) and was unchanged if MgCI 2 (5 mM) was substituted for MnC12, or in the absence of ADP. The apparent S0. 5 for DL-isocitrate was unchanged by the addition of 2 mM ATP in the presence or absence of ADP (conditions 1 mM N A D , 2 mM MnCI2). N A D H was a potent inhibitor of the enzyme. In the absence of ADP the 150 for N A D H was 3.7 + 0.5 /~M (conditions 2 mM MnCI 2, 10 mM DL-isocitrate); 1 mM ADP strong-
ly reversed this inhibition and increased the 150 approximately 10 fold to 32 _+ 2.5 p.M. The activation by citrate ,was weak compared to that by ADP. Citrate (at 5 raM) increased the activity by about 21% (conditions 1 mM NAD +, 10 mM DL-isocitrate, 2 mM MnCI 2) in the presence of 1 mM ADP, and by 50o/0 in the absence of ADP. CaCI 2 was also found to activate trout heart N A D + - I D H . The activation occurred only in the presence of ADP. The greatest degree o f activation was about' 50~ (conditions 1 mM NAD +, 10 mM ,DL-isocitrate, 2 mM MnCI2.) at 0.1 mM CaCI 2.
Discussion
The conflicting reports on the presence and absence o f N A D + - I D H in tissues of fish (Moon and Hochachka 1971; Moon and Ouellet 1979; Alp et al. 1976; Mourika 1983; McCormack and Denton 1981) may well be explained by the inherent instability of the enzyme. The present study demonstrates the presence of N A D + - I D H in several tissues of two species of fish. In all cases the enzyme was quite unstable and required a complex homogenization buffer containing ADP, Mg 2+, citrate and glycerol for stability and retention of enzyme activity for the assays. Thus, the fish enzyme appears to be considerably less stable than the equivalent enzyme from other animal sources. The activities in trout tissues varied considerably, but were generally consistent with the aerobic versus anaerobic nature of tissue function. The brain and heart (very aerobic tissues) had the highest activities, gill, liver and kidney showed intermediate activities, and white muscle, whose function is largely powered by anaerobic glycolysis, had very low activity. These findings are very similar to those of an earlier study on heart and white muscle of this species (Alp et al. 1976). Eel tissues showed a similar result with activity in the liver being greater than that in muscle. Activity of N A D P * - I D H in fish tissues exceeds that of N A D + - I D H in all instances, the ratio N A D P + : N A D + activity in trout tissues ranging from a high of about 100:1 in heart to 20:1 in liver, and 2:1 in brain (Table 1) (Moon and Hochachka
1971 ; Alp et al. 1976). These ratios are considerably higher than equivalent values for other vertebrate species (Alp et al. 1976) and this, plus the earlier inability to detect N A D + - I D H in fish liver (Moon and Hochachka 1971; Moon and Ouellet 1979), lead to the initial proposal that the role of N A D + - I D H in the tricarboxylic acid cycle was replaced in fish tissues by mitochondrial NADP +IDH. The present study, by documenting NAD +IDH activity in trout and eel tissues, suggests that there is no need to propose an altered route of isocitrate oxidation in fish; the tricarboxylic acid cycle, as seen in other animals, is complete. However, the distribution o f N A D P * - I D H in eel liver is intriguing: mitochondrial NADP * - I D H activity exceeds cytoplasmic activity by 3-fold, exactly the opposite to that reported in rat liver (Moon and Ouellet 1979). The adaptive significance of this deserves further attention. Further insight into the functions of N A D * IDH in fish tissues can be obtained from the kinetic and regulatory properties of the enzyme which has not previously been studied in fish. Trout heart N A D + - I D H requires a divalent cation for catalysis. The requirement can be met by Mg 2+ or Mn 2+. The latter was found to be the more effective co-factor; S0. 5 for Mn 2+ was lower than for Mg 2 + and, in the presence of ADP, the enzyme showed greater activity with Mn 2+ than with Mg 2+. ADP increased the affinity for both substrates, and markedly changed the saturation kinetics for DL-isocitrate from sigmoidal to hyperbolic. O f the three activators of trout heart N A D + - I D H , ADP was the most effective. The enzyme from all fish tissues was activated by ADP. The degree of activation varied between tissues of the trout, suggesting that there might be tissuespecific, multiple molecular forms of the enzyme in fish. Citrate and Ca 2+ were not as effective as ADP in activating the enzyme, but it should be noted that Ca 2 + was more effective than citrate in the presence of ADP. Of the inhibitors found, 2-oxoglutarate and CoA would seem to have little physiological relevance because the concentrations required for 50% inhibition would be quite high and beyond the physiological range (Tischler et al. 1977). On the other
h a n d , the strong i n h i b i t i o n by N A D H a n d A T P might be extremely significant in regulating the activity o f the e n z y m e in vivo, a n d this could have very p o t e n t effects on oxidative m e t a b o l i s m in the mitochondria. M i t o c h o n d r i a l oxidative m e t a b o l i s m is activated by A D P a n d Ca 2§ in vitro ( H a n s f o r d 1975; H a n s ford a n d J o h n s o n 1975a, b; J o h n s o n a n d Hansfc~rd 1975; M c C o r m a c k a n d D e n t o n 1984). Both o f these s u b s t a n c e s activate N A D + - I D H by increasing the a f f i n i t y for isocitrate a n d r e d u c i n g the i n h i b i t i o n by N A D H , N A D P H , a n d A T P (Chen and P l a u t 1963; P l a u t a n d Aogaichi 1968; W i l l s o n a n d T i p t o n 1980; M c C o r m a c k a n d D e n t o n 1981; G a b r i e l a n d Plaut 1984; G a b r i e l et aL 1985). This c o m p l e m e n t s the studies o f changes in m i t o c h o n d r i a l m e t a b o l i t e levels which have s h o w n that there is a crossover between isocitrate a n d 2 - o x o g l u t a r a t e d u r i n g metabolic a c t i v a t i o n ( L a N o u e et al. 1970; H a n s f o r d 1974; H a n s f o r d a n d J o h n s o n 1975a, b; J o h n s o n a n d H a n s f o r d 1975). C o n s i d e r i n g that N A D § I D H is f o u n d in low activities in fish tissues as c o m p a r e d to citrate synthase, a n d that is has similar regulatory properties to the m a m m a l i a n e n z y m e , it is p r o b a b l e that N A D + - I D H has the same critical role in the r e g u l a t i o n o f the tricarboxylic acid cycle in fish m i t o c h o n d r i a as it does in m a m m a l i a n systems. F u t u r e studies can n o w a t t e m p t to c o n f i r m this a n d focus o n the c o n t r o l o f m i t o c h o n d r i a l oxidative m e t a b o l i s m by Ca 2+ a n d other regulatory m e c h a n i s m s (e.g. covalent m o d i f i c a t i o n ) acting at the level o f N A D + - I D H .
Acknowledgements T h e a u t h o r s t h a n k Dr. T . W . M o o n , University of O t t a w a for s t i m u l a t i n g their interest in this project a n d for helpful discussions. We t h a n k Dr. M o o n a n d C o m m a n d a n t Properties Ltd., M o n t e b e l l o , P . Q . for p r o v i d i n g fish. S u p p o r t e d by an o p e r a t i n g grant from N . S . E . R . C . C a n a d a to KBS.
References cited Alp, P.R., Newsholme, E.A. and Zammit, V.A. 1976. Activi-
ties of citrate synthase and NAD-linked isocitrate dehydrogenase from vertebrates and invertebrates. Biochem. J. 154: 689-700. Ballantyne, J.S. and Storey, K.B. 1985. Characterization of mitochondria from the freezing tolerant larvae of the gall fly, Eurosta solidaginis: Substrate preferences, salt and pH effects on warm and cold acclimited animals. Can. J. Zool. 63: 373-379. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. Chen, R.F. and Plaut, G.W.E. 1963. Activation and inhibition of DPN-linked isocitrate dehydrogenase of mammalian liver. Biochemistry 2: 1023-1032. Cohen, P.F. and Colman, R.F. 1972. Diphosphopyridine nucleotide dependent isocitrate dehydrogenase from pig heart. Characterization of the active substrates and modes of regulation. Biochemistry 8: 1501-1508. Gabriel, J.L., Milner, R. and Plaut, G.W.E. 1985. Inhibition and activation of bovine heart NAD-specific isocitrate dehydrogenase by ATP. Arch. Biochem. Biophys. 240: 128-134. Gabriel, J.L. and Plaut, G.W.E. 1984a. Citrate activation of NAD-specific isocitrate dehydrogenase from bovine heart. J. Biol. Chem. 259: 1622-1628. Gabriel, J.L. and Plaut, G.W.E. 1984b. Inhibition of bovine heart NAD-specific isocitrate dehydrogenase by reduced pyridine nucleotides: modulation of inhibition by ADP, NAD, Ca2 ~, citrate and isocitrate. Biochemistry 23: 2773-2778. Goebel, H. and Kilingenberg, M. 1964. DPN-spezifische Isocitrate-Dehydrogenase der Mitochondrien. I. Kinetische Eigenschaften, Vorkommen und Funktion der DPN-spezifischen lsocitrate-Dehydrogenase. Biochem. Z. 340: 441-464. Hansford, R.G. 1972. Some properties of pyruvate and 2-oxoglutarate oxidation by blowfly flight-muscle mitochondria. Biochem. J. 127: 271-283. Hansford, R.G. 1972. The control of tricarboxylate-cycle oxidations in the blowfly flight muscle. The steady-state concentrations of coenz,yme A, acetyl-coenzyme A and succinyl-coenzymeA in flight muscle and isolated mitochondria. Biochem. J. 142: 509-519. Hansford, R.G. 1975. The control of tricarbbxylate-cycleoxidations in the blowfly flight muscle. The oxidized and reduced nicotinamide-adenine dinucleotide content of the flight muscle and isolated mitochondria, the adenosine triphosphate and adenosine diphosphate content of mitochondria, and the energy status of the mitochondria during controlled respiration. Biochem. J. 146: 537-547. Hansford, R.G. and Johnson, R.N. 1975a. The nature and control of the tricarboxylate cycle in beetle flight muscle. Biochem. J. 148: 389-401. Hansford, R.G. and Johnson, R.N. 1975b. The steady state concentration of coenzyme A-SH and coenzyme A thioester, citrate and isocitrate during tricarboxylate oxidations in rabbit heart mitochondria. J. Biol. Chem. 251: 8361-8375. Job, D., Cochet, C., Dhein, A. and Chambaz, E.M. 1978. A rapid method for screening inhibitor effects: Determination
of I~ and its standard deviation. Anal. Biochem. 84: 68-77. Johnson, R.N. and Hansford, R.G. 1975. The control of tyicarboxylate-cycle oxidations, in blowfly flight muscle: the steady-state concentrations of citrate, isocitrate, 2-oxoglutarate and malate in flight muscle and isolated mitochondria. Biochem. J. 146: 527-535. LaNoue, K., Nicklas, W.J. and Williamson, J.R. 1970. Control of citric acid cycle activity in rat heart mitochondria. J. Biol. Chem 245:102-111. McCormack, J.G. and Denton, R.M. 1981. A comparative study of the regulation by Ca 2+ of the activation of the 2-oxoglutarate dehydrogenase complex and NAD-isocitrate dehydrogenase from a variety of sources. Biochem. J. 196: 619-624. McCormack, J.G. and Denton, R.M. 1984. Role of Ca 2" ions in the regulation of intramitochondrial metabolism in rat heart. Evidence from studies with isolated mitochondria that adrenaline activates the pyruvate dehydrogenase and 2-oxoglutarate complexes by increasing the intramitochondrial concentration of Ca 2" . Biochem. J. 218: 235-247. Moon, T.W. and Hochach~a, P.W. 1971. Temperature and the kinetic analysis of trout isocitrate dehydrogenases. Comp. Biochem. Physiol. 42B: 725-730. Moon, T.W. and Ouellet, G. 1979. The oxidation of tricarboxylic acid cycle intermediates, with particular reference to
isocitrate, by intact mitochondria isolated from the liver of the American eel, Anguilla rostrata LeSueur. Arch. Biochem. Biophys. 195: 438-452. Mourika, J. 1983. Oxidations in the tricarboxylic acid cycle by intact mitochondria isolated from the lateral red muscle of goldfish (Carassius auratus k.). Effects of anoxia on the oxidation of pyruvate and glutamate. Comp. Biochem. Physiol. 76B: 851-859. Plaut, G.W.E. and Aogaichi, T. 1968. Purification and properties of diphosphopyridine nucleotide-linked isocitrate dehydrogenase of mammalian liver. J. Biol. Chem. 243: 5572-5583. Tischler, M.E., Hecht, P. and Williamson, J.R. 1977. Determination of mitochondrial/cytosolic metabolite gradients in isolated rat liver cells by cell disruption. Arch. Biochem. Biophys. 181: 222-236. Wilson, W.J.C. and Tipton, K.F. 1980. Allosteric properties of ox brain nicotinamide-adenine dinucleotide dependent isocitrate dehydrogenase. J. Neurochem. 34: 793-799. Zammit, V.A. and Newsholme, E.A. 1976. Effects of calcium ions and adenosine diphosphate on the activities of NADlinked isocitrate dehydrogenase from the radular muscles of the whelk and the flight muscles of insects. Biochem. J. 154: 677-687.
Kugler Publications, Amsterdam/Berkeley
NAD +-linked isocitrate dehydrogenase in fish tissues Kenneth B. Storey I and Jeremy H . A . Fields 2
l lnstitute o f Biochemistry and Department o f Biology, Carleton University, Ottawa, Ontario, Canada KIS 5B6 2Department of Biology, University of San Diego, San Diego, California 92110, U.S.A.
Abstract
N A D + - l i n k e d isocitrate dehydrogenase was found in the brain, heart, gills, kidney, liver and muscle of trout, and in the liver and muscle of eel. A complex homogenization buffer containing 1 mM A D P , 5 mM MgSO 4, 5 mM citrate and 40~ glycerol is required for retrieval of significant amounts of stable enzyme. The highest activities were found in brain of trout and the lowest in white muscle of trout and eel. The enzyme was partially purified from frozen trout heart to a final activity of 0.04 ~ . M / m i n / m g protein, and the kinetic properties of this partially purified enzyme were studied. The enzyme requires either Mn 2+ or Mg 2+ for activity, higher activities being observed with Mn 2§ . Saturation kinetics for DL-isocitrate were sigmoidal, apparent S0. 5 = 8.2 + 0.6 mM and n H = 1.8 _+ 0.2, in the absence of A D P , changing to hyperbolic, apparent So. 5 = 1.4 _+ 0.3 mM and n H = 1.0, with 1 mM A D P added. Citrate and Ca 2+ were found to activate the enzyme to a small extent. N A D H strongly inhibited the enzyme, 150 = 3.7 + 0.5 ~M. A T P was also found to be an inhibitor, 150 = 7.2 +_ 1.4 mM. These properties are consistent with the role of the enzyme as a m a j o r control site of the tricarboxylic acid cycle.
Introduction
The tricarboxylic acid cycle is the m a j o r aerobic pathway involved in the catabolism of organic compounds to provide metabolic energy to living organisms. Because of its central role in energy metabolism, this cycle is tightly coupled to an organism's metabolic needs and shows appropriate biological control. The control of the tricarboxylic acid cycle during various metabolic transitions is complex, but one enzyme, N A D § isocitrate dehydrogenase (EC 1.1.1.41) ( N A D + - I D H ) , has been shown to be a m a j o r control site for flux
through the cycle. This has been established by studies on oxidative metabolism in isolated mitochondria (LaNoue et al. 1970; Hansford 1975; Hansford and Johnson 1975a, b; Johnson and Hansford 1975). The effects of various metabolites on the flux through the tricarboxylic acid cycle are well correlated with their effects on N A D + - I D H (LaNoue et al. 1970; Hansford 1975; Hansford and Johnson 1975a, b; Johnson and Hansford 1975 ; Gabriel and Ptaut 1984b). Several studies have established that A D P is a potent activator of this enzyme (Chen and Plaut 1963; Goebel and Kilingenberg 1964; Plaut and Aogaichi 1968; Cohen and
Correspondence and reprint requeststo: Dr. K.B. Storey, Department of Biology, Carleton University, Ottawa, Ontario, Canada KIS 5B6.
Colman 1972; Zammit and Newsholme 1976), while citrate and Ca 2+ activate to a lesser extent, and N A D H , N A D P H and A T P inhibit the enzyme (Chen and Plaut 1963; Plaut and Aogaichi 1968; Cohen and Colman 1972; Gabriel and Plaut 1984a, b; Gabriel et al. 1985). The tricarboxylic acid cycle is presumed to be ubiquitous in free living organisms that spend most of their lives in an aerobic environment. It is surprising, therefore, to find occasional claims that this key enzyme is missing. Several workers have a t t e m p t e d to measure the activity of this enzyme in tissues of fish with little success (Moon and Hochachka 1971; Moon and Ouellet 1979). Moon and Ouellet (1979) found that isolated mitochondria from liver of the eel Anguilla rostrata could oxidize added isocitrate and possessed N A D P + linked isocitrate dehydrogenase (EC 1.1.1.42) ( N A D P + - I D H ) as well as an active N A D P H : N A D transhydrogenase (EC 1.6.1.1). These authors concluded, therefore, that the oxidation of isocitrate in mitochondria from eel liver probably proceeded via the N A D P + - I D H and then electrons were transferred to N A D + by the transhydrogenase for entry into the normal electron transfer system. A complete lack of N A D + - I D H in selected species of fish would be exceptional in the animal kingdom, particularly since other vertebrates possess the enzyme, and it has been reported from heart and muscle of trout and dogfish (Alp et al. 1976; McCormack and Denton 1981). Rather than accept the view that fish have made adaptive changes to utilize N A D P + - I D H rather than N A D + - I D H in mitochondrial oxidations, we chose to re-examine the situation with the premise that N A D +IDH is probably present but highly unstable. For this purpose we employed several different homogenization media and assay conditions to search for N A D + - I D H in tissues of trout and eel. Fish N A D + - I D H proved to be a highly unstable enzyme requiring a complex homogenization buffer. However, using a buffer which contained several stabilizers of the enzyme, we found low activities of the enzyme in all tissues tested. We then partially purified N A D + - I D H from trout heart, because this tissue was a m o n g those with the highest activity of the enzyme, and studied its kinetic and regula-
tory properties in order to further understand its role in the energy metabolism of fish tissue.
Materials and methods Live rainbow trout (Sahno gairdneri) and American eels (Anguilla rostrata) were obtained from the Department of Biology, University of Ottawa. Fish were killed by a blow to the head and tissues were rapidly dissected out and stored on ice for a few minutes if used fresh, or frozen and stored at -20~ for future use. Additional trout tissues were obtained frozen from C o m m a n d a n t Properties Ltd., Montebello, P.Q. and kept frozen at - 2 0 ~ for future use in enzyme purification. Biochemicals were obtained from Sigma Chemical Co., St. Louis; all other chemicals were reagent grade.
Buffers used Buffer A: 20 mM imidazole-HCl, pH 7.2, 20 mM 2-mercaptoethanol, 1 mM A D P , 40~ (v/v) glycerol, Buffer B: 20 mM imidazole-HCl, pH 7.2, 20 mM 2-mercaptoethanol, 1 mM A D P , 5 mM citrate, 5 mM MgSO4, Buffer C: 20 mM imidazole-HCl, pH 7.2, 20 mM 2-mercaptoethanol, 1 mM A D P , 5 mM citrate, 5 mM MgSO 4, 4007o (v/v) glycerol, Buffer D: 100 mM Tris-HCl, pH 7.5, 5 mM citrate, 5 mM MgSO4, 2 mM E G T A , 2.8 mM 2-mercaptoethanol, 2 mM E G T A , 1 mM ADP, Buffer E: 20 mM imidazole-HCl, pH 7.5, 5 mM citrate, 5 mM MgSO 4, 2 mM E G T A , 2.8 mM 2-mercaptoethanol, 20O7o (v/v) glycerol.
Preparation of tissue extracts Trout tissues were homogenized in buffer in a ratio of 1:3 w/v and eel tissues 1:5 w / v using a Polytron
3 P T I 0 homogenizer. Three homogenizing buffers (A, B, and C) were tested. Phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor, was added immediately before homogenization (at a final concentration of 2 raM) to all eel tissues and previously frozen tissues, and to fresh trout liver and kidney. H o m o g e n a t e s were centrifuged at 30,000 x g for 20 min at 4~
and used for kinetic analyses without further purification.
Protein assay The concentration of protein in the extracts was measured by Coomassie blue binding (Bradford 1976) using the BioRad Laboratories prepared reagent and bovine g a m m a globulin as the standard.
Assa.v o f en~vme activity Standard assay conditions were 50 mM imidazoleHCI, pH 7.0, 5 mM DL-isocitrate, 2 mM MnCI 2, 1 mM N A D + and 1 mM A D P . The enzyme was assayed at 23~ by measuring the increase in A340 with a Pye Unicam SP 8 - 100 or SP 1800 recording spectrophotometer. Assays were started by the addition of enzyme. One unit of enzyme activity is defined as the amount of enzyme producing 1 micromole N A D H per minute. Affinity constants, S0. 5, were determined from Hill plots. 150 values were determined by the method of Job et al. (1978).
Partial purification o f the enzyme from trout heart Frozen trout hearts were thawed and homogenized (1:5 w/v) in Buffer D with the addition of 2 m m PMSF just before homogenization. The homogenate was centrifuged at 30,000 x g for 20 min at 2~ The supernatant was treated with 0.08 g / m l of polyethylene glycol 8000. The suspension was then stirred on ice for 30 min, then centrifuged at 30,000 x g for 20 rain. The supernatant was then treated with the further addition of polyethylene glycol 8000 to a final level of 0.12 g / m l , stirred on ice and centrifuged as before. The pellet, containing N A D + - I D H , was dissolved in a small volume (about 4 ml) of Buffer E. It was applied to a 4 • 1 cm column of DEAE-Sephadex A-50 that had been equilibrated with Buffer E. N A D + - I D H was then eluted from the column with a 0 - 1 M gradient of KCI in a total volume of 50 ml Buffer E; the elution peak was between about 350 and 500 mM KCI. Fractions containing enzyme activity were pooled
Preparation o f mitochondria Cytosolic and mitochondrial fractions were prepared from trout liver, gill and heart using the method of Ballantyne and Storey (1985).
Results
Activities o f N A D +-IDH in various tissues N A D + - I D H activity was found in all trout tissues examined, although in most instances the activity was very low. No activity was found in eel liver or muscle when the tissue was homogenized in Buffer A or Buffer B, but when Buffer C was used the standard assay conditions produced activities of 0.6 unit/g wet weight in liver and 0.15 unit/g wet weight in white muscle. The data for trout tissues are given in Tabl~ 1. Invariably, when the tissues were homogenized in Buffer A or Buffer B no activity or very low activity was found. When homogenization was in Buffer C, however, all tissues showed substantial NAD + - I D H activity. Previously frozen tissues had lower enzyme activities than fresh tissues in most cases. The standard assay conditions appeared to give optimal activity in most cases. In all tissues, the addition of I mM A D P activated the enzyme; the degree of activation ranged from 80% (trout brain) to 260% (gill and heart). The presence of aconitase in crude preparations could potentially reduce the observed activity of N A D + - I D H through competition for the c o m m o n substrate, isocitrate. Therefore, activities were also measured in the presence
4 Table I. Activities (unit/g wet wt) of N A D §
Tissue
dehydrogenase in fresh and previously frozen trout tissues Fresh
Frozen
t
Gill Liver Kidney Heart j Brain White muscle
Buffer A
Buffer B
Buffer C
Buffer C
0.19 0 n.m, 0.47 n,m, 0.01
0.11 0 n.m. 0.39 n.m. 0
0.25 0.24 0.30 0.99 1.35 0.02
0.17 0.14 0.46 n.m. 0.85 0.01
iAssayed with 35 mM m a g n e s i u m citrate added to the standard assay medium; n.m. = not measured.
Table 2. Purification of N A D " -isocitrate dehydrogenase from trout heart
30,000 x g Supernatant Polyethylene glycol DEAE-Sephadex
Total activity (#M/min)
Yield (%)
Specific activity ( # M / m i n / m g protein)
Fold purification
1.68 1.44 0.41
100 86 24
0.007 0.039 0.041
1 5.6 5.9
of 35 mM Mg.citrate (a 7:1 ratio citrate:isocitrate). Trout heart showed a small increase in N A D § 1DH activity under these conditions, but was the only tissue to do so.
Subcellular distribution o f N A D +- I D H
Crude mitochondrial and cytosolic fractions were prepared from trout heart, liver, and gill. In all three tissues, N A D § was found only in the mitochondrial fraction; activity in the cytosolic fraction was below the sensitivity of the assay. Polytron homogenization in Buffer C released all N A D + - I D H activity to the supernatant fraction; no activity was found in cell debris when centrifuged pellets were re-homogenized in Buffer D, which contains no glycerol.
Kinetic properties o f N A D +-IDH f r o m trout heart
Trout heart N A D + - I D H was purified about 6 fold to a final specific activity of approximately 0.04 u n i t / m g protein. Purification steps are summarized in Table 2. Activities of both aconitase and
N A D P + - I D H were absent from the partially purified preparation. The N A D § preparation was quite unstable in the absence of A D P , losing about 50~ of the total activity in 24 hours. Addition of A D P to a final concentration of 1 mM stabilized the enzyme somewhat, but activity was still diminished by 25~ after 24 hours. The data for substrate and co-factor saturation studies are summarized in Table 3. The enzyme required Mn 2§ or Mg 2§ for activity. In the absence of A D P the maximal activity with either co-factor was approximately equal, although the affinity for Mn 2§ was higher than that for Mg 2§ In the presence of 1 mM A D P the enzyme showed a 5 fold greater affinity for Mn z§ than for Mg 2+ and a higher velocity. A D P activation was more pronounced if Mn 2+ was the co-factor (apparent K a = 0.17 _ 0.025 mM under conditions of 0.5 mM N A D § 5 mM DL-isocitrate, 2 mM MnCI2) than if Mg 2§ was the co-factor (apparent K a = 0.8 + 0.12 mM under conditions of 0.5 mM N A D § 5 mM DLisocitrate, 5 mM MgCI2). In the absence of A D P the saturation kinetics for DL-isocitrate were sigmoidal (n H= 1.8 + 0.2) (Fig. 1), but 1 mM A D P changed this to hyperbolic form and also reduced the apparent S0. 5 for DL-isocitrate approximately
Table 3. Apparent S0. 5 and Hill coefficient, n H, values for substrates and co-factors of trout heart N A D §
isocitrate dehydro-
genase no A D P
plus I mM A D P nH
S0. 5 (mM)
nH
So. 5 (mM)
Substrate NAD +
0.52 + 0.04
DL-isocitrate
8.2
1.0
___ 0.6
0.25 + 0.05
1.0
1.8 • 0.2
1.4
1.0
2.3 _+ 0.5 2.3 _ 0.3
0.23 • 0.07 1.3 • 0.4
_+ 0.3
Co-factor MnCI 2 MgCI 2
0.56 + 0.06 2.1 +_ 0.4
1.0 1.0
Values are means • S.D. for determinations on 3 individual preparations of the partially purified enzyme; conditions: 50 mM imidazole-HCl buffer pH 7.5, 23~ plus: for N A D § saturation, 10 mM DL-isocitrate, 2 mM MnCI2; for isocitrate saturation, 1.0 mM N A D § , 2 mM MnCI2; for MnC12 and MgCI 2 saturation, 1.0 mM N A D §
10 mM DL-isocitrate.
12 1.0
4 %
0,9
x
"T 3 0.6 04
~2
log 02 0 t 210 10 [ DL-isocitrate] rnM
o12
0'4
0'6
0 ' 8 ) i 10 1.2 14 1'6 9/ log [DL-isocitrote]
-02
i
3O -04 -05 -04
9 ./
,b
Fig. 1. a. Enzyme velocity versus concentration of DL-isocitrate for N A D § -IDH partially purified from trout heart. Assay conditions are as in Table 3 with no A D P added; b. Data replotted as a Hill plot.
6 fold (Table 3). The apparent S0. 5 for N A D + was also reduced by about one half by 1 mM ADP. The following metabolites were tested for their effect on N A D + - I D H : citrate, malate, succinate, 2-oxoglutarate, ATP and A M P (all at 5 mM), 0.1 mM N A D H , 0.5 mM CoA and 0.05 mM acetyl CoA. Of these, citrate activated slightly, ATP and N A D H were fairly good inhibitors (about 20~ inhibition by CoA and about 10~ inhibition by 2-oxoglutarate at 1 mM N A D , 10 mM DLisocitrate, 2 mM MnCI 2) in the absence of ADP.
The I50 for ATP was 7.2 + 1.4 mM (conditions I mM N A D +, 10 mM DL-isocitrate, 1 mM ADP, 2 mM MnCI 2) and was unchanged if MgCI 2 (5 mM) was substituted for MnC12, or in the absence of ADP. The apparent S0. 5 for DL-isocitrate was unchanged by the addition of 2 mM ATP in the presence or absence of ADP (conditions 1 mM N A D , 2 mM MnCI2). N A D H was a potent inhibitor of the enzyme. In the absence of ADP the 150 for N A D H was 3.7 + 0.5 /~M (conditions 2 mM MnCI 2, 10 mM DL-isocitrate); 1 mM ADP strong-
ly reversed this inhibition and increased the 150 approximately 10 fold to 32 _+ 2.5 p.M. The activation by citrate ,was weak compared to that by ADP. Citrate (at 5 raM) increased the activity by about 21% (conditions 1 mM NAD +, 10 mM DL-isocitrate, 2 mM MnCI 2) in the presence of 1 mM ADP, and by 50o/0 in the absence of ADP. CaCI 2 was also found to activate trout heart N A D + - I D H . The activation occurred only in the presence of ADP. The greatest degree o f activation was about' 50~ (conditions 1 mM NAD +, 10 mM ,DL-isocitrate, 2 mM MnCI2.) at 0.1 mM CaCI 2.
Discussion
The conflicting reports on the presence and absence o f N A D + - I D H in tissues of fish (Moon and Hochachka 1971; Moon and Ouellet 1979; Alp et al. 1976; Mourika 1983; McCormack and Denton 1981) may well be explained by the inherent instability of the enzyme. The present study demonstrates the presence of N A D + - I D H in several tissues of two species of fish. In all cases the enzyme was quite unstable and required a complex homogenization buffer containing ADP, Mg 2+, citrate and glycerol for stability and retention of enzyme activity for the assays. Thus, the fish enzyme appears to be considerably less stable than the equivalent enzyme from other animal sources. The activities in trout tissues varied considerably, but were generally consistent with the aerobic versus anaerobic nature of tissue function. The brain and heart (very aerobic tissues) had the highest activities, gill, liver and kidney showed intermediate activities, and white muscle, whose function is largely powered by anaerobic glycolysis, had very low activity. These findings are very similar to those of an earlier study on heart and white muscle of this species (Alp et al. 1976). Eel tissues showed a similar result with activity in the liver being greater than that in muscle. Activity of N A D P * - I D H in fish tissues exceeds that of N A D + - I D H in all instances, the ratio N A D P + : N A D + activity in trout tissues ranging from a high of about 100:1 in heart to 20:1 in liver, and 2:1 in brain (Table 1) (Moon and Hochachka
1971 ; Alp et al. 1976). These ratios are considerably higher than equivalent values for other vertebrate species (Alp et al. 1976) and this, plus the earlier inability to detect N A D + - I D H in fish liver (Moon and Hochachka 1971; Moon and Ouellet 1979), lead to the initial proposal that the role of N A D + - I D H in the tricarboxylic acid cycle was replaced in fish tissues by mitochondrial NADP +IDH. The present study, by documenting NAD +IDH activity in trout and eel tissues, suggests that there is no need to propose an altered route of isocitrate oxidation in fish; the tricarboxylic acid cycle, as seen in other animals, is complete. However, the distribution o f N A D P * - I D H in eel liver is intriguing: mitochondrial NADP * - I D H activity exceeds cytoplasmic activity by 3-fold, exactly the opposite to that reported in rat liver (Moon and Ouellet 1979). The adaptive significance of this deserves further attention. Further insight into the functions of N A D * IDH in fish tissues can be obtained from the kinetic and regulatory properties of the enzyme which has not previously been studied in fish. Trout heart N A D + - I D H requires a divalent cation for catalysis. The requirement can be met by Mg 2+ or Mn 2+. The latter was found to be the more effective co-factor; S0. 5 for Mn 2+ was lower than for Mg 2 + and, in the presence of ADP, the enzyme showed greater activity with Mn 2+ than with Mg 2+. ADP increased the affinity for both substrates, and markedly changed the saturation kinetics for DL-isocitrate from sigmoidal to hyperbolic. O f the three activators of trout heart N A D + - I D H , ADP was the most effective. The enzyme from all fish tissues was activated by ADP. The degree of activation varied between tissues of the trout, suggesting that there might be tissuespecific, multiple molecular forms of the enzyme in fish. Citrate and Ca 2+ were not as effective as ADP in activating the enzyme, but it should be noted that Ca 2 + was more effective than citrate in the presence of ADP. Of the inhibitors found, 2-oxoglutarate and CoA would seem to have little physiological relevance because the concentrations required for 50% inhibition would be quite high and beyond the physiological range (Tischler et al. 1977). On the other
h a n d , the strong i n h i b i t i o n by N A D H a n d A T P might be extremely significant in regulating the activity o f the e n z y m e in vivo, a n d this could have very p o t e n t effects on oxidative m e t a b o l i s m in the mitochondria. M i t o c h o n d r i a l oxidative m e t a b o l i s m is activated by A D P a n d Ca 2§ in vitro ( H a n s f o r d 1975; H a n s ford a n d J o h n s o n 1975a, b; J o h n s o n a n d Hansfc~rd 1975; M c C o r m a c k a n d D e n t o n 1984). Both o f these s u b s t a n c e s activate N A D + - I D H by increasing the a f f i n i t y for isocitrate a n d r e d u c i n g the i n h i b i t i o n by N A D H , N A D P H , a n d A T P (Chen and P l a u t 1963; P l a u t a n d Aogaichi 1968; W i l l s o n a n d T i p t o n 1980; M c C o r m a c k a n d D e n t o n 1981; G a b r i e l a n d Plaut 1984; G a b r i e l et aL 1985). This c o m p l e m e n t s the studies o f changes in m i t o c h o n d r i a l m e t a b o l i t e levels which have s h o w n that there is a crossover between isocitrate a n d 2 - o x o g l u t a r a t e d u r i n g metabolic a c t i v a t i o n ( L a N o u e et al. 1970; H a n s f o r d 1974; H a n s f o r d a n d J o h n s o n 1975a, b; J o h n s o n a n d H a n s f o r d 1975). C o n s i d e r i n g that N A D § I D H is f o u n d in low activities in fish tissues as c o m p a r e d to citrate synthase, a n d that is has similar regulatory properties to the m a m m a l i a n e n z y m e , it is p r o b a b l e that N A D + - I D H has the same critical role in the r e g u l a t i o n o f the tricarboxylic acid cycle in fish m i t o c h o n d r i a as it does in m a m m a l i a n systems. F u t u r e studies can n o w a t t e m p t to c o n f i r m this a n d focus o n the c o n t r o l o f m i t o c h o n d r i a l oxidative m e t a b o l i s m by Ca 2+ a n d other regulatory m e c h a n i s m s (e.g. covalent m o d i f i c a t i o n ) acting at the level o f N A D + - I D H .
Acknowledgements T h e a u t h o r s t h a n k Dr. T . W . M o o n , University of O t t a w a for s t i m u l a t i n g their interest in this project a n d for helpful discussions. We t h a n k Dr. M o o n a n d C o m m a n d a n t Properties Ltd., M o n t e b e l l o , P . Q . for p r o v i d i n g fish. S u p p o r t e d by an o p e r a t i n g grant from N . S . E . R . C . C a n a d a to KBS.
References cited Alp, P.R., Newsholme, E.A. and Zammit, V.A. 1976. Activi-
ties of citrate synthase and NAD-linked isocitrate dehydrogenase from vertebrates and invertebrates. Biochem. J. 154: 689-700. Ballantyne, J.S. and Storey, K.B. 1985. Characterization of mitochondria from the freezing tolerant larvae of the gall fly, Eurosta solidaginis: Substrate preferences, salt and pH effects on warm and cold acclimited animals. Can. J. Zool. 63: 373-379. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. Chen, R.F. and Plaut, G.W.E. 1963. Activation and inhibition of DPN-linked isocitrate dehydrogenase of mammalian liver. Biochemistry 2: 1023-1032. Cohen, P.F. and Colman, R.F. 1972. Diphosphopyridine nucleotide dependent isocitrate dehydrogenase from pig heart. Characterization of the active substrates and modes of regulation. Biochemistry 8: 1501-1508. Gabriel, J.L., Milner, R. and Plaut, G.W.E. 1985. Inhibition and activation of bovine heart NAD-specific isocitrate dehydrogenase by ATP. Arch. Biochem. Biophys. 240: 128-134. Gabriel, J.L. and Plaut, G.W.E. 1984a. Citrate activation of NAD-specific isocitrate dehydrogenase from bovine heart. J. Biol. Chem. 259: 1622-1628. Gabriel, J.L. and Plaut, G.W.E. 1984b. Inhibition of bovine heart NAD-specific isocitrate dehydrogenase by reduced pyridine nucleotides: modulation of inhibition by ADP, NAD, Ca2 ~, citrate and isocitrate. Biochemistry 23: 2773-2778. Goebel, H. and Kilingenberg, M. 1964. DPN-spezifische Isocitrate-Dehydrogenase der Mitochondrien. I. Kinetische Eigenschaften, Vorkommen und Funktion der DPN-spezifischen lsocitrate-Dehydrogenase. Biochem. Z. 340: 441-464. Hansford, R.G. 1972. Some properties of pyruvate and 2-oxoglutarate oxidation by blowfly flight-muscle mitochondria. Biochem. J. 127: 271-283. Hansford, R.G. 1972. The control of tricarboxylate-cycle oxidations in the blowfly flight muscle. The steady-state concentrations of coenz,yme A, acetyl-coenzyme A and succinyl-coenzymeA in flight muscle and isolated mitochondria. Biochem. J. 142: 509-519. Hansford, R.G. 1975. The control of tricarbbxylate-cycleoxidations in the blowfly flight muscle. The oxidized and reduced nicotinamide-adenine dinucleotide content of the flight muscle and isolated mitochondria, the adenosine triphosphate and adenosine diphosphate content of mitochondria, and the energy status of the mitochondria during controlled respiration. Biochem. J. 146: 537-547. Hansford, R.G. and Johnson, R.N. 1975a. The nature and control of the tricarboxylate cycle in beetle flight muscle. Biochem. J. 148: 389-401. Hansford, R.G. and Johnson, R.N. 1975b. The steady state concentration of coenzyme A-SH and coenzyme A thioester, citrate and isocitrate during tricarboxylate oxidations in rabbit heart mitochondria. J. Biol. Chem. 251: 8361-8375. Job, D., Cochet, C., Dhein, A. and Chambaz, E.M. 1978. A rapid method for screening inhibitor effects: Determination
of I~ and its standard deviation. Anal. Biochem. 84: 68-77. Johnson, R.N. and Hansford, R.G. 1975. The control of tyicarboxylate-cycle oxidations, in blowfly flight muscle: the steady-state concentrations of citrate, isocitrate, 2-oxoglutarate and malate in flight muscle and isolated mitochondria. Biochem. J. 146: 527-535. LaNoue, K., Nicklas, W.J. and Williamson, J.R. 1970. Control of citric acid cycle activity in rat heart mitochondria. J. Biol. Chem 245:102-111. McCormack, J.G. and Denton, R.M. 1981. A comparative study of the regulation by Ca 2+ of the activation of the 2-oxoglutarate dehydrogenase complex and NAD-isocitrate dehydrogenase from a variety of sources. Biochem. J. 196: 619-624. McCormack, J.G. and Denton, R.M. 1984. Role of Ca 2" ions in the regulation of intramitochondrial metabolism in rat heart. Evidence from studies with isolated mitochondria that adrenaline activates the pyruvate dehydrogenase and 2-oxoglutarate complexes by increasing the intramitochondrial concentration of Ca 2" . Biochem. J. 218: 235-247. Moon, T.W. and Hochach~a, P.W. 1971. Temperature and the kinetic analysis of trout isocitrate dehydrogenases. Comp. Biochem. Physiol. 42B: 725-730. Moon, T.W. and Ouellet, G. 1979. The oxidation of tricarboxylic acid cycle intermediates, with particular reference to
isocitrate, by intact mitochondria isolated from the liver of the American eel, Anguilla rostrata LeSueur. Arch. Biochem. Biophys. 195: 438-452. Mourika, J. 1983. Oxidations in the tricarboxylic acid cycle by intact mitochondria isolated from the lateral red muscle of goldfish (Carassius auratus k.). Effects of anoxia on the oxidation of pyruvate and glutamate. Comp. Biochem. Physiol. 76B: 851-859. Plaut, G.W.E. and Aogaichi, T. 1968. Purification and properties of diphosphopyridine nucleotide-linked isocitrate dehydrogenase of mammalian liver. J. Biol. Chem. 243: 5572-5583. Tischler, M.E., Hecht, P. and Williamson, J.R. 1977. Determination of mitochondrial/cytosolic metabolite gradients in isolated rat liver cells by cell disruption. Arch. Biochem. Biophys. 181: 222-236. Wilson, W.J.C. and Tipton, K.F. 1980. Allosteric properties of ox brain nicotinamide-adenine dinucleotide dependent isocitrate dehydrogenase. J. Neurochem. 34: 793-799. Zammit, V.A. and Newsholme, E.A. 1976. Effects of calcium ions and adenosine diphosphate on the activities of NADlinked isocitrate dehydrogenase from the radular muscles of the whelk and the flight muscles of insects. Biochem. J. 154: 677-687.
