Oyster citrate synthase: control of carbon entry into the Krebs cycle of a facultative anaerobe
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J. H. A. FIELDS A N D P. W. HOCHACHKA Deparrment of Zoology. University of British Columbia, Vancouver, B.C., Canada V6T I W5 Received December 10, 1975 FIELDS, J. H. A., and P. W. HOCHACHKA. 1976. Oyster citrate synthase: control of carbon entry into the Krebs cycle of a facultative anaerobe. Can. J. Zool. 54: 892-895. Citrate synthase (EC 4.1.3.7) in adductor muscle of the oyster, Crassostrea gigas, occurred in relatively low specific activity, about 1.5 pmol product formed per minute per gram wet weight of tissue. The enzyme activity was essentially independent of pH between pH 7.5 and 9.0. The K , values for acetyl-CoA and oxaloacetate were about 0.005 mM in each case. Catalytic activity was modulated by the adenylates, citrate, and 2-ketoglutarate. all of which were inhibitory. The regulatory properties of the enzyme suggest that during the transition to anoxia oxaloacetate becomes limiting, thus reducing flux through the initial stages of the Krebs cycle. 1976. Oyster citrate synthase: control of carbon entry FIELDS,J . H. A., et P. W. HOCHACHKA. into the Krebs cycle of a facultative anaerobe. Can. J. Zool. 54: 892-895. La citrate-synthetase (EC 4.1.3.7) du muscle adducteur de I'huitre, Crassostrea gigas, se manifeste avec une activite specifique relativement faible, environ 1.5 pmol de produit forme par minute par gramme de poids frais de tissu. L'activitt enzymatique est essentiellement indtpendante du pH entre les valeurs de pH 7.5 et 9.0. Les valeurs de K , pour I'acttyl-CoA et I'oxaloacCtate sont de 0.005 mM dans les deux cas. L'activite catalytique est modulee par les adenylates, le citrate et le 2-cetoglutarate, tous des inhibiteurs. Les proprittes rkgulatrices de l'enzyme permettent de croire que, durant la transition vers I'anoxie, I'oxaloac0tate devient facteur limitant, reduisant ainsi le flux dans les premiers stades du cycle de Krebs. [Traduit par le journal]
Introduction The property of facultative anaerobiosis is displayed by a number of marine invertebrates including bivalve molluscs such as the oyster, Crassostrea gigas. In the evolutionary design of their metabolism, lactate dehydrogenase (EC 1.1.1.27) is usually deleted, and lactate, therefore, is rarely seen as an anaerobic end product. In its stead, compounds such as alanine and succinate accumulate as end products of a simultaneous carbohydrate and amino acid fermentation (Hochachka et al. 1973; Hochachka and Mustafa 1972; Saz 1971). Although the Krebs cycle is apparently functional during aerobic metabolism (Hammen 1969), during anoxia the Krebs cycle reactions appear to be disconnected, probably at the level of oxaloacetate formation and utilization (Mustafa and Hochachka 1973a, 19733). Net flow of carbon in one arm of the cycle is reversed, yielding a sequence (oxaloacetate -+ malate fumarate -+ succinate) that contributes to succinate accumulation during anoxia. The other arm of the Krebs cycle, initiated by citrate synthase (EC 4.1.3.7), -+
either is fully turned off, as for example, in the isolated, anoxic oyster heart (Collicutt 1975), or is held at a reduced activity, supplying ciketoglutarate for a small but significant glutamate synthesis from glucose, which occurs during prolonged anoxia (De Zwaan and van Marrewijk 1973). Citrate synthase, catalyzing the reaction oxaloacetate + acetyl-CoA -+ citrate + CoA,' clearly is positioned at a pivotal locus in such a metabolic organization and we, therefore, addressed ourselves to the question of how it is regulated in the oyster adductor muscle.
Materials and Methods Oysters were purchased from a local seafood supplier and stored in running recirculated seawater until required. Diethylaminoethyl (DEAE) Sephadex was a product of Pharmacia Fine Chemicals; hydroxylapatite was obtained from Bio Rad Laboratories, Richmond, California. Acetyl-CoA was purchased from P-L Biochemicals; all other biochemicals were obtained from Sigma. Other chemicals used were reagent grade. 'ABBREVIATIONS USED: COA, coenzyme A ; DTNB,
5,5'-dithiobis-(2-nitrobenzoic acid); MDH, malate dehydrogenase (EC 1.1.1.37); Tris, tris(hydroxymethy1)aminomethane.
FIELDS AND HOCHACHKA
893
Results and Discussion Spec$c Activity Oyster adductor muscle has a very low activity of citrate synthase, there being about 1.5 units/g wet weight (assayed in fresh homogenates prepared from healthy organisms). Thus the tissue has a very low potential for aerobic metabolism, and this is reflected by the low numbers of mitochondria present (Hanson and Lowy 1961).
Protein Determination Protein concentrations were determined spectrophotometrically at 280 and 260 nm, using the formula below (Layne 1957).
Effect of pH Shortly after shell closure in Crassostrea gigas, the pH of the mantle fluid drops to about 6.5 and then continues to decline slowly to values as low as 5.4 under prolonged anoxia. Intracellular pH presumably follows a similar pattern and has led to the suggestion that a decreasing pH is itself an important controlling element in the aerobicanaerobic transition (Hochachka and Mustafa 1972). For this reason, it was important at the outset to determine the pH dependence of the reaction. Unlike the mammalian enzymes studied thus far (Srere 1974), oyster adductor muscle citrate synthase is essentially pH independent between 7.5 and 9.0, which are the practical limits of the DTNB-assay system (Srere 1969), a result which reduces the possibility of pH having a significant role in regulating carbon flow at this metabolic branch point. Since the assay prevented detailed studies down to the pH 6.0 range, no definitive conclusion here can be made. It was decided to characterize the enzyme at pH 8.0 to facilitate direct comparison with studies on citrate synthase from other sources.
Can. J. Zool. Downloaded from www.nrcresearchpress.com by University of Otago on 01/06/15 For personal use only.
Enzyme Assay All assays were performed at 25 "C using a Unicam SP 1800 UV spectrophotometer equipped with a constanttemperature cell holder. The enzyme was assayed by following the production of CoA with DTNB, according to the method of Srere (1969). Routine assays were performed with 0.05 mM acetyl-CoA, 0.05 m M oxaloacetate, 0.25 m M DTNB in 50 mM Tris HCI, pH 8.0, at 25 "C. One unit of enzyme activity was defined as the amount catalyzing the formation of l pmol CoA/min at 25 "C.
Protein (mg/ml) = 1.55 ODz8,, - 0.76 ODz6,,.
Purification of Enzyme All procedures were performed at 0-4 "C. Adductor muscle (10 g) was dissected free of surrounding tissues, blotted dry with filter paper, and homogenized in 5 volumes of 0.1 M sodium phosphate buffer, pH 7.4. The suspension was centrifuged at 20 000 g for 20 min, the resulting supernatant set aside, the pellet rehomogenized in a further 4 volumes of the same buffer, and recentrifuged as above. The supernatants were combined, this solution being the crude homogenate. Ammonium sulphate 0.209 g/ml was slowly added with continuous stirring to the crude homogenate, the suspension centrifuged at 20 OOOg for 30 min, and the pellet discarded. The supernatant was then treated with a further 0.2 g/ml of ammonium sulphate, centrifuged, and the supernatant discarded. The pellet obtained was dissolved in a minimal volume of 5 m M sodium phosphate buffer, pH 7.4, dialyzed against two changes of 1 litre of the same buffer for 2 h each, and applied to a 10 x 1.6 cm column of DEAE Sephadex A-50 previously equilibrated with 5 m M sodium phosphate, pH 7.4. A linear gradient of 5-100 m M sodium phosphate, pH 7.4, was applied to the column, the enzyme being eluted as a single peak. Fractions containing enzyme activity were pooled and applied to a 10 x 1.6 cm column of hydroxylapatite in 20 m M sodium phosphate, pH 7.4, washed with the same buffer, and then washed with 80 mM sodium phosphate, pH 7.4, until MDH activity had dropped to a low level. The column was then washed with 100 m M sodium phosphate buffer, fractions of 2 ml being collected in the final wash. Fractions containing activity of 0.2 units/ml, or greater, were pooled and this partially purified enzyme was used for most of the kinetic studies. This procedure usually yielded an enzyme with a specific activity of 6.7 units/mg protein and contained a small amount of MDH. To remove this MDH, a 10-ml aliquot was dialyzed against 2 litres of 10 m M imidazole HCI buffer, pH 7.2, (nicotinamide and applied to an agarose-NAD adenine dinucleotide) affinity column (P-L Biochemicals). Fractions containing citrate synthase but no measurable MDH were collected and used to study NADH (reduced NADt) inhibition of citrate synthase. +
Substrate Afinities Like most animal citrate synthases thus far studied, the oyster adductor enzyme follows normal Michaelis-Menten kinetics for both substrates over the concentration ranges studied (3-60 pM for acetyl-CoA; 3-50 p M for oxaloacetate). The K,,, for acetyl-CoA is 5.5 p M and for oxaloacetate it is 5.0 pM. The values for both acetyl-CoA and oxaloacetate are similar to those reported for other citrate synthases (Srere 1974). Regulation by Adenylates As with other citrate synthases, the adenylates are potential inhibitors of adductor muscle citrate synthase (Table 1). Adenosine triphosphate (ATP) is the most effective (Ki= 0.5 mM),
CAN. J. ZOOL. VOL. 54, 1976
TABLE1 . Effects of various inhibitors on citrate synthase Type of inhibition
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Inhibitor ATP ADP AMP MgATP NADPH NADH Na2S04 MgSO, Citrate 2-ketoalutarate
Vs. acetyl-CoA
Vs. oxaloacetate
K,,m M
Competitive Competitive Competitive Competitive Competitive Competitive Competitive Competitive Mixed Mixed
Noncompetitive Noncompetitive Noncompetitive
0.5 1.6 3.5 1.1 0.9 2.0 9.7 1.7 3.0 6.0
Mixed Mixed
FIG.1 . Effects of MgSO,, ATP, and MgATP on acetyl-CoA saturation kinetics, 11 V versus 11s plot. A control; 2 mM MgSO,; 1 m M ATP or 2.0 m M MgATP; A 2 mM ATP. Concentration of oxaloacetate 0.05 mM.
while adenosine monophosphate (AMP) is the least effective ( K , = 3.5 mM). The inhibition in all cases is competitive with respect to acetylCoA and noncompetitive with respect to oxaloacetate. Inhibition by ATP can be greatly reduced, but not abolished by the addition of MgSO, (Fig. l), as previously shown for the pig heart enzyme (Kosicki and Lee 1966). It should be noted, however, that MgSO, and Na,SO, are also inhibitory, being competitive with respect to acetyl-CoA, as previously shown for mammalian enzymes. MgSO, has a lower Kithan Na2S04, presumably as a result of interactions of the cation Mg2+ with the polyphosphate group of acetyl-CoA.
citrate synthase is strongly inhibited by NADH and this has also been found to be the case for the enzyme from squid mantle muscle (Hochachka et al. 1975). During anoxia, one would anticipate large alterations in the NADH/NADf ratio, which theoretically could provide a sensitive signal for reducing citrate synthase activity during anoxia. It is, therefore, particularly interesting that the adductor enzyme is quite refractory to both NADH and NAD' at physiological concentrations. In fact, NADt at a concentration of 2 m M had no effect, while the K i for NADH was found to be 2 mM, which is clearly higher than the levels expected under the most severe conditions.
Redox Regulation In some bacteria (Johnson and Hanson 1974)
Metabolite Regulation Of a large series of amino, dicarboxylic, and
FIELDS AND HOCHACHKA
Can. J. Zool. Downloaded from www.nrcresearchpress.com by University of Otago on 01/06/15 For personal use only.
tricarboxylic acids tested, only citrate and 2ketoglutarate were found to have any significant effect on citrate synthase activity. Both the latter compounds yield a mixed inhibition pattern with respect to oxaloacetate and acetyl-CoA. It is presumed that these compounds are in fact competitive with oxaloacetate, but the presence of counter ions gives an overall mixed pattern, as has been shown for mammalian enzymes (Srere 1974). Citrate and Zketoglutarate are both metabolized further in the Krebs cycle, or can be used for other biosynthetic purposes; hence, their effects on citrate synthase can be viewed as classical negative feedback control of an earlier step involved in their formation.
Functional Signijicance Perhaps the most surprising outcome of this study is how 'normal' the oyster adductor citrate synthase appears to be. Like other animal citrate synthases, its activity would appear to be controlled by the combination of three factors, namely availability of substrate, 'energy charge' of the cell, and the levels of citrate and 2-ketoglutarate (Srere 1974). For an aerobic system this arrangement would be entirely satisfactory, increasing citrate synthase activity when the energy charge is low or when citrate and 2ketoglutarate are being used. However, in a facultative anaerobe, these conditions probably also exist during anoxia, and potentiating the citrate synthase activity under these conditions would be extremely disadvantageous because it would lead to activity of a basically aerobic system during anaerobic conditions. Furthermore, as pointed out earlier, oxaloacetate is channelled primarily towards succinate during anoxia, making it even more necessary to curtail citrate synthase activity. The most probable point at which control is achieved is the availability of oxaloacetate, which is known to be significant in regulating mammalian citrate synthase in vivo (Olson and Williamson 1971). Because the primary source of oxaloacetate is malate, it is highly probable that a reduction in mitochondrial MDH activity is the method used for 'disconnecting' the oxaloacetate + 2-ketoglutarate arm of the Krebs cycle. Indeed, if oyster adductor muscle mitochondria1 MDH is as
895
sensitive to NADH inhibition as the cytoplasmic isoenzyme has been shown to be (Fields and Hochachka, in preparation), then the levels of oxaloacetate would be very closely regulated by the NADH/NAD+ ratio, and this would preclude the necessity for citrate synthase to be regulated directly by the NADH/NAD+ ratio. COLLICUTT, J. 1975. Anaerobic metabolism in the isolated oyster heart. M.Sc. Thesis, University of British Columbia, Vancouver. DE ZWAAN,A., and J. A. VAN MARREWIJK.1973. Anaerobic glucose degradation in the sea mussel My rilus edulis L. Comp. Biochem. Physiol. 44B: 429439. HAMMEN,C. S. 1969. Metabolism of the oyster, Crassostrea virginica. Am. Zool. 9: 309-318. HANSON,J., and J. LOWY. 1961. The structure of the muscle fibres in the translucent part of the oyster Crassosrrea angulata. Proc. R. Soc. London, 154B: 173-193. HOCHACHKA, P. W., J. H. A. FIELDS,and T. MUSTAFA. 1973. Animal life without oxygen: basic biochemical mechanisms. Am. Zool. 13: 543-555. HOCHACHKA, P. W., and T. MUSTAFA.1972. Invertebrate facultative anaerobiosis. Science, 178: 10561060. P. W., K. B. STOREY,and J. BALDWIN. HOCHACHKA, 1975. Squid muscle citrate synthase: control of carbon entry into the Krebs cycle. Comp. Biochem. Physiol. 52B: 193-199. 1974. Bacterial citrate JOHNSON, D. E., and R. S. HANSON. synthase: purification, molecular weight and kinetic mechanisms. Biochim. Biophys. Acta, 350: 336-353. KOSICKI. G. W., and L. P. K. LEE. 1966. Effect of divalent metal ions on nucleotide inhibition of pig heart citrate synthase. J. Biol. Chem. 241: 3571-3574. LAYNE,E. 1957. Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymol. 3: 447454. MUSTAFA, T., and P. W. HOCHACHKA. 1973a. Enzymes in facultative anaerobiosis of molluscs. 11. Basic catalytic properties of phosphoenolpyruvate carboxykinase in oyster adductor muscle. Comp. Biochem. Physiol. 45B: 639-656. 19736. Enzymes in facultative anaerobiosis of molluscs. 111. Phosphoenolpyruvate carboxykinase and its role in the aerobic-anaerobic transition. Comp. Biochem. Physiol. 45B: 657-668. 1971. Regulation of OLSON,M. S., and J. R. WILLIAMSON. citrate synthesis in isolated rat liver mitochondria. J. Biol. Chem. 246: 77947803. SAZ, H. J. 1971. Facultative anaerobiosis in the invertebrates: pathways and control systems. Am. Zool. 11: 125-135. SRERE,P. A. 1969. Citrate synthase. Methods Enzymol. 13: 3-16. 1974. Controls of citrate synthase activity. Life Sci. 15: 1695-1710.
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Can. J. Zool. Downloaded from www.nrcresearchpress.com by University of Otago on 01/06/15 For personal use only.
J. H. A. FIELDS A N D P. W. HOCHACHKA Deparrment of Zoology. University of British Columbia, Vancouver, B.C., Canada V6T I W5 Received December 10, 1975 FIELDS, J. H. A., and P. W. HOCHACHKA. 1976. Oyster citrate synthase: control of carbon entry into the Krebs cycle of a facultative anaerobe. Can. J. Zool. 54: 892-895. Citrate synthase (EC 4.1.3.7) in adductor muscle of the oyster, Crassostrea gigas, occurred in relatively low specific activity, about 1.5 pmol product formed per minute per gram wet weight of tissue. The enzyme activity was essentially independent of pH between pH 7.5 and 9.0. The K , values for acetyl-CoA and oxaloacetate were about 0.005 mM in each case. Catalytic activity was modulated by the adenylates, citrate, and 2-ketoglutarate. all of which were inhibitory. The regulatory properties of the enzyme suggest that during the transition to anoxia oxaloacetate becomes limiting, thus reducing flux through the initial stages of the Krebs cycle. 1976. Oyster citrate synthase: control of carbon entry FIELDS,J . H. A., et P. W. HOCHACHKA. into the Krebs cycle of a facultative anaerobe. Can. J. Zool. 54: 892-895. La citrate-synthetase (EC 4.1.3.7) du muscle adducteur de I'huitre, Crassostrea gigas, se manifeste avec une activite specifique relativement faible, environ 1.5 pmol de produit forme par minute par gramme de poids frais de tissu. L'activitt enzymatique est essentiellement indtpendante du pH entre les valeurs de pH 7.5 et 9.0. Les valeurs de K , pour I'acttyl-CoA et I'oxaloacCtate sont de 0.005 mM dans les deux cas. L'activite catalytique est modulee par les adenylates, le citrate et le 2-cetoglutarate, tous des inhibiteurs. Les proprittes rkgulatrices de l'enzyme permettent de croire que, durant la transition vers I'anoxie, I'oxaloac0tate devient facteur limitant, reduisant ainsi le flux dans les premiers stades du cycle de Krebs. [Traduit par le journal]
Introduction The property of facultative anaerobiosis is displayed by a number of marine invertebrates including bivalve molluscs such as the oyster, Crassostrea gigas. In the evolutionary design of their metabolism, lactate dehydrogenase (EC 1.1.1.27) is usually deleted, and lactate, therefore, is rarely seen as an anaerobic end product. In its stead, compounds such as alanine and succinate accumulate as end products of a simultaneous carbohydrate and amino acid fermentation (Hochachka et al. 1973; Hochachka and Mustafa 1972; Saz 1971). Although the Krebs cycle is apparently functional during aerobic metabolism (Hammen 1969), during anoxia the Krebs cycle reactions appear to be disconnected, probably at the level of oxaloacetate formation and utilization (Mustafa and Hochachka 1973a, 19733). Net flow of carbon in one arm of the cycle is reversed, yielding a sequence (oxaloacetate -+ malate fumarate -+ succinate) that contributes to succinate accumulation during anoxia. The other arm of the Krebs cycle, initiated by citrate synthase (EC 4.1.3.7), -+
either is fully turned off, as for example, in the isolated, anoxic oyster heart (Collicutt 1975), or is held at a reduced activity, supplying ciketoglutarate for a small but significant glutamate synthesis from glucose, which occurs during prolonged anoxia (De Zwaan and van Marrewijk 1973). Citrate synthase, catalyzing the reaction oxaloacetate + acetyl-CoA -+ citrate + CoA,' clearly is positioned at a pivotal locus in such a metabolic organization and we, therefore, addressed ourselves to the question of how it is regulated in the oyster adductor muscle.
Materials and Methods Oysters were purchased from a local seafood supplier and stored in running recirculated seawater until required. Diethylaminoethyl (DEAE) Sephadex was a product of Pharmacia Fine Chemicals; hydroxylapatite was obtained from Bio Rad Laboratories, Richmond, California. Acetyl-CoA was purchased from P-L Biochemicals; all other biochemicals were obtained from Sigma. Other chemicals used were reagent grade. 'ABBREVIATIONS USED: COA, coenzyme A ; DTNB,
5,5'-dithiobis-(2-nitrobenzoic acid); MDH, malate dehydrogenase (EC 1.1.1.37); Tris, tris(hydroxymethy1)aminomethane.
FIELDS AND HOCHACHKA
893
Results and Discussion Spec$c Activity Oyster adductor muscle has a very low activity of citrate synthase, there being about 1.5 units/g wet weight (assayed in fresh homogenates prepared from healthy organisms). Thus the tissue has a very low potential for aerobic metabolism, and this is reflected by the low numbers of mitochondria present (Hanson and Lowy 1961).
Protein Determination Protein concentrations were determined spectrophotometrically at 280 and 260 nm, using the formula below (Layne 1957).
Effect of pH Shortly after shell closure in Crassostrea gigas, the pH of the mantle fluid drops to about 6.5 and then continues to decline slowly to values as low as 5.4 under prolonged anoxia. Intracellular pH presumably follows a similar pattern and has led to the suggestion that a decreasing pH is itself an important controlling element in the aerobicanaerobic transition (Hochachka and Mustafa 1972). For this reason, it was important at the outset to determine the pH dependence of the reaction. Unlike the mammalian enzymes studied thus far (Srere 1974), oyster adductor muscle citrate synthase is essentially pH independent between 7.5 and 9.0, which are the practical limits of the DTNB-assay system (Srere 1969), a result which reduces the possibility of pH having a significant role in regulating carbon flow at this metabolic branch point. Since the assay prevented detailed studies down to the pH 6.0 range, no definitive conclusion here can be made. It was decided to characterize the enzyme at pH 8.0 to facilitate direct comparison with studies on citrate synthase from other sources.
Can. J. Zool. Downloaded from www.nrcresearchpress.com by University of Otago on 01/06/15 For personal use only.
Enzyme Assay All assays were performed at 25 "C using a Unicam SP 1800 UV spectrophotometer equipped with a constanttemperature cell holder. The enzyme was assayed by following the production of CoA with DTNB, according to the method of Srere (1969). Routine assays were performed with 0.05 mM acetyl-CoA, 0.05 m M oxaloacetate, 0.25 m M DTNB in 50 mM Tris HCI, pH 8.0, at 25 "C. One unit of enzyme activity was defined as the amount catalyzing the formation of l pmol CoA/min at 25 "C.
Protein (mg/ml) = 1.55 ODz8,, - 0.76 ODz6,,.
Purification of Enzyme All procedures were performed at 0-4 "C. Adductor muscle (10 g) was dissected free of surrounding tissues, blotted dry with filter paper, and homogenized in 5 volumes of 0.1 M sodium phosphate buffer, pH 7.4. The suspension was centrifuged at 20 000 g for 20 min, the resulting supernatant set aside, the pellet rehomogenized in a further 4 volumes of the same buffer, and recentrifuged as above. The supernatants were combined, this solution being the crude homogenate. Ammonium sulphate 0.209 g/ml was slowly added with continuous stirring to the crude homogenate, the suspension centrifuged at 20 OOOg for 30 min, and the pellet discarded. The supernatant was then treated with a further 0.2 g/ml of ammonium sulphate, centrifuged, and the supernatant discarded. The pellet obtained was dissolved in a minimal volume of 5 m M sodium phosphate buffer, pH 7.4, dialyzed against two changes of 1 litre of the same buffer for 2 h each, and applied to a 10 x 1.6 cm column of DEAE Sephadex A-50 previously equilibrated with 5 m M sodium phosphate, pH 7.4. A linear gradient of 5-100 m M sodium phosphate, pH 7.4, was applied to the column, the enzyme being eluted as a single peak. Fractions containing enzyme activity were pooled and applied to a 10 x 1.6 cm column of hydroxylapatite in 20 m M sodium phosphate, pH 7.4, washed with the same buffer, and then washed with 80 mM sodium phosphate, pH 7.4, until MDH activity had dropped to a low level. The column was then washed with 100 m M sodium phosphate buffer, fractions of 2 ml being collected in the final wash. Fractions containing activity of 0.2 units/ml, or greater, were pooled and this partially purified enzyme was used for most of the kinetic studies. This procedure usually yielded an enzyme with a specific activity of 6.7 units/mg protein and contained a small amount of MDH. To remove this MDH, a 10-ml aliquot was dialyzed against 2 litres of 10 m M imidazole HCI buffer, pH 7.2, (nicotinamide and applied to an agarose-NAD adenine dinucleotide) affinity column (P-L Biochemicals). Fractions containing citrate synthase but no measurable MDH were collected and used to study NADH (reduced NADt) inhibition of citrate synthase. +
Substrate Afinities Like most animal citrate synthases thus far studied, the oyster adductor enzyme follows normal Michaelis-Menten kinetics for both substrates over the concentration ranges studied (3-60 pM for acetyl-CoA; 3-50 p M for oxaloacetate). The K,,, for acetyl-CoA is 5.5 p M and for oxaloacetate it is 5.0 pM. The values for both acetyl-CoA and oxaloacetate are similar to those reported for other citrate synthases (Srere 1974). Regulation by Adenylates As with other citrate synthases, the adenylates are potential inhibitors of adductor muscle citrate synthase (Table 1). Adenosine triphosphate (ATP) is the most effective (Ki= 0.5 mM),
CAN. J. ZOOL. VOL. 54, 1976
TABLE1 . Effects of various inhibitors on citrate synthase Type of inhibition
Can. J. Zool. Downloaded from www.nrcresearchpress.com by University of Otago on 01/06/15 For personal use only.
Inhibitor ATP ADP AMP MgATP NADPH NADH Na2S04 MgSO, Citrate 2-ketoalutarate
Vs. acetyl-CoA
Vs. oxaloacetate
K,,m M
Competitive Competitive Competitive Competitive Competitive Competitive Competitive Competitive Mixed Mixed
Noncompetitive Noncompetitive Noncompetitive
0.5 1.6 3.5 1.1 0.9 2.0 9.7 1.7 3.0 6.0
Mixed Mixed
FIG.1 . Effects of MgSO,, ATP, and MgATP on acetyl-CoA saturation kinetics, 11 V versus 11s plot. A control; 2 mM MgSO,; 1 m M ATP or 2.0 m M MgATP; A 2 mM ATP. Concentration of oxaloacetate 0.05 mM.
while adenosine monophosphate (AMP) is the least effective ( K , = 3.5 mM). The inhibition in all cases is competitive with respect to acetylCoA and noncompetitive with respect to oxaloacetate. Inhibition by ATP can be greatly reduced, but not abolished by the addition of MgSO, (Fig. l), as previously shown for the pig heart enzyme (Kosicki and Lee 1966). It should be noted, however, that MgSO, and Na,SO, are also inhibitory, being competitive with respect to acetyl-CoA, as previously shown for mammalian enzymes. MgSO, has a lower Kithan Na2S04, presumably as a result of interactions of the cation Mg2+ with the polyphosphate group of acetyl-CoA.
citrate synthase is strongly inhibited by NADH and this has also been found to be the case for the enzyme from squid mantle muscle (Hochachka et al. 1975). During anoxia, one would anticipate large alterations in the NADH/NADf ratio, which theoretically could provide a sensitive signal for reducing citrate synthase activity during anoxia. It is, therefore, particularly interesting that the adductor enzyme is quite refractory to both NADH and NAD' at physiological concentrations. In fact, NADt at a concentration of 2 m M had no effect, while the K i for NADH was found to be 2 mM, which is clearly higher than the levels expected under the most severe conditions.
Redox Regulation In some bacteria (Johnson and Hanson 1974)
Metabolite Regulation Of a large series of amino, dicarboxylic, and
FIELDS AND HOCHACHKA
Can. J. Zool. Downloaded from www.nrcresearchpress.com by University of Otago on 01/06/15 For personal use only.
tricarboxylic acids tested, only citrate and 2ketoglutarate were found to have any significant effect on citrate synthase activity. Both the latter compounds yield a mixed inhibition pattern with respect to oxaloacetate and acetyl-CoA. It is presumed that these compounds are in fact competitive with oxaloacetate, but the presence of counter ions gives an overall mixed pattern, as has been shown for mammalian enzymes (Srere 1974). Citrate and Zketoglutarate are both metabolized further in the Krebs cycle, or can be used for other biosynthetic purposes; hence, their effects on citrate synthase can be viewed as classical negative feedback control of an earlier step involved in their formation.
Functional Signijicance Perhaps the most surprising outcome of this study is how 'normal' the oyster adductor citrate synthase appears to be. Like other animal citrate synthases, its activity would appear to be controlled by the combination of three factors, namely availability of substrate, 'energy charge' of the cell, and the levels of citrate and 2-ketoglutarate (Srere 1974). For an aerobic system this arrangement would be entirely satisfactory, increasing citrate synthase activity when the energy charge is low or when citrate and 2ketoglutarate are being used. However, in a facultative anaerobe, these conditions probably also exist during anoxia, and potentiating the citrate synthase activity under these conditions would be extremely disadvantageous because it would lead to activity of a basically aerobic system during anaerobic conditions. Furthermore, as pointed out earlier, oxaloacetate is channelled primarily towards succinate during anoxia, making it even more necessary to curtail citrate synthase activity. The most probable point at which control is achieved is the availability of oxaloacetate, which is known to be significant in regulating mammalian citrate synthase in vivo (Olson and Williamson 1971). Because the primary source of oxaloacetate is malate, it is highly probable that a reduction in mitochondrial MDH activity is the method used for 'disconnecting' the oxaloacetate + 2-ketoglutarate arm of the Krebs cycle. Indeed, if oyster adductor muscle mitochondria1 MDH is as
895
sensitive to NADH inhibition as the cytoplasmic isoenzyme has been shown to be (Fields and Hochachka, in preparation), then the levels of oxaloacetate would be very closely regulated by the NADH/NAD+ ratio, and this would preclude the necessity for citrate synthase to be regulated directly by the NADH/NAD+ ratio. COLLICUTT, J. 1975. Anaerobic metabolism in the isolated oyster heart. M.Sc. Thesis, University of British Columbia, Vancouver. DE ZWAAN,A., and J. A. VAN MARREWIJK.1973. Anaerobic glucose degradation in the sea mussel My rilus edulis L. Comp. Biochem. Physiol. 44B: 429439. HAMMEN,C. S. 1969. Metabolism of the oyster, Crassostrea virginica. Am. Zool. 9: 309-318. HANSON,J., and J. LOWY. 1961. The structure of the muscle fibres in the translucent part of the oyster Crassosrrea angulata. Proc. R. Soc. London, 154B: 173-193. HOCHACHKA, P. W., J. H. A. FIELDS,and T. MUSTAFA. 1973. Animal life without oxygen: basic biochemical mechanisms. Am. Zool. 13: 543-555. HOCHACHKA, P. W., and T. MUSTAFA.1972. Invertebrate facultative anaerobiosis. Science, 178: 10561060. P. W., K. B. STOREY,and J. BALDWIN. HOCHACHKA, 1975. Squid muscle citrate synthase: control of carbon entry into the Krebs cycle. Comp. Biochem. Physiol. 52B: 193-199. 1974. Bacterial citrate JOHNSON, D. E., and R. S. HANSON. synthase: purification, molecular weight and kinetic mechanisms. Biochim. Biophys. Acta, 350: 336-353. KOSICKI. G. W., and L. P. K. LEE. 1966. Effect of divalent metal ions on nucleotide inhibition of pig heart citrate synthase. J. Biol. Chem. 241: 3571-3574. LAYNE,E. 1957. Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymol. 3: 447454. MUSTAFA, T., and P. W. HOCHACHKA. 1973a. Enzymes in facultative anaerobiosis of molluscs. 11. Basic catalytic properties of phosphoenolpyruvate carboxykinase in oyster adductor muscle. Comp. Biochem. Physiol. 45B: 639-656. 19736. Enzymes in facultative anaerobiosis of molluscs. 111. Phosphoenolpyruvate carboxykinase and its role in the aerobic-anaerobic transition. Comp. Biochem. Physiol. 45B: 657-668. 1971. Regulation of OLSON,M. S., and J. R. WILLIAMSON. citrate synthesis in isolated rat liver mitochondria. J. Biol. Chem. 246: 77947803. SAZ, H. J. 1971. Facultative anaerobiosis in the invertebrates: pathways and control systems. Am. Zool. 11: 125-135. SRERE,P. A. 1969. Citrate synthase. Methods Enzymol. 13: 3-16. 1974. Controls of citrate synthase activity. Life Sci. 15: 1695-1710.
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