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The effect of calcium ions on subcellular localization of aldolase–FBPase complex in skeletal muscle
FEBS 29340
FEBS Letters 579 (2005) 1607–1612
The effect of calcium ions on subcellular localization of aldolase–FBPase complex in skeletal muscle Piotr Mamczura, Dariusz Rakusa, Agnieszka Gizaka, Danuta Dusb, Andrzej Dzugaja,* a
Department of Animal Physiology, Institute of Zoology, Wroclaw University, Cybulskiego 30, 50-205 Wroclaw, Poland Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Science, Wroclaw, Poland
b
Received 4 November 2004; revised 25 January 2005; accepted 28 January 2005 Available online 14 February 2005 Edited by Amy McGough
Abstract In skeletal muscles, FBPase–aldolase complex is located on a-actinin of the Z-line. In the present paper, we show evidence that stability of the complex is regulated by calcium ions. Real time interaction analysis, confocal microscopy and the protein exchange method have revealed that elevated calcium concentration decreases association constant of FBPase–aldolase and FBPase-a-actinin complex, causes fast dissociation of FBPase from the Z-line and slow accumulation of aldolase within the I-band and M-line. Therefore, the release of Ca2+ during muscle contraction might result, simultaneously, in the inhibition of glyconeogenesis and in the acceleration of glycolysis. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: FBPase; Aldolase; Calcium; a-Actinin; Glyconeogenesis; Co-localization
1. Introduction Muscle fructose 1,6-bisphosphatase (FBPase) [EC 3.1.3.11], a key enzyme of glyconeogenesis, catalyzes the irreversible reaction of hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. Muscle aldolase [EC 4.1.2.13] catalyzes the reversible reaction of synthesis of fructose 1,6-bisphosphate from glyceraldehyde 3-phosphate and dihydroxyacetone phosphate and, in glyconeogenesis, supplies the substrate for FBPase. Muscle FBPase is very sensitive to AMP inhibition and in the presence of physiological concentrations of this effector in muscle fibres FBPase should be almost completely inactive [1,2]. Recently, we have presented evidence that, in vitro, muscle aldolase binds to muscle FBPase and the binding results in dramatic changes in allosteric properties of the latter enzyme, desensitizing it towards AMP inhibition [2,3] and enabling the intermediate channeling between the enzymes [4]. Investigating subcellular localization of the two enzymes we have found that, in striated muscles, FBPase and aldolase colocalize on the Z-line (with the strong binding of the former en* Corresponding author. Fax: +48 71 328 82 46. E-mail address: [email protected] (A. Dzugaj).
Abbreviations: FBPase, fructose 1,6-bisphosphatase; FITC, fluorescein isothiocyanate; TRITC, rhodamine isothiocyanate; EGTA, ethylene glycol-bis(b-aminoethyl ether)-N,N,N 0 ,N 0 -tetraacetic acid; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; PEG, polyethylene glycol; HEPES, N-[2-hydroxyethyl]piperazine-N 0 [2-ethanesulfonic acid]; DTT, dithiothreitol
zyme to a-actinin) [5] and we have postulated the existence of glyconeogenic metabolon around the Z-line [5]. Structural separation of glyconeogenesis and glycolysis (the glycolytic complex is known to be located on actin [6–8]) may protect the muscle cell against the loss of the energy via futile cycles. Calcium is the main regulator of muscle contraction [9,10]. It also affects the number of other cellular activities including the metabolism of carbohydrates [7,11–13]. Quite recently, we have found that calcium is a potent inhibitor muscle FBPase and, in cardiomyocytes, causes dissociation of the enzyme from the Z-line [14]. The primary aim of the present study was to investigate the effect of calcium ions on the stability, activity and subcellular localization of aldolase–FBPase complex. In the running report, the evidence is presented that calcium decreases affinity of FBPase to aldolase and a-actinin and causes disintegration of aldolase–FBPase complex.
2. Materials and methods Phosphocellulose P-11 was purchased from Whatman (England), ethanolamine was from Merck (Germany), and BIAcore sensor chip CM5 was from Biacore AB (Sweden). Alexa Fluor 546 anti-mouse antibodies were from Molecular Probes (USA). Other reagents were from Sigma (USA). 2.1. Enzyme purification and activity determination Rabbit muscle FBPase and aldolase were purified according to [1] and [15], respectively. The activities of FBPase and aldolase were determined as described by Rakus and Dzugaj [1] and Rakus et al. [16], respectively. The concentration of the enzymes was determined spectrophotometrically and the purity of aldolase and FBPase was checked by 10% SDS–PAGE [17]. The effect of calcium ions on the velocity of the reaction catalyzed by free FBPase and aldolase–FBPase complex was measured as was described previously [3,14]. Spectrophotometric measurements were performed with an Agilent 8453 diode array spectrophotometer. 2.2. Fluorescent labeling Fluorescently labeled FBPase (FITC-FBPase) and aldolase (TRITC-aldolase) were obtained by modification of the proteins with fluorescein isothiocyanate (FITC) and tetramethyl-rhodamine isothiocyanate (TRITC), respectively, as described by Goding [18]. The lack of proteolysis of fluorescently labeled proteins was checked by 10% SDS–PAGE [17]. The number of fluorochrome molecules conjugated to the enzymes was estimated spectrophotometrically. 2.3. The protein exchange Single skeletal muscle fibres were prepared as described previously [8]. The stabilization of the striation pattern of the skinned muscle fibres was achieved as described by Brenner et al. [19]. The protein
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.01.071
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exchange method, previously described by Kraft et al. [8] and Rakus et al. [5], was used to localize and co-localize FBPase and aldolase. Before the incubation with muscle fibres, both enzymes were dialyzed against relaxing buffer (10 mM imidazol, 2 mM MgCl2, 1 mM EGTA, 20 mM creatine phosphate, 2 mM DTT, 106 mM potassium propionate; pH 7.0, t = 4 C). In localization experiments, the skinned muscle fibres were incubated overnight with FITC-FBPase (0.2 mg/ml), or with TRITC-aldolase (1 mg/ml) for 4 h. To co-localize both enzymes, the muscle fibres were incubated overnight with FITC-FBPase (0.2 mg/ml) and subsequently with TRITC-aldolase (1 mg/ml) for 4 h. To interpret the achieved striation pattern, skinned fibres were incubated with anti-a-actinin antibodies, secondary antibodies labeled with Alexa Fluor 546 and then with FITC-FBPase (Fig. 1D–F). To test the effect of calcium ions on localization and co-localization of aldolase and FBPase, the fibres containing associated fluorescently labeled proteins were transferred into the activating solution (10 mM imidazol, 0.1 mM CaCl2, 2 mM MgCl2, 20 mM creatine phosphate, 2 mM DTT, 106 mM potassium propionate; pH 7.0, t = 4 C). All fibres were mounted on microscopic slides and examined by Confocal microscopy. The fluorescent images were obtained with a Bio-Rad MRC 1024ES confocal scanner unit and Zeiss Axiovert S100 inverted microscope. The degree of co-localization, expressed in PearsonÕs correlation coefficient, which calculates the proportion of all red intensities that have green components among all red intensities, was assessed by the colocalization analysis function in the AutoVisualise/AutoDeblur v 9.3 free 30-day demo software (AutoQuant Imaging Inc.).
ibrated with the running buffer (10 mM HEPES, 100 mM KCl, 1 mM DTT, 2 mM MgCl2; pH 7.4, t = 25 C) until a stable baseline was obtained. The level of immobilization was 4426 and 3079 RU for aldolase and a-actinin, respectively. All binding experiments were carried out at 25 C with a constant flow rate of 10 ll/min in the running buffer. To determine the affinity of FBPase to a-actinin and aldolase, 50 ll aliquots of FBPase (270, 675 and 1350 nM) in the running buffer in the absence and in the presence of 10 lM CaCl2 or 100 lM CaCl2 were injected over the aldolase-immobilized and a-actinin-immobilized sensor chip, followed by a 3-min washing with the running buffer supplemented with appropriate concentration of calcium. The binding constants (KA) were determined both by global fitting of the sensograms to a binding models using BIAevaluation 3.1 software (Pharmacia Biosensor AB) as well as by equilibrium binding analysis with GraFit 4.0.12 program (Erithacus Software Ltd., R.J. Leatherbarrow). The global analysis of the interaction between FBPase and a-actinin was performed using a single-site binding model with drifting base-line. The best fitted model describing the interaction between FBPase and aldolase accounts for the existence of one binding site on the surface of the FBPase molecule and two conformational states of the complex [5]. The equilibrium binding constants were obtained by non-linear regression analysis using GraFit 4.0.12 program and the following equation:
2.4. Effect of calcium on aldolase and FBPase release from subcellular structures of muscle fibres Skeletal muscles dissected from a freshly killed rabbit were homogenized in the buffer containing: 20 mM Tris, 0.4 mM EDTA, 1 mM PMSF (pH 7.4, t = 4 C) in the absence and in the presence of 5% PEG 8000 as well as in the presence of 5% PEG 8000 and 0.01 or 0.1 mM CaCl2. The ratio of tissue to buffer (wt/vol) was 1:2. Homogenates were centrifuged at 25 000 · g for 20 min at 4 C and the supernatants were used for the determination of aldolase and FBPase activity [1,16]. The pellets were homogenized in 10 volumes of homogenization buffer with 0.5% Triton X-100, incubated for 30 min on ice, centrifuged at 25 000 · g for 20 min at 4 C and the obtained supernatants were tested for the enzyme activity.
where Req is response in resonance units (RU) at equilibrium, C is the FBPase concentration, Rmax is RU at maximum and KA is the binding constant.
2.5. Real-time interaction analysis by BIAcore The binding of FBPase to immobilized a-actinin and to immobilized aldolase was measured using BIAcore 1000 (Biacore AB, Sweden). The sensor chips CM5 with carboxymethylated dextran matrix were used throughout all experiments. The immobilization of a-actinin was described previously [5]. The immobilization of aldolase was carried out using the standard EDC/NHS chemical procedure for the activation of a working channel. A 5 ll solution of aldolase (0.1 mg/ml in 10 mM acetate buffer; pH 6.5, flow rate 5 ll/min) was injected over the activated sensor surface, followed by a 50 ll solution of 1 M ethanolamine to deactivate the unreacted NHS-esters. The chip was equil-
Req ¼ ðRmax CÞ=ð1=K A þ CÞ
3. Results Fluorescent labeling of the enzymes revealed that FBPase monomer bound on average three molecules of FITC, whereas two molecules of TRITC were bound with aldolase monomer. Although the labeling strongly decreased the specific activity of both enzymes, the modification did not significantly reduce the affinity of these enzymes to each other as well as of FBPase to a-actinin [5]. The protein exchange experiments revealed that FITCFBPase is located on both sides of the Z-line (Fig. 1A) and TRITC-aldolase accumulates within the I-band and around the M-line (Fig. 2A). The same striation pattern for the both enzymes as well as for their co-localization (Fig. 3A) was achieved previously [5]. In control experiments, where muscle fibres were incubated with the respective fluorochrome alone, no staining was observed (data not shown). Co-localization
Fig. 1. The effect of calcium ions on localization of FITC-FBPase: in the absence of Ca2+ (A); after 1 min of incubation with 10 lM Ca2+ (B) and 100 lM Ca2+ (C). Co-localization (D) of FBPase (E) and a-actinin (F) in muscle fibre. Bar: 5 lm.
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Fig. 2. The effect of calcium ions on localization of TRITC-aldolase: in the absence of Ca2+ (A); after 5 min (B), 10 min (C) and 20 min (D) of incubation with 10 lM Ca2+; after 5 min (E), 10 min (F) and 20 min (G) of incubation with 100 lM Ca2+. Bar: 5 lm.
of red and green signals was assessed by the co-localization analysis function in the AutoVisualise/AutoDeblur software. The degree of co-localization, expressed in PearsonÕs correlation coefficient [20] was 0.73, what indicates high degree of the co-localization (Fig. 4A). The addition of 10 and 100 lM calcium to the skinned muscle fibres with bound FITC-FBPase resulted in fast and complete dissociation of the enzyme from the Z-line (Fig. 1B and C). The reverse effect exerted calcium on the localization of aldolase: 10 and 100 lM CaCl2 caused the slow accumulation of TRITC-aldolase in the I-band and around the M-line (Fig. 2B–G). The same effect of Ca2+ as rapid dissociation of FITCFBPase from the Z-line and slow accumulation of TRITCaldolase within the I-band and on the M-line was observed in the co-localization experiment (Fig. 3B and C). In the presence of calcium ions the PearsonÕs correlation coefficient was 0.14, what indicates no co-localization of the proteins [20] (Fig. 4B and C).
The global fitting of the sensograms revealed the binding constant for aldolase–FBPase equal to 8.56 · 106 M1 (Fig. 5A) and for FBPase-a-actinin equal to 1.01 · 107 M1 (Fig. 6A). Similar values of KA were obtained when the data was analyzed by equilibrium binding analysis (Figs. 5A and 6A) and these values are in the range of KA reported previously [3,5,21]. The association of FBPase to aldolase and a-actinin was strongly affected by calcium ions, and 10 lM Ca2+ decreased the affinity of FBPase to aldolase and to a-actinin about 4–5-fold and 10-fold, respectively (Figs. 5A–C and 6A–C). The release of FBPase and aldolase from subcellular structures of muscle was dependent on the presence of the crowding agent imitating the physiological conditions – PEG 8000 (Table 1). The amount of both enzymes associated with structures of muscle cells was about 6–7 times higher in the presence of 5% PEG 8000 than in its absence (Table 1). An addition of 100 lM CaCl2 to the homogenization buffer only slightly affected the amount of FBPase and aldolase associated with
Fig. 3. The effect of calcium ions on co-localization of FITC-FBPase and TRITC-aldolase: in the absence of Ca2+ (A); after 10 min of incubation with 10 lM Ca2+ (B); after 10 min of incubation with 100 lM Ca2+ (C). Bar: 5 lm.
Fig. 4. The graphical representation of the PearsonÕs correlation coefficient showing high degree of the FBPase and aldolase co-localization (yellow points) in the absence of Ca2+ (A); no co-localization in the presence of 10 lM (B) or 100 lM Ca2+ (C). Bar: 5 lm.
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Fig. 5. (A) The binding constants between FBPase and aldolase in the absence and in the presence of calcium ions as determined by the global fitting of the sensograms and the equilibrium binding analysis. (B) Representative sensograms and fitted curves showing Ca2+-dependent binding of FBPase (675 nM) to aldolase. (C) The equilibrium binding analysis of the effect of calcium ions on the affinity of FBPase to aldolase.
subcellular structures (Table 1). On the other hand, an addition of calcium to the homogenization buffer containing PEG 8000 significantly decreased the amount of FBPase associated with subcellular structures and increased the amount of the associated aldolase (Table 1). Ca2+ inhibited the activity of muscle FBPase with I0.5 equal to 0.48 lM (Fig. 7) [14], whereas the inhibition of the activity of aldolase–FBPase complex was much weaker and an apparent inhibitory constant was about 24 lM (Fig. 7). The relative activity of aldolase–FBPase complex in the presence of calcium ions (Fig. 7) roughly correlated with the amount of the aldolase–FBPase complex under the experimental conditions (calculated with Eq. (1)). Thus, it is possible that calcium decreases the concentration of the complex and, as a result, it can inhibit FBPase activity in a classic manner. qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðK A ½AþK A ½F þ1Þ ðK A ½AþK A ½F þ1Þ2 4K 2A ½A½F ½AF ¼ ; 2K A ð1Þ where [AF] – concentration of aldolase–FBPase complex, [F] – total concentration of FBPase (7.15 · 107 M), [A] – total con-
centration of aldolase (6.25 · 1010 M), KA – association constant between aldolase and FBPase (Fig. 5A–C).
4. Discussion For years it was a common belief that lactate produced in a contracting muscle is transported via the blood stream to the liver where is converted to glucose, which subsequently is transported to the muscle again (Cori cycle). In last few years evidence has been accumulated that, in the skeletal muscle, up to 50% of lactate is converted to glycogen (glyconeogenesis) [22]. In such a pathway aldolase as well as FBPase are indispensable. Aldolase not only directly supplies substrate to FBPase [4], but also interacting with FBPase desensitizes it towards AMP inhibition [1,3]. Glucose is the main source of energy for muscle fibres and its level therein is regulated not solely by hormones but also by intracellular calcium. Calcium is involved in the regulation of glycogenolysis by activating glycogen phosphorylase which subsequently causes the release of glucose from glycogen for ATP generation [23,24]. Calcium also activates, in an insulinindependent manner, GLUT4-dependent entrance of glucose
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Fig. 6. (A) The binding constants between FBPase and a-actinin in the absence and in the presence of calcium ions as determined by the global fitting of the sensograms and the equilibrium binding analysis. (B) Representative sensograms and fitted curves showing Ca2+-dependent binding of FBPase (675 nM) to a-actinin. (C) The equilibrium binding analysis of the effect of calcium ions on the affinity of FBPase to a-actinin.
into muscle fibres [25] and accelerates glycolysis affecting binding of glycolytic enzymes to subcellular structures [7,26]. On the other hand, calcium ions are presumed to inhibit glyconeogenesis: Ca2+ is the strong inhibitor of muscle FBPase and during the muscle contraction an elevated level of calcium should almost completely inhibit the enzyme [14]. Moreover, Table 1 The effect of calcium on aldolase and FBPase release from subcellular structures of muscle fibres Buffer
FBPase associated with subcellular structures (%)
Aldolase associated with subcellular structures (%)
Without crowding agent Without crowding agent Ca2+ – 100 lM 5% PEG 5% PEG Ca2+ – 10 lM 5% PEG Ca2+ – 100 lM
7.3 ± 3.8 5.9 ± 2.7
8.5 ± 2.4 11.7 ± 4.1
47 ± 8.6 38 ± 7.4 17 ± 3.2
35 ± 7.9 39 ± 4.2 56 ± 9.2
Each value represents the mean and S.D. of triplicate independent experiments.
Fig. 7. The inhibition of free FBPase (full circles, dashed line) and aldolase–FBPase complex (empty circles, solid line) activity by calcium ions. Black squares represent the saturation of aldolase with FBPase in the presence of 10 and 100 lM Ca2+, as calculated according to Eq. (1).
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in cardiomyocytes, Ca2+ causes dissociation of FBPase from the Z-line [14] disintegrating the hypothetical glyconeogenic metabolon. In the present paper, we have reported that an increased concentration of calcium decreases both the affinity of FBPase to aldolase (Fig. 5) and to a-actinin (Fig. 6). This destabilizatory effect of Ca2+ results in the release of muscle FBPase from the multienzymatic complex located around the Z-line (Table 1, Figs. 1 and 3) and free form of the enzyme is strongly inhibited both by calcium ions (Fig. 7) and AMP [1,3]. Simultaneous to the inhibition of glyconeogenesis, the increased concentration of calcium causes the accumulation of aldolase within I-band (Figs. 2 and 3), the region of the postulated glycolytic complex occurrence [6–8,27]. The reverse regulation of the activity of glycolysis and glyconeogenesis by Ca2+ is in agreement with the data showing that calcium increases the amount of FBPase in the cytosolic extract from muscle fibres, but decreases the amount of aldolase (Table 1). Thus, aldolase catalyzing the reversible reaction of F1,6-P2 synthesis is involved in the formation of the glyconeogenic or glycolytic complex, dependently on the physiological state of the cell. In the process of glycogen synthesis, the enzyme not only supplies the substrate for FBPase and desensitizes it to AMP inhibition [1,3], but also partially desensitizes FBPase to action of calcium – the inhibition of the complex is about 50 times weaker than free muscle FBPase (Fig. 7) [14]. Presumably, Ca2+ does not inhibit directly the activity of FBPase associated with aldolase. It seems that calcium firstly destabilizes the complex and then inhibits the activity of free FBPase. Such a mode of the inhibition by calcium and the desensitization to the inhibitor might enable the activity of glyconeogenesis during the moderate muscle activity. Summarizing: glucostasis is a complex phenomenon in which the glucose supply and its consumption are precisely regulated. In the muscle tissue, glycogen synthesis from carbohydrate precursors is an important part of the maintenance mechanism of the glucose level and calcium ions participate in the regulation of this process. Intracellular muscle calcium activates glycogenolysis, rises up the level of glucose, accelerates glycolysis and inhibits glyconeogenesis. Acknowledgements: This work was supported by Polish Committee for Scientific Research Grant No. 2 PO4A 005 26. We are very grateful to Kamila Wrobel for the linguistic assistance.
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P. Mamczur et al. / FEBS Letters 579 (2005) 1607–1612 [6] Srere, P.A. and Ovadi, J. (1990) Enzyme-enzyme interactions and their metabolic role. FEBS Lett. 268, 360–364. [7] Ovadi, J. and Srere, P.A. (2000) Macromolecular compartmentation and channeling. Int. Rev. Cytol. 192, 255–280. [8] Kraft, T., Hornemann, T., Stolz, M., Nier, V. and Wallimann, T. (2000) Coupling of creatine kinase to glycolytic enzymes at the sarcomeric I-band of skeletal muscle: a biochemical study in situ. J. Muscle Res. Cell. Motil. 21, 691–703. [9] Martonosi, N.A. and Pikula, S. (2003) The network of calcium regulation in muscle. Acta Biochim. Pol. 50, 1–30. [10] Barry, W.H. and Bridge, J.H. (1993) Intracellular calcium homeostasis in cardiac myocytes. Circulation 87, 1806–1815. [11] Singh, P., Salih, M., Leddy, J.J. and Tuana, B.S. (2004) The muscle-specific calmodulin-dependent protein kinase assembles with the glycolytic enzyme complex at the sarcoplasmic reticulum and modulates the activity of glyceraldehyde-3-phosphate dehydrogenase in a Ca2+/calmodulin-dependent manner. J. Biol. Chem. 279, 35176–35182. [12] Wimhurst, M.J. and Manchester, K.L. (1970) Effect of bivalent cations on some enzymes of gluconeogenesis. Biochem. J. 118, 19P. [13] Rice, N.A., Nadeau, O.W., Yang, Q. and Carlson, G.M. (2002) The calmodulin-binding domain of the catalytic gamma subunit of phosphorylase kinase interacts with its inhibitory alpha subunit: evidence for a Ca2+ sensitive network of quaternary interactions. J. Biol. Chem. 277, 14681–14687. [14] Gizak, A., Majkowski, M., Dus, D. and Dzugaj, A. (2004) Calcium inhibits muscle FBPase and affects its intracellular localization in cardiomyocytes. FEBS Lett. 576, 445–448. [15] Penhoet, E.E., Kochman, M. and Rutter, J.W. (1969) Isolation of fructose diphosphate aldolases A, B, and C. Biochemistry 8, 4391–4395. [16] Rakus, D., Skalecki, K. and Dzugaj, A. (2000) Kinetic properties of pig (Sus scrofa domestica) and bovine (Bos Taurus) D -fructose1,6-bisphosphate 1-phosphohydrolase (F1,6Bpase): liver-like isozymes in mammalian lung tissue. Comp. Biochem. Physiol. B 127, 123–134. [17] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. [18] Goding, J.W. (1976) Conjugation of antibodies with fluorochromes: modifications to the standard methods. J. Immunol. Methods 13, 215–226. [19] Brenner, B., Chalovich, J.M., Greene, L.E., Eisenberg, E. and Schoenberg, M. (1986) Stiffness of skinned rabbit psoas fibers in MgATP and MgPPi solution. Biophys. J. 50, 685–691. [20] Manders, M.E., Verbeek, F.J. and Aten, J.A. (1993) Measurement of co-localization of objects in dual-color confocal images. J. Micros. 169, 375–382. [21] Gizak, A., Rakus, D. and Dzugaj, A. (2003) Immunohistochemical localization of human fructose-1,6-bisphosphatase in subcellular structures of myocytes, Histol. Histopathology 18, 135–142. [22] Fournier, P.A., Brau, L., Ferreira, L.D., Fairchild, T., Raja, G., James, A. and Palmer, T.N. (2002) Glycogen resynthesis in the absence of food ingestion during recovery from moderate or high intensity physical activity: novel insights from rat and human studies. Comp. Biochem. Physiol. A 133, 755–763. [23] Davis, L.H. and Kauffman, F.C. (1986) Calcium-dependent activation of glycogen phosphorylase in rat pheochromocytoma PC12 cells by nerve growth factor. Biochem. Biophys. Res. Commun. 138, 917–924. [24] Heilmeyer, L.M., Meyer, F., Haschke, R.H. and Fischer, E.H. (1970) Control of phosphorylase activity in a muscle glycogen particle. II. Activation by calcium. J. Biol. Chem. 245, 6649–6656. [25] Holloszy, J.O. (2003) A forty-year memoir of research on the regulation of glucose transport into muscle. Am. J. Physiol. Endocrinol. Metab. 284, E453–E467. [26] Clarke, F.M. and Masters, C.J. (1975) On the association of glycolytic enzymes with structural proteins of skeletal muscle. Biochim. Biophys. Acta 381, 37–46. [27] Dolken, G., Leisner, E. and Pette, D. (1975) Immunofluorescent localization of glycogenolytic and glycolytic enzyme proteins and of malate dehydrogenase isozymes in cross-striated skeletal muscle and heart of the rabbit. Histochemistry 43, 113– 121.
FEBS Letters 579 (2005) 1607–1612
The effect of calcium ions on subcellular localization of aldolase–FBPase complex in skeletal muscle Piotr Mamczura, Dariusz Rakusa, Agnieszka Gizaka, Danuta Dusb, Andrzej Dzugaja,* a
Department of Animal Physiology, Institute of Zoology, Wroclaw University, Cybulskiego 30, 50-205 Wroclaw, Poland Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Science, Wroclaw, Poland
b
Received 4 November 2004; revised 25 January 2005; accepted 28 January 2005 Available online 14 February 2005 Edited by Amy McGough
Abstract In skeletal muscles, FBPase–aldolase complex is located on a-actinin of the Z-line. In the present paper, we show evidence that stability of the complex is regulated by calcium ions. Real time interaction analysis, confocal microscopy and the protein exchange method have revealed that elevated calcium concentration decreases association constant of FBPase–aldolase and FBPase-a-actinin complex, causes fast dissociation of FBPase from the Z-line and slow accumulation of aldolase within the I-band and M-line. Therefore, the release of Ca2+ during muscle contraction might result, simultaneously, in the inhibition of glyconeogenesis and in the acceleration of glycolysis. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: FBPase; Aldolase; Calcium; a-Actinin; Glyconeogenesis; Co-localization
1. Introduction Muscle fructose 1,6-bisphosphatase (FBPase) [EC 3.1.3.11], a key enzyme of glyconeogenesis, catalyzes the irreversible reaction of hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. Muscle aldolase [EC 4.1.2.13] catalyzes the reversible reaction of synthesis of fructose 1,6-bisphosphate from glyceraldehyde 3-phosphate and dihydroxyacetone phosphate and, in glyconeogenesis, supplies the substrate for FBPase. Muscle FBPase is very sensitive to AMP inhibition and in the presence of physiological concentrations of this effector in muscle fibres FBPase should be almost completely inactive [1,2]. Recently, we have presented evidence that, in vitro, muscle aldolase binds to muscle FBPase and the binding results in dramatic changes in allosteric properties of the latter enzyme, desensitizing it towards AMP inhibition [2,3] and enabling the intermediate channeling between the enzymes [4]. Investigating subcellular localization of the two enzymes we have found that, in striated muscles, FBPase and aldolase colocalize on the Z-line (with the strong binding of the former en* Corresponding author. Fax: +48 71 328 82 46. E-mail address: [email protected] (A. Dzugaj).
Abbreviations: FBPase, fructose 1,6-bisphosphatase; FITC, fluorescein isothiocyanate; TRITC, rhodamine isothiocyanate; EGTA, ethylene glycol-bis(b-aminoethyl ether)-N,N,N 0 ,N 0 -tetraacetic acid; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; PEG, polyethylene glycol; HEPES, N-[2-hydroxyethyl]piperazine-N 0 [2-ethanesulfonic acid]; DTT, dithiothreitol
zyme to a-actinin) [5] and we have postulated the existence of glyconeogenic metabolon around the Z-line [5]. Structural separation of glyconeogenesis and glycolysis (the glycolytic complex is known to be located on actin [6–8]) may protect the muscle cell against the loss of the energy via futile cycles. Calcium is the main regulator of muscle contraction [9,10]. It also affects the number of other cellular activities including the metabolism of carbohydrates [7,11–13]. Quite recently, we have found that calcium is a potent inhibitor muscle FBPase and, in cardiomyocytes, causes dissociation of the enzyme from the Z-line [14]. The primary aim of the present study was to investigate the effect of calcium ions on the stability, activity and subcellular localization of aldolase–FBPase complex. In the running report, the evidence is presented that calcium decreases affinity of FBPase to aldolase and a-actinin and causes disintegration of aldolase–FBPase complex.
2. Materials and methods Phosphocellulose P-11 was purchased from Whatman (England), ethanolamine was from Merck (Germany), and BIAcore sensor chip CM5 was from Biacore AB (Sweden). Alexa Fluor 546 anti-mouse antibodies were from Molecular Probes (USA). Other reagents were from Sigma (USA). 2.1. Enzyme purification and activity determination Rabbit muscle FBPase and aldolase were purified according to [1] and [15], respectively. The activities of FBPase and aldolase were determined as described by Rakus and Dzugaj [1] and Rakus et al. [16], respectively. The concentration of the enzymes was determined spectrophotometrically and the purity of aldolase and FBPase was checked by 10% SDS–PAGE [17]. The effect of calcium ions on the velocity of the reaction catalyzed by free FBPase and aldolase–FBPase complex was measured as was described previously [3,14]. Spectrophotometric measurements were performed with an Agilent 8453 diode array spectrophotometer. 2.2. Fluorescent labeling Fluorescently labeled FBPase (FITC-FBPase) and aldolase (TRITC-aldolase) were obtained by modification of the proteins with fluorescein isothiocyanate (FITC) and tetramethyl-rhodamine isothiocyanate (TRITC), respectively, as described by Goding [18]. The lack of proteolysis of fluorescently labeled proteins was checked by 10% SDS–PAGE [17]. The number of fluorochrome molecules conjugated to the enzymes was estimated spectrophotometrically. 2.3. The protein exchange Single skeletal muscle fibres were prepared as described previously [8]. The stabilization of the striation pattern of the skinned muscle fibres was achieved as described by Brenner et al. [19]. The protein
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.01.071
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exchange method, previously described by Kraft et al. [8] and Rakus et al. [5], was used to localize and co-localize FBPase and aldolase. Before the incubation with muscle fibres, both enzymes were dialyzed against relaxing buffer (10 mM imidazol, 2 mM MgCl2, 1 mM EGTA, 20 mM creatine phosphate, 2 mM DTT, 106 mM potassium propionate; pH 7.0, t = 4 C). In localization experiments, the skinned muscle fibres were incubated overnight with FITC-FBPase (0.2 mg/ml), or with TRITC-aldolase (1 mg/ml) for 4 h. To co-localize both enzymes, the muscle fibres were incubated overnight with FITC-FBPase (0.2 mg/ml) and subsequently with TRITC-aldolase (1 mg/ml) for 4 h. To interpret the achieved striation pattern, skinned fibres were incubated with anti-a-actinin antibodies, secondary antibodies labeled with Alexa Fluor 546 and then with FITC-FBPase (Fig. 1D–F). To test the effect of calcium ions on localization and co-localization of aldolase and FBPase, the fibres containing associated fluorescently labeled proteins were transferred into the activating solution (10 mM imidazol, 0.1 mM CaCl2, 2 mM MgCl2, 20 mM creatine phosphate, 2 mM DTT, 106 mM potassium propionate; pH 7.0, t = 4 C). All fibres were mounted on microscopic slides and examined by Confocal microscopy. The fluorescent images were obtained with a Bio-Rad MRC 1024ES confocal scanner unit and Zeiss Axiovert S100 inverted microscope. The degree of co-localization, expressed in PearsonÕs correlation coefficient, which calculates the proportion of all red intensities that have green components among all red intensities, was assessed by the colocalization analysis function in the AutoVisualise/AutoDeblur v 9.3 free 30-day demo software (AutoQuant Imaging Inc.).
ibrated with the running buffer (10 mM HEPES, 100 mM KCl, 1 mM DTT, 2 mM MgCl2; pH 7.4, t = 25 C) until a stable baseline was obtained. The level of immobilization was 4426 and 3079 RU for aldolase and a-actinin, respectively. All binding experiments were carried out at 25 C with a constant flow rate of 10 ll/min in the running buffer. To determine the affinity of FBPase to a-actinin and aldolase, 50 ll aliquots of FBPase (270, 675 and 1350 nM) in the running buffer in the absence and in the presence of 10 lM CaCl2 or 100 lM CaCl2 were injected over the aldolase-immobilized and a-actinin-immobilized sensor chip, followed by a 3-min washing with the running buffer supplemented with appropriate concentration of calcium. The binding constants (KA) were determined both by global fitting of the sensograms to a binding models using BIAevaluation 3.1 software (Pharmacia Biosensor AB) as well as by equilibrium binding analysis with GraFit 4.0.12 program (Erithacus Software Ltd., R.J. Leatherbarrow). The global analysis of the interaction between FBPase and a-actinin was performed using a single-site binding model with drifting base-line. The best fitted model describing the interaction between FBPase and aldolase accounts for the existence of one binding site on the surface of the FBPase molecule and two conformational states of the complex [5]. The equilibrium binding constants were obtained by non-linear regression analysis using GraFit 4.0.12 program and the following equation:
2.4. Effect of calcium on aldolase and FBPase release from subcellular structures of muscle fibres Skeletal muscles dissected from a freshly killed rabbit were homogenized in the buffer containing: 20 mM Tris, 0.4 mM EDTA, 1 mM PMSF (pH 7.4, t = 4 C) in the absence and in the presence of 5% PEG 8000 as well as in the presence of 5% PEG 8000 and 0.01 or 0.1 mM CaCl2. The ratio of tissue to buffer (wt/vol) was 1:2. Homogenates were centrifuged at 25 000 · g for 20 min at 4 C and the supernatants were used for the determination of aldolase and FBPase activity [1,16]. The pellets were homogenized in 10 volumes of homogenization buffer with 0.5% Triton X-100, incubated for 30 min on ice, centrifuged at 25 000 · g for 20 min at 4 C and the obtained supernatants were tested for the enzyme activity.
where Req is response in resonance units (RU) at equilibrium, C is the FBPase concentration, Rmax is RU at maximum and KA is the binding constant.
2.5. Real-time interaction analysis by BIAcore The binding of FBPase to immobilized a-actinin and to immobilized aldolase was measured using BIAcore 1000 (Biacore AB, Sweden). The sensor chips CM5 with carboxymethylated dextran matrix were used throughout all experiments. The immobilization of a-actinin was described previously [5]. The immobilization of aldolase was carried out using the standard EDC/NHS chemical procedure for the activation of a working channel. A 5 ll solution of aldolase (0.1 mg/ml in 10 mM acetate buffer; pH 6.5, flow rate 5 ll/min) was injected over the activated sensor surface, followed by a 50 ll solution of 1 M ethanolamine to deactivate the unreacted NHS-esters. The chip was equil-
Req ¼ ðRmax CÞ=ð1=K A þ CÞ
3. Results Fluorescent labeling of the enzymes revealed that FBPase monomer bound on average three molecules of FITC, whereas two molecules of TRITC were bound with aldolase monomer. Although the labeling strongly decreased the specific activity of both enzymes, the modification did not significantly reduce the affinity of these enzymes to each other as well as of FBPase to a-actinin [5]. The protein exchange experiments revealed that FITCFBPase is located on both sides of the Z-line (Fig. 1A) and TRITC-aldolase accumulates within the I-band and around the M-line (Fig. 2A). The same striation pattern for the both enzymes as well as for their co-localization (Fig. 3A) was achieved previously [5]. In control experiments, where muscle fibres were incubated with the respective fluorochrome alone, no staining was observed (data not shown). Co-localization
Fig. 1. The effect of calcium ions on localization of FITC-FBPase: in the absence of Ca2+ (A); after 1 min of incubation with 10 lM Ca2+ (B) and 100 lM Ca2+ (C). Co-localization (D) of FBPase (E) and a-actinin (F) in muscle fibre. Bar: 5 lm.
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Fig. 2. The effect of calcium ions on localization of TRITC-aldolase: in the absence of Ca2+ (A); after 5 min (B), 10 min (C) and 20 min (D) of incubation with 10 lM Ca2+; after 5 min (E), 10 min (F) and 20 min (G) of incubation with 100 lM Ca2+. Bar: 5 lm.
of red and green signals was assessed by the co-localization analysis function in the AutoVisualise/AutoDeblur software. The degree of co-localization, expressed in PearsonÕs correlation coefficient [20] was 0.73, what indicates high degree of the co-localization (Fig. 4A). The addition of 10 and 100 lM calcium to the skinned muscle fibres with bound FITC-FBPase resulted in fast and complete dissociation of the enzyme from the Z-line (Fig. 1B and C). The reverse effect exerted calcium on the localization of aldolase: 10 and 100 lM CaCl2 caused the slow accumulation of TRITC-aldolase in the I-band and around the M-line (Fig. 2B–G). The same effect of Ca2+ as rapid dissociation of FITCFBPase from the Z-line and slow accumulation of TRITCaldolase within the I-band and on the M-line was observed in the co-localization experiment (Fig. 3B and C). In the presence of calcium ions the PearsonÕs correlation coefficient was 0.14, what indicates no co-localization of the proteins [20] (Fig. 4B and C).
The global fitting of the sensograms revealed the binding constant for aldolase–FBPase equal to 8.56 · 106 M1 (Fig. 5A) and for FBPase-a-actinin equal to 1.01 · 107 M1 (Fig. 6A). Similar values of KA were obtained when the data was analyzed by equilibrium binding analysis (Figs. 5A and 6A) and these values are in the range of KA reported previously [3,5,21]. The association of FBPase to aldolase and a-actinin was strongly affected by calcium ions, and 10 lM Ca2+ decreased the affinity of FBPase to aldolase and to a-actinin about 4–5-fold and 10-fold, respectively (Figs. 5A–C and 6A–C). The release of FBPase and aldolase from subcellular structures of muscle was dependent on the presence of the crowding agent imitating the physiological conditions – PEG 8000 (Table 1). The amount of both enzymes associated with structures of muscle cells was about 6–7 times higher in the presence of 5% PEG 8000 than in its absence (Table 1). An addition of 100 lM CaCl2 to the homogenization buffer only slightly affected the amount of FBPase and aldolase associated with
Fig. 3. The effect of calcium ions on co-localization of FITC-FBPase and TRITC-aldolase: in the absence of Ca2+ (A); after 10 min of incubation with 10 lM Ca2+ (B); after 10 min of incubation with 100 lM Ca2+ (C). Bar: 5 lm.
Fig. 4. The graphical representation of the PearsonÕs correlation coefficient showing high degree of the FBPase and aldolase co-localization (yellow points) in the absence of Ca2+ (A); no co-localization in the presence of 10 lM (B) or 100 lM Ca2+ (C). Bar: 5 lm.
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Fig. 5. (A) The binding constants between FBPase and aldolase in the absence and in the presence of calcium ions as determined by the global fitting of the sensograms and the equilibrium binding analysis. (B) Representative sensograms and fitted curves showing Ca2+-dependent binding of FBPase (675 nM) to aldolase. (C) The equilibrium binding analysis of the effect of calcium ions on the affinity of FBPase to aldolase.
subcellular structures (Table 1). On the other hand, an addition of calcium to the homogenization buffer containing PEG 8000 significantly decreased the amount of FBPase associated with subcellular structures and increased the amount of the associated aldolase (Table 1). Ca2+ inhibited the activity of muscle FBPase with I0.5 equal to 0.48 lM (Fig. 7) [14], whereas the inhibition of the activity of aldolase–FBPase complex was much weaker and an apparent inhibitory constant was about 24 lM (Fig. 7). The relative activity of aldolase–FBPase complex in the presence of calcium ions (Fig. 7) roughly correlated with the amount of the aldolase–FBPase complex under the experimental conditions (calculated with Eq. (1)). Thus, it is possible that calcium decreases the concentration of the complex and, as a result, it can inhibit FBPase activity in a classic manner. qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðK A ½AþK A ½F þ1Þ ðK A ½AþK A ½F þ1Þ2 4K 2A ½A½F ½AF ¼ ; 2K A ð1Þ where [AF] – concentration of aldolase–FBPase complex, [F] – total concentration of FBPase (7.15 · 107 M), [A] – total con-
centration of aldolase (6.25 · 1010 M), KA – association constant between aldolase and FBPase (Fig. 5A–C).
4. Discussion For years it was a common belief that lactate produced in a contracting muscle is transported via the blood stream to the liver where is converted to glucose, which subsequently is transported to the muscle again (Cori cycle). In last few years evidence has been accumulated that, in the skeletal muscle, up to 50% of lactate is converted to glycogen (glyconeogenesis) [22]. In such a pathway aldolase as well as FBPase are indispensable. Aldolase not only directly supplies substrate to FBPase [4], but also interacting with FBPase desensitizes it towards AMP inhibition [1,3]. Glucose is the main source of energy for muscle fibres and its level therein is regulated not solely by hormones but also by intracellular calcium. Calcium is involved in the regulation of glycogenolysis by activating glycogen phosphorylase which subsequently causes the release of glucose from glycogen for ATP generation [23,24]. Calcium also activates, in an insulinindependent manner, GLUT4-dependent entrance of glucose
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Fig. 6. (A) The binding constants between FBPase and a-actinin in the absence and in the presence of calcium ions as determined by the global fitting of the sensograms and the equilibrium binding analysis. (B) Representative sensograms and fitted curves showing Ca2+-dependent binding of FBPase (675 nM) to a-actinin. (C) The equilibrium binding analysis of the effect of calcium ions on the affinity of FBPase to a-actinin.
into muscle fibres [25] and accelerates glycolysis affecting binding of glycolytic enzymes to subcellular structures [7,26]. On the other hand, calcium ions are presumed to inhibit glyconeogenesis: Ca2+ is the strong inhibitor of muscle FBPase and during the muscle contraction an elevated level of calcium should almost completely inhibit the enzyme [14]. Moreover, Table 1 The effect of calcium on aldolase and FBPase release from subcellular structures of muscle fibres Buffer
FBPase associated with subcellular structures (%)
Aldolase associated with subcellular structures (%)
Without crowding agent Without crowding agent Ca2+ – 100 lM 5% PEG 5% PEG Ca2+ – 10 lM 5% PEG Ca2+ – 100 lM
7.3 ± 3.8 5.9 ± 2.7
8.5 ± 2.4 11.7 ± 4.1
47 ± 8.6 38 ± 7.4 17 ± 3.2
35 ± 7.9 39 ± 4.2 56 ± 9.2
Each value represents the mean and S.D. of triplicate independent experiments.
Fig. 7. The inhibition of free FBPase (full circles, dashed line) and aldolase–FBPase complex (empty circles, solid line) activity by calcium ions. Black squares represent the saturation of aldolase with FBPase in the presence of 10 and 100 lM Ca2+, as calculated according to Eq. (1).
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in cardiomyocytes, Ca2+ causes dissociation of FBPase from the Z-line [14] disintegrating the hypothetical glyconeogenic metabolon. In the present paper, we have reported that an increased concentration of calcium decreases both the affinity of FBPase to aldolase (Fig. 5) and to a-actinin (Fig. 6). This destabilizatory effect of Ca2+ results in the release of muscle FBPase from the multienzymatic complex located around the Z-line (Table 1, Figs. 1 and 3) and free form of the enzyme is strongly inhibited both by calcium ions (Fig. 7) and AMP [1,3]. Simultaneous to the inhibition of glyconeogenesis, the increased concentration of calcium causes the accumulation of aldolase within I-band (Figs. 2 and 3), the region of the postulated glycolytic complex occurrence [6–8,27]. The reverse regulation of the activity of glycolysis and glyconeogenesis by Ca2+ is in agreement with the data showing that calcium increases the amount of FBPase in the cytosolic extract from muscle fibres, but decreases the amount of aldolase (Table 1). Thus, aldolase catalyzing the reversible reaction of F1,6-P2 synthesis is involved in the formation of the glyconeogenic or glycolytic complex, dependently on the physiological state of the cell. In the process of glycogen synthesis, the enzyme not only supplies the substrate for FBPase and desensitizes it to AMP inhibition [1,3], but also partially desensitizes FBPase to action of calcium – the inhibition of the complex is about 50 times weaker than free muscle FBPase (Fig. 7) [14]. Presumably, Ca2+ does not inhibit directly the activity of FBPase associated with aldolase. It seems that calcium firstly destabilizes the complex and then inhibits the activity of free FBPase. Such a mode of the inhibition by calcium and the desensitization to the inhibitor might enable the activity of glyconeogenesis during the moderate muscle activity. Summarizing: glucostasis is a complex phenomenon in which the glucose supply and its consumption are precisely regulated. In the muscle tissue, glycogen synthesis from carbohydrate precursors is an important part of the maintenance mechanism of the glucose level and calcium ions participate in the regulation of this process. Intracellular muscle calcium activates glycogenolysis, rises up the level of glucose, accelerates glycolysis and inhibits glyconeogenesis. Acknowledgements: This work was supported by Polish Committee for Scientific Research Grant No. 2 PO4A 005 26. We are very grateful to Kamila Wrobel for the linguistic assistance.
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