Is there a pyruvate kinase in pig liver mitochondria?

Is there a pyruvate kinase in pig liver mitochondria? Roberto Pizzuto 1, Gianluca Paventi 1, Gabriella Chieppa 1, Anna Atlante 2, Salvatore Passarella 1 1 Dipartimento di Scienze per la Salute, Università del Molise, Campobasso, Italy 2 Istituto di Biomembrane e Bioenergetica, CNR, Bari, Italy Abstract. In order to ascertain whether mammalian mitochondria possess their own pyruvate kinase, we isolated mitochondria from liver of Large White pig and investigated pyruvate kinase occurrence both via immunological analysis and by assaying photometrically the pyruvate kinase reaction. We show that mitochondria contain pyruvate kinase located in the inner compartments; the pyruvate kinase reaction shows hyperbolic dependence on the substrate concentration, is inhibited by malonate and shows maximum activity at ph between 7-7.6 and Ea equal to 33 kj/mol. Key words: Mitochondria, pyruvate kinase, phosphoenolpyruvate INTRODUCTION Pyruvate kinase (EC 2.7.1.40) (PK) is an enzyme of the Embden-Meyerhof glycolytic pathway and catalyses the anaerobic conversion of phosphoenolpyruvate (PEP) and ADP to pyruvate and ATP. Pyruvate kinase, is allosterically inactivated by ATP, alanine and acetyl-coa, allosterically activated by fructose 1,6BP, and is inactivated by covalent modification (phosphorylation) from the kinase cascade [1]. There are 4 different isozymes expressed in mammals with each one showing a tissue-specific distribution and exhibiting different kinetic properties [2-5]. PKs are generally referred to as a cytosolic enzymes, however, we have already shown that PEP can be metabolized by Jerusalem artichoke mitochondria (JAM) by virtue of the presence of the mitochondrial PK; consistently, JAM can take up externally added PEP in a carrier-mediated uptake and the addition of PEP causes pyruvate and ATP production and appearance of citrate and oxaloacetate outside mitochondria [6]. This prompted us to investigate whether mammalian mitochondria in particular those isolated from pig liver (PLM) possess their own PK. We show here the existence of PK in PLM, as investigated both immunologically by using anti-pk antibodies and by monitoring photometrically the PK reaction. Since PK activity is revealed only in solubilized mitochondria, we conclude that a mitochondrial pyruvate kinase (mpk) is localized in the inner mitochondrial compartments. MATERIALS AND METHODS Materials All reagents were from SIGMA with the exception of sucrose, Triton X-100, HEPES, and Tris (Baker). All chemicals were of purest grade available and were used as Tris salts at ph 7.0-7.4 adjusted with Tris or HCl. None of the compounds used in this work affects the enzymes used to monitor pyruvate kinase activity. Isolation of PLM and preparation of the cytosolic fraction PLM were isolated as described in [7] using a medium consisting of 0.25 M sucrose, 20 mm Tris/HCl ph 7.2. The final mitochondrial pellet was suspended in the same medium to obtain 60-70 mg of protein/ml. Mitochondrial protein content was determined by the method of Lowry as in Harris [8], using BSA as a standard. The cytosolic fraction (CF) was obtained by centrifuging (105,000 g for 60 min at 4 C) the supernatant obtained during isolation of PLM. Immunoblot analysis Immunoblot analysis was performed on both total mitochondrial and total cytosolic protein by using antibodies raised against PK, COX IV and -tubulin. Polyclonal antibodies recognizing COX IV and tubulin were used as markers of mitochondria and cytosol, respectively. Both purified PLM and cytosolic protein were solubilised in 1% Triton X-100, 500 mm NaCl, 50 mm Tris/HCl (ph 7.5), 1 mm EGTA, 1 mm EDTA, 0.5 mm dithiothreitol and 0.1 mm phenylmethanesulfonyluo- 270 The Italian Journal of Biochemistry Vol. 56 (4) 2007

ride for 30 min on ice. Protein content was determined using the Bradford reagent (Bio-Rad Laboratories, Hercules, CA, USA), with BSA as a standard. Solubilized proteins (40 and 80 g) were subjected to electrophoresis on 12% SDS-polyacrylamide gel [9]. Following electrophoresis, protein blots were transferred to a poly(vinylidenedifluoride) membrane. The membrane was blocked with 5% non fat milk in Tris buffer solution, and incubated overnight with the corresponding primary antibodies in the blocking solution at 4 C. After being washed three times with Tris buffer solution plus Tween-20 (0.3%), the membrane was incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibody. The detected protein signals were visualized with enhanced chemiluminescence western blotting reagents (Amersham, ECL, Little Chalfont, UK). Relative absorbances and areas of bands were quantified using a GS-700 Imaging Densitometer implemented with molecular analyst software (Bio- Rad Laboratories). Pyruvate kinase assay To assay Triton X-100-solubilized PLM were incubated at 25 C in 2 ml of the standard medium consisting of 0.25 M sucrose,10 mm KCl, 20 mm Hepes-Tris ph 7.2, 1 mm MgCl 2. The PK assay was performed photometrically at 340 nm by means of a Jasco V-560 spectrophotometer (Tokyo, Japan) by measuring either the pyruvate production via LDH (2 e.u.) and NADH 0.2 mm, or the ATP production via the ATP detecting system as in [6]. The activity was expressed as nmol NADH oxidized or NADP + reduced /min x mg protein. RESULTS In order to ascertain whether PLM contain their own PK, an immunological analysis was carried out by using anti-pk antibodies (Fig. 1A). PK was found both in cytoplasm and in the mitochondrial fraction. As a control, mitochondria were also subjected to Western blotting using antibodies specific for subunit IV of the cytochrome c oxidase (COX IV) a mitochondrial marker, and -tubulin, used as a cytosolic marker. PLM were found to be essentially free of cytosolic contamination since no significant amount of -tubulin was detected (see also below). Notice that -tubulin proved to bind mitochondria isolated from certain sources [10]. Conversely, the immunological analysis of the cytosolic fraction of the same preparation, showed the presence of both cytosolic PK and -tubulin, but not that COX IV. The existence of the mpk was confirmed by checking photometrically whether externally added PEP can produce pyruvate, this resulting in absorbance decrease due to NADH oxidation in the presence of lactate dehydrogenase. As a result of PEP addition to intact PLM no significant absorbance change was found, this indicating the absence of PK in the outer membrane, in the intermembrane space and on the outer side of the mitochondrial inner membrane (Fig. 1B). As expected, when Triton X-100 (0.1 %) was added to PLM to dissolve the mitochondrial membranes, NADH oxidation was found at a very low rate. As a result of PEP (5 mm) addition a strong increase of the NADH oxidation rate was found up to 5.2 nmol NADH oxidized/min mg of mitochondrial protein (Fig. 1C). Malonate (3 mm), inhibitor of a variety of PKs, including mpk of Jerusalem artichoke [6] was found to inhibit PK reaction by 30% (Fig. 1D). Control was made that malonate was ineffective in inhibiting the lactate dehydrogenase. As expected, no PK activity was measured when mitochondrial sample were boiled to denaturate the proteins (not shown). In a parallel experiment, the occurrence of the mpk was confirmed by measuring the ATP formation with the ATP detecting system [6], consisting of glucose, hexokinase, NADP + and glucose-6-pdehydrogenase: ATP formation was found occurring at the same rate as that of pyruvate production measured as above, in a manner inhibited by malonate (not shown). The dependence of the rate of NADH oxidation on either ADP (at fixed 10 mm PEP) (Fig. 2A) or PEP (at fixed 2.5 mm ADP) increasing concentrations (Fig. 2B) was investigated. Saturation characteristics were found with K m(adp) and K m(pep) values equal to 0.54 mm and 1.5 mm respectively; V max values were about 7 nmol/min mg of sample protein in both cases. In another set of experiment, the ph and the temperature dependence of the PK reaction were investigated using 5 mm PEP (Figs. 3A and B): the mpk displayed a ph profile with a wide maximum between ph 7 and 7.6; the activation energy obtained by the Arrhenius plot was 33 kj/mol as determined in 10-40 C temperature range. DISCUSSION In this paper, we first show that mammalian mitochondria, in particular those from Large White pig liver, possess their own PK. Such a conclusion derives from the immunological analysis and by kinetic studies in which we show saturation characteristics for the PK reaction, malonate inhibition, ph and temperature dependence. Since the PK activity can be assayed only in solubilized PLM, mpk must be localized on the inner side of the mitochondrial The Italian Journal of Biochemistry Vol. 56 (4) 2007 271

Figure 1 Immunodetection of mitochondrial PK and its activity assay in PLM. (A) Solubilized proteins (40 and 80 g) from both mitochondrial and cytosolic fractions were analyzed by western blot as described in Materials and methods. Membrane blots were incubated with polyclonal anti-pk, anti-cox IV and anti- -tubulin. COX IV and -tubulin were used as mitochondrial and cytosolic markers, respectively. (B) PLM (0.5 mg mitochondrial protein) were incubated at 25 C in 2 ml of standard medium in the presence of NADH (0.2 mm). At the arrow, LDH (2 e.u.), ADP (2.5 mm) and PEP (5 mm) were added. (C) PLM (0.5 mg mitochondrial protein) were incubated at 25 C in 2 ml of standard medium in the presence of NADH (0.2 mm). At the arrow, LDH (2 e.u.), ADP (2.5 mm), Triton X-100 (0,1 %) and PEP (5 mm) were added. (D) PLM (0.5 mg mitochondrial protein) were incubated at 25 C in 2 ml of standard medium in the presence of NADH (0.2 mm), LDH (2 e.u.), ADP (2.5 mm), and Triton X-100 (0.1 %). At the arrow PEP (5 mm) was added either in the absence or in the presence of malonate (MALO) (3 mm). inner membrane or in the matrix space. The choice of pig liver mitochondria merits some discussion. Since pigs of different breeds can differ one from another with respect to fat amount and since in initial investigation we have found evidence that PEP is involved in fatty acid synthesis [6], we will investigate mpk occurrence and activity in Large White pig, showing a low fat content, and in other pigs with high fat content. In this regard, although the role of mitochondrial PK must be at present only a matter of speculation, we might propose that, as a result of the PEP metabolism via the putative mitochondrial PK, reduction in ADP dependent isocitrate dehydrogenase activity could occur, with consequent increase of matrix citrate concentration and consequent citrate efflux outside mitochondria to trigger fatty acid synthesis. Interestingly, citrate appearance in the extramitochondrial phase was found as a result of PEP addition to JAM [6]. Finally this paper shows that the actual scenario of PEP metabolism in mammalian, at least in pig, must change. We believe that as in [6] when studying PEP metabolism in mammalian, PEP uptake by mitochondria, PEP intramitochondrial metabolism and export of newly synthesised metabolites must be considered in the future. ACKNOWLEDGEMENTS This work was partially financed by FIRB RBNE03B8KK_003 (to S.P.). 272 The Italian Journal of Biochemistry Vol. 56 (4) 2007

Figure 2 The dependence of pyruvate production on increasing concentrations of either PEP or ADP. (A) PLM (0.5 mg mitochondrial protein) were incubated in 2 ml of standard medium in the presence of NADH (0.2 mm), LDH (2 e.u.), Triton X-100 (0.1 %) and PEP (10 mm). ADP were added at the indicated concentrations and PK reaction was measured. (B) PLM (0.5 mg mitochondrial protein) were incubated in 2 ml of standard medium in the presence of NADH (0.2 mm), LDH (2 e.u.), Triton X-100 (0.1 %) and ADP (2.5 mm). PEP were added at the indicated concentrations and PK reaction was measured. In both cases the rate (v o ) of NADH oxidation was calculated as the tangent to the initial part of the progress curve (as described in fig. 1C) and expressed as nmol NADH oxidized/min mg of sample protein. Figure 3 ph and the temperature dependence of the PK reaction. (A) PLM (0.5 mg) were incubated in 2 ml of the standard medium whose ph was adjusted to the indicated values with either Tris or HCl and the dependence of pyruvate production on increasing concentrations of PEP was measured as in fig. 2B. Effect of ph on obtained Vmax values for PK reaction was reported. (B) Temperature dependence of PK reaction: PLM (0.5 mg) were incubated in 2 ml of the standard medium in the presence of NADH (0.2 mm), LDH (2 e.u.), ADP (2.5 mm), Triton X-100 (0.1 %) and PEP (10 mm) and the dependence of the rate of pyruvate production on increasing temperature was measured as in fig. 1C. The Italian Journal of Biochemistry Vol. 56 (4) 2007 273

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