Novel isoenzyme of 2-oxoglutarate dehydrogenase is identified in brain, but not in heart Victoria Bunik 1,2 , Thilo Kaehne 3 , Dmitry Degtyarev 1 , Tatiana Shcherbakova 2 and Georg Reiser 4 1 Bioengineering and Bioinformatics Department, Lomonosov Moscow State University, Russia 2 Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Russia 3 Institute of Experimental Internal Medicine, Otto-von-Guericke University Magdeburg, Germany 4 Institute of Neurobiochemistry, Medical Faculty, Otto-von-Guericke University Magdeburg, Germany The 2-oxoglutarate dehydrogenase complex (OGDHC) is a key regulator of a branch point in the tricarboxylic acid cycle. It belongs to the family of 2-oxo acid dehy- drogenase complexes which comprise multiple copies of the three catalytic enzyme components: E1, thia- mine diphosphate (ThDP)-dependent 2-oxo acid dehy- drogenase (in OGDHC it is E1o); E2, dihydrolipoyl acyltransferase with the covalently bound lipoic acid Keywords 2-oxoglutarate dehydrogenase isoenzyme; mitochondrial membrane; multienzyme complex; thiamine; tricarboxylic acid cycle Correspondence V. Bunik, Belozersky Institute of Physico- Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia Fax: +7 495 939 31 81 Tel: +7 495 939 44 84 E-mail: bunik@belozersky.msu.ru G. Reiser, Institut fu ¨ r Neurobiochemie, Medizinische Fakulta ¨ t, Otto-von-Guericke- Universita ¨ t Magdeburg, Leipziger Straße 44, 39120 Magdeburg, Germany Fax: +49 391 67 13097 Tel:+49 391 67 13088 E-mail: georg.reiser@med.ovgu.de (Received 17 April 2008, revised 5 July 2008, accepted 8 August 2008) doi:10.1111/j.1742-4658.2008.06632.x 2-Oxoglutarate dehydrogenase (OGDH) is the first and rate-limiting com- ponent of the multienzyme OGDH complex (OGDHC) whose malfunction is associated with neurodegeneration. The essential role of this complex in the degradation of glucose and glutamate, which have specific significance in brain, raises questions about the existence of brain-specific OGDHC iso- enzyme(s). We purified OGDHC from extracts of brain or heart mitochon- dria using the same procedure of poly(ethylene glycol) fractionation, followed by size-exclusion chromatography. Chromatographic behavior and the insufficiency of mitochondrial disruption to solubilize OGDHC revealed functionally significant binding of the complex to membrane. Components of OGDHC from brain and heart were identified using nano- high performance liquid chromatography electrospray tandem mass spec- trometry after trypsinolysis of the electrophoretically separated proteins. In contrast to the heart complex, where only the known OGDH was deter- mined, the band corresponding to the brain OGDH component was found to also include the novel 2-oxoglutarate dehydrogenase-like (OGDHL) pro- tein. The ratio of identified peptides characteristic of OGDH and OGDHL was preserved during purification and indicated comparable quantities of the two proteins in brain. Brain OGDHC also differed from the heart com- plex in the abundance of the components, lower apparent molecular mass and decreased stability upon size-exclusion chromatography. The func- tional competence of the novel brain isoenzyme and different regulation of OGDH and OGDHL by 2-oxoglutarate are inferred from the biphasic dependence of the overall reaction rate versus 2-oxoglutarate concentra- tion. OGDHL may thus participate in brain-specific control of 2-oxogluta- rate distribution between energy production and synthesis of the neurotransmitter glutamate. Abbreviations E1, 2-oxo acid dehydrogenase; E2, dihydrolipoyl acyl transferase; E3, dihydrolipoyl dehydrogenase; nanoLC-MS ⁄ MS, nano-high performance liquid chromatography–electrospray tandem mass spectrometry; OGDH (E1o), 2-oxoglutarate dehydrogenase; OGDHC, 2-oxoglutarate dehydrogenase complex; OGDHL, 2-oxoglutarate dehydrogenase-like protein; ROS, reactive oxygen species; ThDP, thiamin diphosphate. 4990 FEBS Journal 275 (2008) 4990–5006 ª 2008 The Authors Journal compilation ª 2008 FEBS residue (in OGDHC it is E2o); and the terminal com- ponent E3, FAD-dependent dihydrolipoyl dehydroge- nase, which is common to all complexes. The consecutive action of these components within the multienzyme complex provides for the multistep pro- cess of oxidative decarboxylation of a 2-oxo acid (R = -CH 2 -CH 2 -COOH for 2-oxoglutarate; R = -CH 3 for pyruvate): According to reaction (1), oxidative decarboxylation of 2-oxoglutarate produces energy in the form of NADH and a macroergic acyl thioester bond of succi- nyl-CoA. Essential for aerobic energy production in all tissues, the reaction also involves the important branch-point metabolites 2-oxoglutarate and succinyl- CoA and may thus be subject to differential regulation according to the tissue-specific metabolic network. In particular, succinyl-CoA, which in mammalian mito- chondria may be used for the substrate-level phosphor- ylation of GDP or ADP, is preferentially transformed into ATP in brain [1]. 2-Oxoglutarate is generated both within the tricarboxylic acid cycle and through gluta- mate transamination and oxidative deamination. The ensuing role of OGDHC in the degradation of gluta- mate, which is neurotoxic in excess, is in accordance with the known association between reduced OGDHC activity and neurodegeneration, both age-related [2] and inborn [3,4]. Furthermore, 2-oxoglutarate takes part in metabolic signaling [5–10], and therefore its degradation by OGDHC may affect signal transduc- tion. Regulated by thioredoxin, OGDHC is at the intercept of not only energy production and glutamate turnover, but also mitochondrial production ⁄ scaveng- ing of reactive oxygen species (ROS) [11]. To tune these pathways to the specific demands of the brain, the featured integration of OGDHC into the cell- specific metabolic network is required. This may be achieved through the expression of isoenzymes, their structural differences providing for specificity in both regulation and protein–protein interactions. However, no tissue-specific isoenzymes of the OGDHC compo- nents have been isolated to date. Moreover, the insta- bility of brain OGDHC during purification interferes with obtaining the brain complex in a homogeneous state [12]. In addition to general problems known to arise upon enzyme purification from fat-rich brain tis- sue, the isolation of functional 2-oxo acid dehydro- genase multienzyme complexes poses additional challenges regarding the preservation of non-covalent protein–protein interactions which determine the native structure of such megadalton systems. In this study, we therefore aimed at structural characterization of brain OGDHC using approaches that do not require the complex to be purified to homogeneity. In parti- cular, MS analysis is used to identify the individual proteins and their relative abundance in complex pro- tein mixtures [13–15]. Using this technique, we ana- lyzed a preparation of brain OGDHC which was purified to an extent that enabled kinetic study of the complex. As a result, the structure and function of brain OGDHC were characterized under conditions that preserved the native state of the complex. Specific features of brain OGDHC were revealed by compari- son with OGDHC from heart. We show that, in contrast to heart, the brain preparation comprises comparable amounts of both the known 2-oxogluta- rate dehydrogenase and its novel isoenzyme, a hith- erto hypothetical 2-oxoglutarate dehydrogenase-like (OGDHL) protein, with the isoenzyme ratio preserved during the purification of OGDHC by different proce- dures. Although the existence of OGDHL has been inferred from nucleic acid data, with recent structure– function analysis predicting it to be a novel OGDH isoenzyme [16], the protein has not been reported in mammalian mitochondrial proteomes [17–19]. We show that the presence in brain of the novel isoenzyme of the first component of OGDHC is accompanied by a different supramolecular organization and stability of the complex. Our kinetic study corroborates the cat- alytic competence of the novel isoenzyme in the overall OGDHC reaction predicted previously [16], and also reveals specific regulation of the two isoenzymes by 2-oxoglutarate, which may have implications for brain glutamate metabolism. Results Solubilization and partial purification of OGDHC from rat brain and heart mitochondria The 2-oxo acid dehydrogenase complexes are presumed to be enzymes of the mitochondrial matrix. Accordingly, given that the mitochondria were disrupted, their purifi- cation was carried out without detergents [20,21]. Later, it was found that detergents may improve the solubiliza- tion of both the pyruvate and 2-oxoglutarate dehydro- genase complexes from mammalian tissues at different stages of purification [22–25], although the mecha- nism(s) of their solubilizing action on the complexes have not been systematically studied. In order to better preserve native enzyme regulation and protein–protein interactions, we attempted to obtain detergent-free OGDHC from isolated brain mitochondria using soni- cation only. Solubilization was controlled by following V. Bunik et al. Novel 2-oxoglutarate dehydrogenase FEBS Journal 275 (2008) 4990–5006 ª 2008 The Authors Journal compilation ª 2008 FEBS 4991 the distribution of OGDHC activity between the supernatant and the detergent extract of the broken mitochondria pellet. Mitochondrial disruption with the probe sonicator did not reproducibly solubilize OGDHC activity. Although disruption was evident from the appearance in the supernatant of the activity of the third component of the mitochondrial 2-oxo acid dehydrogenase complexes, dihydrolipoyl dehydroge- nase, overall OGDHC activity (Reaction 1) remained in the broken mitochondria pellet and was solubilized from the pellet only in the presence of detergent (6%Tri- ton X-100 or 1% Chaps). By contrast, sonication using ‘Bioruptor’ enabled reproducible solubilization of the majority (90%) of OGDHC activity from brain mitochondria without detergents. This preparation is further referred to as ‘soluble’ OGDHC. A similar procedure with heart mitochondria left significant amounts of OGDHC in the pellet. Hence, 1% Chaps was used to fully solubilize the heart complex from the pellet. Detergent extraction was also used for brain OGDHC when its solubilization by sonication was not efficient or complete. Such preparations are further called ‘detergent-extracted’ OGDHC. Independent of the OGDHC extraction details, the majority of the complex from the two tissues solubilized together with the integral membrane proteins, such as mitochondrial ADP ⁄ ATP translocase and other transporters (voltage- dependent anion channel, tricarboxylate, 2-oxoglutarate and phosphate carriers). These membrane proteins were identified by nanoLC-MS ⁄ MS in bands 8 and 9 of Fig. 1. Thus, our data on the solubilization of OGDHC activity and the accompanying proteins indicate that in mitochondria from both brain and heart OGDHC inter- acts rather strongly with the membrane fraction. Unlike the heart complex [23], OGDHC from brain was much more prone to lose its activity under gel- filtration conditions which fully resolved it from the pyruvate dehydrogenase complex. Because of this, rela- tively rapid gel-filtration on Sephacryl HR300 16 ⁄ 60 or Sephacryl S300 12 ⁄ 30 columns was used to purify the OGDHC-enriched fraction of the 2-oxo acid dehydro- genase complexes (molecular mass in the range 10 6 – 10 7 Da) from the proteins of a lower molecular mass (10 5 –10 6 Da). Although this fraction contained compo- nents of the pyruvate dehydrogenase complex, as shown below, the activity peak of the latter complex was shifted to lower elution volumes compared with OGDHC. Being rather low even at its peak, the pyru- vate dehydrogenase reaction rate in the OGDHC- enriched fraction did not exceed 10% of the rate of the 2-oxoglutarate dehydrogenase reaction. Impor- tantly, the elution profile of the common E3 compo- nent of the two complexes coincided with the elution of OGDHC, indicating that there was no significant contribution of the pyruvate dehydrogenase complex- bound E3 to the E3 content of our OGDHC-enriched preparation. The latter fraction also lacked the branched chain 2-oxo acid dehydrogenase complex, as neither component of the complex was identified by MS analysis, nor was the activity with 2-oxoisovaleric acid detected. Components of the glycine cleavage system, which also includes E3, were not identified in the OGDHC-enriched fraction. No co-elution of the glycine cleavage system in the high molecular mass fraction comprising the pyruvate and 2-oxoglutarate dehydrogenase complexes was expected, as this com- plex is much smaller and dissociates easily into its components [26]. ABC Fig. 1. Comparison of the SDS electrophoretic patterns of OGDHC preparations from brain and heart mitochondria upon separation on 10% (A, C) and 7% (B) gels. Molecular mass markers (kDa) are indicated on the right, lane numbers are given in the upper row, protein bands are numbered on the left. (A) Brain OGDHC solubilized using ‘Bioruptor’ sonication (lane 1); 1% Chaps extract of the pellet from ‘Bioruptor’- sonicated mitochondria (lane 2); heart OGDHC solubilized by 1% Chaps after ‘Bioruptor’ sonication (lane 3); markers (lane 4). (B) Heart OGDHC solubilized by 1% Chaps after ‘Bioruptor’ sonication (lane 1); brain OGDHC solubilized by ‘Bioruptor’ sonication (lane 2); markers (lane 3). (C) Brain OGDHC solubilized by the probe sonicator (lane 1); markers (lane 3). Novel 2-oxoglutarate dehydrogenase V. Bunik et al. 4992 FEBS Journal 275 (2008) 4990–5006 ª 2008 The Authors Journal compilation ª 2008 FEBS A comparison of the SDS electrophoretic patterns of partially purified heart and brain complexes is shown in Fig. 1A,B. Varying the concentration of the separat- ing gel (10% in Fig. 1A and 7% in Fig. 1B) allowed for a better resolution of some proteins, in particular, those in band 6. The SDS electrophoretic pattern of our preparation from rat heart mitochondria (Fig. 1A, lane 3; Fig. 1B, lane 1) agrees with the known mobility of the components of bovine heart complexes isolated from total heart extract [23]. According to the molecu- lar mass values for the mature proteins, the components of the 2-oxoglutarate and pyruvate dehydrogenase complexes were ascribed to the major protein bands of our preparation as follows: E1o (band 1), E2p (band 2), E3 (band 3), E2o and the E3-binding component of the pyruvate dehydrogenase complex (protein X; band 4), E1pa (band 6), E1pb (band 8). This was confirmed by nanoLC-MS ⁄ MS identification of the components in the protein bands (Table 1). Our study also showed that there are two isoenzymes of pyruvate dehydrogenase kinases in brain (band 6a). Isoenzymes 2 and 3 were distinguished by three and five specific peptides out of four and six total peptides identified, respectively (Table 1). Interaction of OGDHC with membraneous proteo-lipid particles and its functional significance With the sonication parameters fixed, later elution on size-exclusion chromatography on a Sephacryl HR300 column was observed for OGDHC extracted using detergent compared with OGDHC solubilized by soni- cation only. V e decreased reproducibly, from 47 to 44 mL for brain OGDHC and from 44 to 42 mL for heart OGDHC, with standard deviations in V e between different chromatographies of a certain prepa- ration type of < 1 mL. Concomitantly, the shift in V e was observed for the high molecular mass opalescent peak eluted between the column void volume (V 0 = 38 mL) and the OGDHC activity peak (V e between 42 and 47 mL) (Fig. 2A). Elution near the void volume of the column (Fig. 2A), high opalescence at a relatively low protein level and the dependence of V e on both the detergent and the sonication mode sug- gest that this peak comprises membraneous particles. Membrane vesicles that form spontaneously during homogenization are known as the microsomal fraction [27]. A strong dependence of the elution volume of OGDHC on the elution volume of the opalescent peak (Fig. 2B, correlation coefficient 1.13) points to OG- DHC binding to these membrane particles, with their complex disrupted by the chromatography-accom- plished trapping of the dissociated intermediates. Table 2 shows that the better the separation of OGDHC from microsomes, the more E1o and E3 dissociate from the complex, accompanied by a loss of total OGDHC activity when subjected to chromatogra- phy. Increasing dissociation was obvious from the appearance of the well-defined peak for the compo- nent activities (DV e „ 0; Table 2), which follows the Table 1. MS identification of known components of the 2-oxo acid dehydrogenase complexes from brain. Proteins of the bands shown in Fig. 1 were identified through an NCBI search using MASCOT as described in Experimental procedures. The data for a representative experi- ment are given. Components of the 2-oxoglutarate dehydrogenase complex were also identified in heart. Unless indicated otherwise, matches to rat sequences were found. Molecular mass corresponds to the precursor proteins as given in NCBI. NCBI-provided molecular mass of dihydrolipoyllysine acetyltransferase refers to an incomplete sequence, therefore the true molecular mass from the Expasy data- base, which corresponds to that in the SDS-electrophoresis (Fig.1), is added (marked by asterisk). NA, not analyzed. Band in Fig. 1 Component of the 2-oxo acid dehydrogenase complexes NCBI identifier Molecular mass (Da) Brain Heart Protein score No. peptides matched Protein score No. peptides matched 1 2-Oxoglutarate dehydrogenase (E1o) 62945278 117 419 1131 28 1647 60 2 Dihydrolipoyllysine acetyltransferase (E2p) 220838 57 645 67 166* 443 16 NA 3 Dihydrolipoyl dehydrogenase (E3) 40786469 54 574 579 12 975 36 4 Dihydrolipoyl succinyl transferase (E2o) 55742725 49 236 400 7 709 28 4 Component X 28201978 mus 54 250 126 2 279 7 6a Pyruvate dehydrogenase kinase, isoenzyme 3 21704122 mus 48 064 196 6 NA 6a Pyruvate dehydrogenase kinase 2 subunit variant p45 8895958 44 198 151 4 NA 6b Pyruvate dehydrogenase alpha subunit (E1pa) 57657 43 853 716 20 NA 8 Pyruvate dehydrogenase beta subunit (E1pb) 56090293 39 299 519 26 NA V. Bunik et al. Novel 2-oxoglutarate dehydrogenase FEBS Journal 275 (2008) 4990–5006 ª 2008 The Authors Journal compilation ª 2008 FEBS 4993 overall OGDHC activity peak, and an increased ratio of dissociated to complex-bound activities for E3 and E1o at the corresponding elution volumes. Importantly, the chromatography-induced dissociation into compo- nents and the accompanying loss of total OGDHC activity were dependent on the separation from micro- somes rather than on the protein applied (Table 2; experiment N 1 versus 3). Because of the higher analy- tical sensitivity of the E3-catalyzed NAD + reduction compared with ferricyanide reduction by E1o, the E3-catalyzed reaction allowed a better comparison of the significantly different levels of the component activ- ities obtained in these experiments. However, a similar trend was observed for the two components (Table 2), in good agreement with the known formation of the E1o–E3 subcomplex upon OGDHC dissociation [28]. Separation from microsomes decreases both the total and the specific (lmolÆmin )1 Æmg )1 of protein) activity of OGDHC in the peak. Table 2 shows that purifica- tion of OGDHC by chromatography led to a 30-fold increase in specific activity with a low degree of separation from microsomes (experiment 1), but full separation (experiment 3) resulted in no increase in spe- cific activity, despite the OGDHC fractions containing fewer contaminant proteins. Thus, disruption of the interaction between OGDHC and the microsomal frac- tion during chromatography destabilizes the complex structure and function. At a comparable protein concentration in the column eluate, the fraction of applied OGDHC activ- ity found in the eluate differed dramatically for heart (70%) and brain (10%) complexes. The greater loss of brain OGDHC activity (90%) compared with that from heart complex (30%) was not due to a higher degree of purification, because more proteins co-eluted with OGDHC from brain. This was evident from the additional bands on SDS electrophoresis (bands 6a, 7, 8a in Fig. 1) and the greater heterogeneity indicated by nanoLC-MS ⁄ MS analysis of common bands 1, 3, 4, 5. The tissue specificity of the heterogeneity was mostly due to synaptosomal proteins in the brain preparation, Fig. 2. Gel filtration of brain OGDHC on a Sephacryl HR300 16 ⁄ 60 column. (A) Elution profile, showing attenuance at 280 nm (D 280 ) and the OGDHC activity in arbitrary units (A). (B) Dependence of V e of OGDHC on V e of membraneous fraction, the line is drawn according to the equation: y=1.13x ) 3.05. Table 2. Dependence of the OGDHC activity yield on the separation of OGDHC from microsomes. Partially purified from ‘Bioruptor’-soni- cated mitochondria, OGDHC (40–60 mgÆmL )1 ) was applied to the 12 ⁄ 30 column with Sephacryl S-300. The separation varied due to the dif- ferences in the sample volume and ⁄ or relative content of the microsomes. The interference of the elution volumes of OGDHC and microsomes, I, was calculated from the elution profiles as the percentage of the microsome-including OGDHC fractions to the total number of the OGDHC-containing fractions. Separation of E3 or E1o from the complex upon chromatography was characterized by the difference between the elution volumes, DV e , of the peaks of E3 or E1o and OGDHC and the ratio of the component activities at these V e (A non-bound E3 ⁄ E1o ⁄ A bound E3 ⁄ E1o ). The OGDHC activity yield is the ratio of the total activity of OGDHC in the eluate to the total activity of the OGDHC applied to the column. ND, not determined. No. Total protein applied (mg) Separation of OGDHC and microsomes, (100 ) I ) (%) Dissociation of E1o from OGDHC Dissociation of E3 from OGDHC Total OGDHC activity yield (%) Specific OGDHC activity increase (%) DV e A non-bound E1o A bound E1o DV e A non-bound E3 A bound E3 1 80 25 0 0.3 0 0.8 66 3000 2 40 56 3 0.9 3 1.9 24 300 3 80 100 ND ND 3 2.7 9 100 Novel 2-oxoglutarate dehydrogenase V. Bunik et al. 4994 FEBS Journal 275 (2008) 4990–5006 ª 2008 The Authors Journal compilation ª 2008 FEBS pointing to the presence of synaptosome-derived microsomes in the membraneous fraction accompany- ing brain OGDHC. Structural differences between OGDHC from brain and heart An essential difference between brain and heart OGDHC was revealed by nanoLC-MS ⁄ MS analysis of band 1. In the brain preparation, this band contained both OGDH and OGDHL, a hypothetical isoenzyme of OGDH predicted from the nucleic acid data [16]. Our analysis of 10 band 1 samples from 9 different brain preparations identified the structures of 10–17 peptides which were specific for OGDH and 5–10 pep- tides specific for OGDHL (Table 3, Fig. 3). Although direct quantification of proteins from the nanoLC- MS ⁄ MS peak intensities is difficult, there is a general correlation between the number of protein peptides identified and the amount of protein present in the mixture, if protein size is normalized [13–15]. For OGDH and OGDHL, which have similar molecular masses, the ratio of identified peptides may be taken as an estimate of the relative abundance of the isoen- zymes in the analyzed sample. We calculated this ratio for OGDH and OGDHL, using either the number of all peptides identified or only those specific for the sequences and that were non-redundant (when peptides with the same primary sequence were counted as one). The latter excludes a possible bias due to common peptides, and is thus a better measure of the specific sequence coverage. However, with the high sequence coverage for each of the isoenzymes (Fig. 3), both cal- culations give a similar ratio. The ratio points to a comparable amount of the two isoenzymes in the brain preparation ( 60% OGDH and 40% OGDHL; Table 3). No reproducible enrichment of OGDHC with one of the isoenzymes could be detected in the different OGDHC preparations, for example, isolated with or without detergents, before or after gel-filtra- tion, precipitated by either poly(ethylene glycol) or pH, and collected from different pools of column elu- ate, which may vary in the OGDHC saturation by peripheral components E1o and E3 (Table 3). It is worth noting that the same isoenzyme ratio was observed in both the crude poly(ethylene glycol) frac- tion of the mitochondrial extract and the chromatogra- phy-purified OGDHC (Table 3). Co-purification of the novel isoenzyme with the high molecular mass OGDHC fraction points to OGDHL being the com- plex component, in good agreement with predictions based on the structural analysis [16]. Table 3. Ratio of the peptides characteristic of OGDH and OGDHL isoenzymes in different preparations of brain OGDHC. Samples isolated under the indicated conditions (details in Experimental procedures) were subjected to SDS electrophoresis, and the OGDH ⁄ OGDHL band of 110 kDa was analyzed using nanoLC-MS ⁄ MS. The indicated number of specific peptides refers to the non-redundant peptides only, i.e. the same peptide modified or of a reduced length was not counted. The total number of peptides found by MASCOT search, as described in Experimental procedures is given in parentheses. Isolation conditions OGDH-specific (total) peptides OGDHL-specific (total) peptides Specific (total) peptide ratio (% OGDH : OGDHL) ‘Bioruptor’ + PEG before chromatography 10 (17) 6 (11) 60 : 40 (60 : 40) ‘Bioruptor’ + PEG V e = 43–45 mL 17 (27) 5 (10) 80 : 20 (70 : 30) V e = 43–46 mL 14 (23) 6 (13) 70 : 30 (60 : 40) V e = 43–48 mL 17 (28) 7 (14) 70 : 30 (70 : 30) ‘Bioruptor’ + pH V e = 44–48 mL 12 (20) 10 (16) 50 : 50 (55 : 45) ‘Bandelin’ + PEG V e = 43–46 mL 8 (17) 8 (15) 50 : 50 (50 : 50) OGDHC solubilized by sonication only 8–17 (17–28) average 13 (22) 5–10 average 7 (13) Average 65 : 35 (60 : 40) (50 : 50 to 80 : 20) ‘Bandelin’, 6% Triton X-100 extract + pH V e = 42–48 mL 12 (23) 9 (19) 60 : 40 (55 : 45) ‘Bioruptor’, 1% Chaps extract + pH V e = 45–48 mL 13 (21) 6 (13) 70 : 30 (60 : 40) ‘Bioruptor’, 1% Chaps extract + PEG V e = 45–48 mL 11 (17) 7 (12) 60 : 40 (60 : 40) Detergent-solubilized OGDHC 11–13 (17–23) average 12 (20) 6–9 (12–19) average 7 (15) Average 60 : 40 (60 : 40) (60 : 40 to 70 : 30) V. Bunik et al. Novel 2-oxoglutarate dehydrogenase FEBS Journal 275 (2008) 4990–5006 ª 2008 The Authors Journal compilation ª 2008 FEBS 4995 OGDHL_rat_h MSQLRLLLFRLGP QARKLLATRDIAAFG GRRRSSGPPTTIPRSRGGVSPSYVEEMYFAWLENPQSVHKSWDNFF 74 OGDH_rat_h MFHLRTCAAKLRPLTASQTVKTFSQNKPAAIRTFQQIRCYSAPVAAEPFLSGTSSNYVEEMYCAWLENPKSVHK SWDIFF 80 OGDH_rat_b MFHLRTCAAKLRPLTASQTVKTFSQNKPAAIRTFQQIRCYSAPVAAEPFLSGTSSNYVEEMYCAWLENPKSVHK SWDIFF 80 OGDHL_rat_b MSQLRLLLFRLGP QARKLLATRDIAAFG GRRRSSGPPTTIPRSRGGVSPSYVEEMYFAWLENPQSVHKSWDNFF 74 OGDHL_rat_h QRATKEASVGPAQPQPP AVIQESRASVSSCTKTSKLVEDHLAVQSLIRAYQIRGHHVAQLDPLGILDADLDSF 147 OGDH_rat_h RNTNAGAPPGTAYQSPLSLSRSSLATMAHAQSLVEAQPNVDKLVEDHLAVQSLIRAYQIRGHHVAQLDPLGILDADLDSS 160 OGDH_rat_b RNTNAGAPPGTAYQSPLSLSRSSLATMAHAQSLVEAQPNVDKLVEDHLAVQSLIRAYQIRGHHVAQLDPLGILDADLDSS 160 OGDHL_rat_b QRATKEASVGPAQPQPP AVIQESRASVSSCTKTSKLVEDHLAVQSLIRAYQIRGHHVAQLDPLGILDADLDSF 147 OGDHL_rat_h VPSDLITTIDKLAFYDLQEADLDKEFRLPTTTFIGGSENTLSLREIIRRLESTYCQHIGLEFMFINDVEQCQWIRQKFET 227 OGDH_rat_h VPADIISSTDKLGFYGLHESDLDKVFHLPTTTFIGGQEPALPLREIIRRLEMAYCQHIGVEFMFINDLEQCQWIR QKFET 240 OGDH_rat_b VPADIISSTDKLGFYGLHESDLDKVFHLPTTTFIGGQEPALPLREIIRRLEMAYCQHIGVEFMFINDLEQCQWIRQKFET 240 OGDHL_rat_b VPSDLITTIDKLAFYDLQEADLDKEFRLPTTTFIGGSENTLSLREIIRRLESTYCQHIGLEFMFINDVEQCQWIRQKFET 227 OGDHL_rat_h PGVMKFSIEEKRTLLARLVRSMRFEDFLARKWSSEKRFGLEGCEVMIPALKTIIDKSSEMGVENVILGMPHRGR LNVLAN 307 OGDH_rat_h PGIMQFTNEEKRTLLARLVRSTRFEEFLQRKWSSEKRFGLEGCEVLIPALKTIIDMSSANGVDYVIMGMPHRGRLNVLAN 320 OGDH_rat_b PGIMQFTNEEKRTLLARLVRSTRFEEFLQRKWSSEKRFGLEGCEVLIPALKTIIDMSSANGVDYVIMGMPHRGRLNVLAN 320 OGDHL_rat_b PGVMKFSIEEKRTLLARLVRSMRFEDFLARKWSSEKRFGLEGCEVMIPALKTIIDKSSEMGVENVILGMPHRGRLNVLAN 307 OGDHL_rat_h VIRKDLEQIFCQFDPKLEAADEGSGDVKYHLGMYHERINRVTNRNITLSLVANPSHLEAVDPVVQGKTKAEQFYRGDAQG 387 OGDH_rat_h VIRKELEQIFCQFDSKLEAADEGSGDMKYHLGMYHRRINRVTDRNITLSLVANPSHLEAADPVVMGKTKAEQFYCGDTEG 400 OGDH_rat_b VIRKELEQIFCQFDSKLEAADEGSGDMKYHLGMYHRRINRVTDRNITLSLVANPSHLEAADPVVMGKTK AEQFYCGDTEG 400 OGDHL_rat_b VIRKDLEQIFCQFDPKLEAADEGSGDVKYHLGMYHERINRVTNRNITLSLVANPSHLEAVDPVVQGKTKAEQFYRGDAQG 387 OGDHL_rat_h RKVMSILVHGDAAFAGQGVVYETFHLSDLPSYTTNGTVHVVVNNQIGFTTDPRMAR SSPYPTDVARVVNAPIFHVNADDP 467 OGDH_rat_h KKVMSILLHGDAAFAGQGIVYETFHLSDLPSYTTHGTVHVVVNNQIGFTTDPRMAR SSPYPTDVARVVNAPIFHVNSDDP 480 OGDH_rat_b KKVMSILLHGDAAFAGQGIVYETFHLSDLPSYTTHGTVHVVVNNQIGFTTDPRMARSSPYPTDVARVVNAPIFHVNSDDP 480 OGDHL_rat_b RKVMSILVHGDAAFAGQGVVYETFHLSDLPSYTTNGTVHVVVNNQIGFTTDPRMAR SSPYPTDVARVVNAPIFHVNADDP 467 OGDHL_rat_h EAVIYVCSVAAEWRNTFNKDVVVDLVCYRRRGHNEMDEPMFTQPLMYKQIHKQVPVLKKYADKLIAEGTVTLQEFEEEIA 547 OGDH_rat_h EAVMYVCKVAAEWRNTFHKDVVVDLVCYRRNGHNEMDEPMFTQPLMYKQIRKQKPVLQKYAELLVSQGVVNQPEYEEEIS 560 OGDH_rat_b EAVMYVCKVAAEWRNTFHKDVVVDLVCYRRNGHNEMDEPMFTQPLMYKQIRKQKPVLQKYAELLVSQGVVNQPEYEEEIS 560 OGDHL_rat_b EAVIYVCSVAAEWRNTFNKDVVVDLVCYRRRGHNEMDEPMFTQPLMYKQIHKQVPVLKKYADKLIAEGTVTLQEFEEEIA 547 OGDHL_rat_h KYDRICEEAYGRSKDKKILHIKHWLDSPWPGFFNVDGEPKSMTYPTTGIPEDTLSHIGNVASSVPLEDFKIHTGLSRILR 627 OGDH_rat_h KYDKICEEAFTRSKDEKILHIKHWLDSPWPGFFTLDGQPRSMTCPSTGLEEDILTHIGNVASSVPVENFTIHGGLSRILK 640 OGDH_rat_b KYDKICEEAFTRSKDEKILHIKHWLDSPWPGFFTLDGQPRSMTCPSTGLEEDILTHIGNVASSVPVENFTIHGGLSRILK 640 OGDHL_rat_b KYDRICEEAYGRSKDKKILHIKHWLDSPWPGFFNVDGEPKSMTYPTTGIPEDTLSHIGNVASSVPLEDFKIHTGLSRILR 627 OGDHL_rat_h GRADMTKKRTVDWALAEYMAFGSLLKEGIHVRLSGQDVERGTFSHRHHVLHDQDVDRRTCVPMNHLWPDQAPYTVCNSSL 707 OGDH_rat_h TRRELVTNRTVDWALAEYMAFGSLLKEGIHVRLSGQDVERGTFSHRHHVLHDQNVDKRTCIPMNHLWPNQAPYTVCNSSL 720 OGDH_rat_b TRRELVTNRTVDWALAEYMAFGSLLKEGIHVRLSGQDVERGTFSHRHHVLHDQNVDKRTCIPMNHLWPNQAPYTVCNSSL 720 OGDHL_rat_b GRADMTKKRTVDWALAEYMAFGSLLKEGIHVRLSGQDVERGTFSHRHHVLHDQDVDRRTCVPMNHLWPDQAPYTVCNSSL 707 OGDHL_rat_h SEYGVLGFELGYAMASPNALVLWEAQFGDFHNTAQCIIDQFISTGQAKWVRHNGIVLLLPHGMEGMGPEHSSARPERFLQ 787 OGDH_rat_h SEYGVLGFELGFAMASPNALVLWEAQFGDFNNMAQCIIDQFICPGQAKWVRQNGIVLLLPHGMEGMGPEHSSARPERFLQ 800 OGDH_rat_b SEYGVLGFELGFAMASPNALVLWEAQFGDFNNMAQCIIDQFICPGQAKWVRQNGIVLLLPHGMEGMGPEHSSARPERFLQ 800 OGDHL_rat_b SEYGVLGFELGYAMASPNALVLWEAQFGDFHNTAQCIIDQFISTGQAKWVRHNGIVLLLPHGMEGMGPEHSSARPERFLQ 787 OGDHL_rat_h MSNDDSDAYP-VFTEDFEVSQLYDCNWIVVNCSTPASYFHVLRRQVLLPFR KPLIVFTPKSLLRHPDAKSSFDQMVSGTS 866 OGDH_rat_h MCNDDPDVLPNLQEENFDISQLYDCNWIVVNCSTPGNFFHVLRRQILLPFRKPLIVFTPKSLLRHPEARTSFDEMLPGTH 880 OGDH_rat_b MCNDDPDVLPNLQEENFDISQLYDCNWIVVNCSTPGNFFHVLRRQILLPFR KPLIVFTPKSLLRHPEARTSFDEMLPGTH 880 OGDHL_rat_b MSNDDSDAYP-VFTEDFEVSQLYDCNWIVVNCSTPASYFHVLRRQVLLPFR KPLIVFTPKSLLRHPDAKSSFDQMVSGTS 866 OGDHL_rat_h FQRMIPEDGPAAQSPERVERLIFCTGKVYYDLVKERSSQGLEKQVAITRLEQISPFPFDLIMREAEKYSGAELVWCQEEH 946 OGDH_rat_h FQRVIPEDGPAAQNPDKVKRLLFCTGKVYYDLTRERKARDMAEEVAITRIEQLSPFPFDLLLKEAQKYPNAELAWCQEEH 960 OGDH_rat_b FQRVIPEDGPAAQNPDKVKRLLFCTGKVYYDLTRERKARDMAEEVAITRIEQLSPFPFDLLLKEAQKYPNAELAWCQEEH 960 OGDHL_rat_b FQRMIPEDGPAAQSPERVERLIFCTGKVYYDLVKERSSQGLEKQVAITRLEQISPFPFDLIMREAEKYSGAELVWCQEEH 946 OGDHL_rat_h KNMGYYDYISPRFMTLLGHSRPIWYVGREPAAAPATGNKNTHLVSLRKFLDTAFNLKAFEGKTF 1010 OGDH_rat_h KNQGYYDYVKPRLRTTIDRAKPVWYAGRDPAAAPATGNKKTHLTELQRFLDTAFDLDAFKK FS- 1023 OGDH_rat_b KNQGYYDYVKPRLRTTIDRAKPVWYAGRDPAAAPATGNKKTHLTELQRFLDTAFDLDAFKKFS- 1023 OGDHL_rat_b KNMGYYDYISPRFMTLLGHSRPIWYVGREPAAAPATGNKNTHLVSLRKFLDTAFNLKAFEGKTF 1010 * Fig. 3. Sequence alignment of rat OGDH and OGDHL showing (in color) the peptides identified by nanoLC-MS ⁄ MS in the OGDHC prepara- tion from heart (two upper sequences marked by ‘h’) and brain (two lower sequences marked by ‘b’). Common peptides for the two sequences are shown in red. The sequence-specific peptides are in bold: pink for the OGDH and blue for the OGDHL. The N-terminal cleav- age site, as determined by the sequencing of the truncated bovine E1o [35], is marked by an asterisk above the alignment. Novel 2-oxoglutarate dehydrogenase V. Bunik et al. 4996 FEBS Journal 275 (2008) 4990–5006 ª 2008 The Authors Journal compilation ª 2008 FEBS In contrast to brain OGDHC, no peptide specific for OGDHL was identified in the heart complex, despite the higher protein load and the purity of the E1o band (band 1, lane 3 versus lane 1; Fig. 1A), which resulted in an increase in the sequence coverage (26–36 non-redundant or 45–60 total peptides in inde- pendent determinations). As shown in Fig. 3, heart preparation exhibits either OGDH-specific peptides (pink) or peptides common to the two proteins (red), but OGDHL-specific peptides (blue) were found in the brain preparation only. Thus, whereas only the known OGDH component coded by chromosome 7 in humans [29,30] was identified by nanoLC-MS ⁄ MS in OGDHC from heart, brain complex, purified using the same procedure, contained comparable amounts of both OGDH (chromosome 7) and OGDHL (human chromosome 10) [31–33] proteins (Table 3, Fig. 3), which were identified even at lower purifica- tion yields. Another structural feature of brain 2-oxo acid dehy- drogenase complexes is seen from SDS electrophoresis. The E3 component (band 3), the majority of which is associated with OGDHC as shown above, is hardly visible in the brain preparation (Fig. 1A, lanes 1–2) compared with the heart preparation (Fig. 1A, lane 3). Despite the low E3 level, under standard assay condi- tions we did not observe any activation of brain OGDHC in the presence of or following preincubation with at least a 10-fold protein excess of E3 (commer- cial bovine enzyme). Thus, even the low levels of E3 seen in the brain preparation were able to support maximal OGDHC reaction rates. This is in accordance with published data on the rate-limiting role of the E1o component in Reaction (1) catalyzed by the com- plex [34]. It is known that the binding of E3 to OG- DHC is mediated by E1o, with the proteolytic removal of a small N-terminal fragment of E1o impairing bind- ing [28,35]. However, the lower E3 level in brain OGDHC was not due to E1o proteolysis, because several peptides preceding the cleavage site (marked by asterisk in Fig. 3) were identified in both isoenzymes by MS analysis. This was in good agreement with the mobility of the E1o band on SDS electrophoresis (Fig. 1), which corresponded to the molecular mass of non-proteolysed E1o (110 kDa), being higher than that of truncated E1o with an apparent molecular mass of 94 kDa [28,35]. Because full extraction of the OGDHC activity from heart mitochondria required 1% Chaps, we checked whether the E3 deficiency of brain OGDHC could be due to the membrane binding of its E3. Figure 1A shows that 1% Chaps extract of the pellet fraction (lane 2) obtained after removal of E3-deficient OGDHC (lane 1) did not contain E3. By contrast, when the activity of E3 and OGDHC was followed in parallel upon sonication, a significant portion of the E3 activity solubilized before the overall activity of OGDHC. Taken together, these findings indicate that the E3 deficiency of brain 2-oxo acid dehydrogenase complexes (Fig. 1) is not due to mem- brane binding of the E3 component. Compared with heart complexes, easier dissociation of this component appears to occur upon sonication of brain mitochon- dria. Indeed, E3 was better presented in complexes that were detergent-extracted after a less efficient soni- cation (Fig. 1C). Sonication by ‘Bioruptor’ (Fig. 1A,B) was nevertheless preferred for the isolation, because it gave reproducible results and did not lead to the high molecular mass aggregates (150–300 kDa) observed in the SDS electrophoresis of OGDHC solubilized with the probe sonicator (Fig. 1C). A different supramolecular organization for OG- DHC from brain and heart was further supported by size-exclusion chromatography, in which proteins of a higher molecular mass are eluted more rapidly, i.e. at a lower elution volume V e . As mentioned above, under the same sonication conditions the activity peak of OGDHC from brain eluted later than that of OGDHC from heart: 44 versus 42 mL for soluble OGDHC and 47 versus 44 mL for Chaps-extracted OGDHC. The later elution corresponds to a lower molecular mass for the purified brain complex, which agrees with its lower saturation with peripheral E3 component, as dis- cussed above. As inferred from both SDS electropho- resis (Fig. 1A,B) and size-exclusion chromatography, the different supramolecular organization of heart and brain OGDHC was further supported by the MS-based estimate of the relative abundance of the complex components in the preparation (Table 4). Abundance coefficients were calculated as described in Experimental procedures according to the previously developed approach of comparative proteomics [13– 15]. As indicated by the standard deviation values for the preparations from one tissue, these ratios showed good agreement in different experiments. However, the values were clearly different for OGDHC from heart and brain. Table 4 shows that in OGDHC from brain the E2o ⁄ E1o and E3 ⁄ E1o ratios (40 and 70%, respec- tively) were no more than half those in the heart com- plex (120 and 140%, respectively). Because the heart preparation did not possess OGDHL, we also com- pared the abundance coefficients for brain OGDHC when based on the OGDH content only. The brain ratios remained lower than those of heart (Table 4). Thus, compared with the heart complex, OGDHC isolated from brain showed an excess of the first component over the second and third. The decrease in V. Bunik et al. Novel 2-oxoglutarate dehydrogenase FEBS Journal 275 (2008) 4990–5006 ª 2008 The Authors Journal compilation ª 2008 FEBS 4997 the MS-based abundance of E2o and E3 components in brain OGDHC correlated with the low intensity of the E3 band in SDS electrophoresis and a lower molecular mass of OGDHC from brain versus heart upon size-exclusion chromatography. Thus, the data obtained using the three independent approaches sug- gest differences in the supramolecular organization of OGDHC isolated from heart and brain. Saturation of brain OGDHC with 2-oxoglutarate Kinetic analysis of the dependence of the overall activ- ity of brain OGDHC on the saturation with 2-oxoglut- arate agrees with the presence in the preparation of two isoenzymes of 2-oxoglutarate dehydrogenase which are functionally competent in Reaction (1). Sol- ubilized with or without detergents, brain OGDHC did not exhibit standard Michaelis–Menten kinetics (Fig. 4). That is, simulations using the parameters yielded by the double reciprocal linearization of the experimental data showed a systematic shift in the the- oretical curves to lower rates at high 2-oxoglutarate saturation (Fig. 4A). In view of the identification of the second isoenzyme of OGDH by nanoLC-MS ⁄ MS, we introduced a second saturation function into the equation. As shown in Fig. 4B, this abolished the inconsistencies between the experiment and the simula- tion, resulting in a satisfactory description of the sys- tem behavior at both low and high substrate saturation. The better correspondence between the experimental data and the two-saturation model is obvious not only from visual inspection of the coinci- dence between the experimental points and theoretical curves in Fig. 4B compared with Fig. 4A, but also from an increase in the correlation coefficients (from 0.804 and 0.918 in Fig. 4A to 0.986 and 0.997 in Fig. 4B). The biphasic saturation parameters provided in the legend to Fig. 4 show that K m,1 and K m,2 values, as well as the contributions of V 1 and V 2 to the maximal reaction rate (V=V 1 + V 2 ), were similar for soluble and Chaps-extracted OGDHC. Based on three independent experiments, the following parameters were obtained: K m,1 = 0.07 ± 0.02 mm; K m,2 =0.40 ± 0.07 mm; V 1 ⁄ (V 1 + V 2 ) = 45 ± 4%; V 2 ⁄ (V 1 + V 2 ) = 55 ± 4%. It is worth noting that the simulation-derived partial contributions of V 1 and V 2 to the overall V value are close to the relative abun- dance of the isoenzymes as determined by nanoLC- MS ⁄ MS (35–40% of OGDHL and 60–65% of OGDH; Table 3). Moreover, detergents are known to desensi- tize cooperative and allosteric enzymes to effectors, but they do not significantly change the kinetic param- eters of brain OGDHC (Fig. 4), in accordance with the lack of change in the isoenzyme ratio caused by detergents (Table 3). Thus, the parameters obtained by simulation of the v(S) dependence according to the model suggested by the MS identification of the two isoenzymes are reproducible and in a good agreement with the MS-based abundance of the isoenzymes in the OGDHC preparation. Taken together, the kinetic and MS data support functional competence of the novel isoenzyme in the overall OGDHC reaction and differ- ent saturation of the two isoenzymes of OGDH with 2-oxoglutarate. Discussion Identification of novel OGDH isoform and its implication in brain metabolism Distinguishing proteins with highly similar primary structures, such as the products of alternative splic- ing or of different genes (isoforms or isoenzymes), represents one of the challenges in characterizing the cellular proteome [15]. The modern development of MS analysis provides strong advantages over immu- nological approaches to address this challenge, because determination of isoform-specific peptides distinguishes unambiguously between isoforms which may show cross-reactivity to antibodies [36]. In this study, we successfully applied nanoLC-MS ⁄ MS to identify both the known OGDH and the hypotheti- cal OGDHL in OGDHC partially purified from brain mitochondria. At the same time, only the Table 4. Relative abundance of the OGDHC components in the preparations. Abundance index, A, corresponds to the number of peptides detected by nanoLC-MS ⁄ MS, normalized to the molecular mass of the OGDHC component (see Experimental procedures). The E2o and E3 abundance indexes were related to that of either E1o (the sum of OGDH + OGDHL) or OGDH taken as 100%. The data are presented as the average values ± SD. Tissue E1o (OGDH + OGDHL) OGDH E2o E3 A % E1o A % OGDH A % E1o ⁄ %OGDH A % E1o ⁄ %OGDH Brain 0.34 ± 0.03 100 0.22 ± 0.04 100 0.14 ± 0.05 40 ⁄ 60 0.23 ± 0.01 70 ⁄ 100 Heart 0.48 ± 0.1 100 0.57 ± 0.01 120 0.66 ± 0.18 140 Novel 2-oxoglutarate dehydrogenase V. Bunik et al. 4998 FEBS Journal 275 (2008) 4990–5006 ª 2008 The Authors Journal compilation ª 2008 FEBS known OGDH was determined in a similar prepara- tion of the complex from heart. Expression of the novel OGDHL component of OGDHC in brain is in accord with the isolation of OGDHL cDNA from brain tissue [31–33], whereas earlier cloning of the OGDH gene used a fetal liver cDNA library [29]. Thus, apart from housekeeping OGDH, OGDHL is synthesized in brain. Integration of the OGDHL isoenzyme into the complex, which was predicted by our structure– function analysis [16], is evident from the constant ratio of OGDH and OGDHL during the purification of brain OGDHC (Table 1), elution of OGDHL in the high molecular mass fraction corresponding to OGDHC, and biphasic saturation with 2-oxoglutarate (Fig. 4), indicative of a functional competence of the two isoenzymes in Reaction (1) catalyzed by the complex. Identification of the two isoenzymes of OGDHC by MS was taken into account in the kinetic modeling of the dependence of the overall reaction rate of brain OGDHC on the 2-oxoglutarate concentration (Fig. 4). Indeed, the dependence can be better described by the sum of two saturation processes than by standard Michaelis–Menten kinetics (Fig. 4), which is in accord with the contribution to the overall reaction rate of the two isoenzymes having different affinities to 2-oxo- glutarate. Moreover, simulation of this model revealed that partial contributions of each of the isoenzymes, V 1 and V 2 into the overall reaction rate V are in a good agreement with the MS-based relative abundance of the isoenzymes (Table 3), suggesting that OGDH and OGDHL have similar catalytic rates. The compat- ibility of parameters derived from kinetic modeling and MS analysis strongly supports the plausibility of the model assuming two isoenzymes for interpretation of the kinetic data. High correlation between the simu- lated dependence and experimental data within this model (Fig. 4B) did not justify further refinement of the model. Thus, kinetic analysis of OGDHC from brain provides experimental evidence in support of an earlier prediction from genome data that OGDHL is a functionally active isoenzyme of OGDH [16]. Further- more, the kinetics is indicative of an approximately sixfold difference between K m,1 and K m,2 characterizing saturation of the two isoenzymes with 2-oxoglutarate. Compared with OGDHC from heart and adrenal glands, which are half-saturated with 2-oxoglutarate at 0.2 mm [37–39], OGDHC from brain requires higher concentrations for full saturation (K m,2 = 0.40 ± 0.06 mm), being sensitive to lower concentrations of 2-oxoglutarate (K m,1 = 0.07 ± 0.02 mm). Possessing the two isoenzymes which provide the different K m values, brain OGDHC may thus respond to an expanded interval in the 2-oxoglutarate levels. The differential regulation of brain OGDH isoenzymes by the substrate may also address the physiological needs of brain tissue to establish different steady-state concentrations of 2-oxoglutarate, depending on cellular conditions, compartment or type. Compared with other tissues, physiological concentrations of glutamate in brain differ not only between regions and cell types, Fig. 4. Kinetic analysis of brain OGDHC saturation with 2-oxogluta- rate. Hollow circles, soluble OGDHC; filled circles, detergent- extracted OGDHC. Dependence of the reaction rate v (arbitrary units of the fluorescence change dFÆmin )1 Æmg )1 of protein) on the 2-oxoglutarate concentration ([S]) was approximated by a single Michaelis–Menten curve v=170*[S] ⁄ (0.07 + [S]), r 2 = 0.804 for soluble OGDHC and v=480*[S] ⁄ (0.09 + [S]) for detergent- extracted OGDHC, r 2 = 0.918 (A) or the sum of the two Michaelis– Menten curves v=110*[S] ⁄ (0.07 + [S]) + 170*[S] ⁄ (0.47 + [S]), r 2 = 0.986 for the soluble OGDHC and v=350*[S] ⁄ (0.09 + [S]) + 320*[S] ⁄ (0.42 + [S]), r 2 = 0.997 for detergent-extracted OGDHC (B). Details of the simulation procedure are given in Experi- mental procedures. V. Bunik et al. Novel 2-oxoglutarate dehydrogenase FEBS Journal 275 (2008) 4990–5006 ª 2008 The Authors Journal compilation ª 2008 FEBS 4999 [...]... differing from that of the heart complex Established differences in the supramolecular structure and stability of brain versus heart OGDHC correlate with the presence in the brain complex of the novel isoenzyme, OGDHL, agree with previous structure–function analysis of protein–protein interactions in OGDHC and provide important insights into physiologically relevant issues A more pronounced dissociation... ratio, which is governed by the brain-specific isoenzymes of glutamate dehydrogenase and isoforms of mitochondrial glutamate carrier [41] Differences in supramolecular organization of brain and heart OGDHC In this study, we characterized the relative abundance of the OGDHC components in partially purified complexes from brain and heart (Table 4) by using MS-based estimates of protein abundance This semiquantitative... pyruvate dehydrogenase complex This is in a good agreement with the E3 deficiency of the rat brain pyruvate dehydrogenase complex, which was observed with purified complex [43] Independently, these and our results indicate that E3 saturation of the 2-oxo acid dehydrogenase complexes is lower in brain than in heart Even if the MS-based abundance of brain OGDHC components (Table 4) is distorted by a loss of. .. a sink for E2o, thus promoting the dissociation of brain OGDHC into components during chromatography-induced separation of OGDHC from the mitochondrial membrane Taken together, the data suggest that interaction with the mitochondrial membrane stabilizes brain OGDHC, whereas substitution of this interaction for that with synaptic membrane interferes with the integrity of the complex This may explain... with the ratio established previously by alternative approaches using highly purified preparations of heart OGDHC That is, the stoichiometry of the enzymatic components determined through the content of cofactors bound to the highly purified OGDHC from heart was shown to be 1 : 1 : 1.5 [42] Taking into account that each subunit of the complex components binds one cofactor, this ratio is in good agreement... E3 binding agrees with the pre-existing structural difference critical for binding the N-terminal domain ( 10 kDa) which has several deletions in OGDHL compared with OGDH (Fig 3) The reduced affinity of brain OGDHC to E3 may provide additional means to regulate the 2-oxoglutarate plus CoA-dependent production of ROS in brain mitochondria because complex-bound E3 is needed for this side reaction of OGDHC... studies of (sub)cellular proteomes [13–15] was very useful in our comparative structural characterization of non-homogeneous OGDHC from heart and brain, because it enabled us to study the complex which is prone to dissociation and inactivation upon purification Applying this approach to partially purified OGDHC from heart, we also showed that the component ratio determined by MS analysis in this study is in. .. Brilliant Blue-stained protein bands of interest were excised and in- gel digested in an adapted manner according to Shevchenko et al [62] Gel pieces were washed twice in 0.1 m NH4HCO3 exchanged with acetonitrile, followed by drying in a vacuum centrifuge The proteins were reduced by rehydrating the gel pieces in 10 mm dithiothreitol for 45 min at 56 °C The thiol groups of the cysteine side chains were subsequently... preparation of 2-oxoglutarate dehydrogenase complex from pig heart muscle J Biol Chem 242, 902–907 43 Koeplin R, Ulrich H & Bisswanger H (1991) Pyruvate dehydrogenase complex from rat brain: characterization, reaction with enantiomers of lipoic acid and distribution in the cerebellum as determined by monoclonal antibodies In Biochemistry and Physiology of Thiamin Diphosphate Enzymes (Bisswanger H &... is known for some 2-oxoglutarate dehydrogenases [49], whereas in heart complex, OGDH dimers are bound to the core [22] The different structure may, in particular, contribute to the difference in the observed Km value of OGDHC for 2-oxoglutarate, because Km is known to be affected by the catalytic steps of the overall reaction, being different from the dissociation constant (KS) of 2-oxoglutarate binding . saturation of the two isoenzymes of OGDH with 2-oxoglutarate. Discussion Identification of novel OGDH isoform and its implication in brain metabolism Distinguishing. Novel isoenzyme of 2-oxoglutarate dehydrogenase is identified in brain, but not in heart Victoria Bunik 1,2 , Thilo Kaehne 3 ,