Phosphorylation of Mitochondrial Elongation Factor Tu in Ischemic Myocardium
Basis for Chloramphenicol-Mediated Cardioprotection
The objective of this study was to identify the mitochondrial proteins that undergo changes in phosphorylation during global ischemia and reperfusion in the isolated rabbit heart. We also assessed whether the cardioprotective intervention of ischemic preconditioning affected mitochondrial protein phosphorylation. We established a reconstituted system using isolated mitochondria and cytosol from control or ischemic hearts. We found that phosphorylation of a 46-kDa protein on a serine residue was increased in ischemia and that phosphorylation was reduced in control or preconditioned hearts. Using 2D gel electrophoresis and mass spectrometry, we have identified the 46-kDa protein as mitochondrial translational elongation factor Tu (EF-Tumt). These data reveal that ischemia and preconditioning modulate the phosphorylation of EF-Tumt and suggest that the mitochondrial protein synthesis machinery may be regulated by phosphorylation. Phosphorylation of mitochondrial EF-Tu has not been previously described; however, in prokaryotes, EF-Tu phosphorylation inhibits protein translation. We hypothesized that phosphorylation of mitochondrial EF-Tu would inhibit mitochondrial protein translation and attempted to reproduce the effect with inhibition of mitochondrial protein synthesis by chloramphenicol. We found that chloramphenicol pretreatment significantly reduced infarct size, suggesting that mitochondrial protein synthesis is one determinant of myocardial injury during ischemia and reperfusion.
Ischemia and reperfusion lead to myocardial injury through a variety of mechanisms. Mitochondrial dysfunction is a characteristic feature of ischemic injury, and recovery of mitochondrial function is essential for myocardial contractility.1 Ischemic preconditioning confers myocardial protection through a brief period of ischemia and reperfusion preceding the more sustained ischemia/reperfusion insult.2 Preconditioning is characterized by earlier recovery of mitochondrial function with more efficient resynthesis of ATP.
Ischemia and reperfusion profoundly affect mitochondria, and preservation of their integrity and function is critical to salvage.3–6 Oxidative phosphorylation is transiently increased after reperfusion but then diminishes; pyridine nucleotides are lost from the mitochondria, and respiration through complex I is impaired; superoxide production is increased, possibly through retrograde electron flow through complex I; the permeability transition pore opens, associated with loss of calcium homeostasis; and cytochrome c is released.1,7–10 However, it is not clear whether these mitochondrial alterations are initiated by an intrinsic response to the low oxygen tension of ischemia or arise in part due to changes in the cytosol. Cytosolic alterations are known to include acidosis, increased inorganic phosphate, elevated calcium, and a rise in long-chain acyl coenzyme A. In addition, a variety of signal transduction pathways are activated during ischemia and reperfusion. We have previously shown that c-Jun NH2-terminal kinase (JNK) translocates from cytosol to mitochondria in response to ischemia/reperfusion, and that in a model of metabolic inhibition in adult rabbit cardiomyocytes, inhibition of JNK is protective.11 It is clear from these diverse studies that ischemia and reperfusion activate cytosolic signals that target the mitochondria to modulate their response during ischemia and reperfusion, and furthermore, that preconditioning also involves signaling from cytosol to mitochondria.
We sought to identify mitochondrial targets of cytosolic signals generated by ischemia, reperfusion, and preconditioning through the use of a reconstituted system consisting of isolated mitochondria and cytosol from control, ischemic, or preconditioned hearts. In the present study, we report that cytosol from ischemic but not control or preconditioned hearts stimulates the phosphorylation of the mitochondrial translational elongation factor Tu (EF-Tumt), by a kinase present in mitochondria. Mitochondria contain an organelle-specific protein-synthesizing system that is essential for the synthesis of the 13 polypeptides encoded by the mitochondrial genome, all of which are components of the electron transfer complexes and the F0F1 ATP synthase located in the mitochondrial inner membrane and matrix, respectively. During mitochondrial protein synthesis, EF-Tumt forms a ternary complex with GTP and mitochondrial aminoacyl-tRNAs (aa-tRNA) and delivers the aa-tRNA to the A-site of the ribosome.12 Studies of prokaryotic EF-Tu have shown that phosphorylation on threonine 382 prevents ternary complex formation and thereby inhibits protein synthesis.13
Materials and Methods
Langendorff Perfusion and Global Ischemia/Reperfusion
The global ischemia protocol was adapted from that of Tsuchida et al.14 All procedures were approved by the Animal Care and Use Committee at The Scripps Research Institute (TSRI). In brief, the heart was excised from the anesthetized rabbit and quickly cannulated onto the Langendorff perfusion apparatus. The heart was perfused with Krebs-Ringer buffer for 15 minutes before ischemia/reperfusion episodes. No-flow ischemia was maintained for 30 minutes and reperfusion was accomplished by restoring flow for 15 minutes (unless otherwise indicated). Ischemic preconditioning was induced by three 5-minute cycles of no-flow ischemia and reperfusion immediately preceding the regular ischemia and reperfusion. The efficacy of these interventions was verified by measurement of creatine kinase release and infarct size measurement using triphenyl tetrazolium chloride (TTC) staining.15,16
Isolation of Mitochondria and Cytosol
Upon completion of global ischemia, the heart was removed from the cannula and the ventricles were minced in 20 mL per heart of ice-cold MSE buffer (in mmol/L, mannitol 225, sucrose 75, EGTA 1, Na3VO4 1, and HEPES-KOH 20 [pH 7.4]). The heart was further polytron-homogenized for 5 seconds at maximal power output by a PowerGen 125 (Fisher Scientific) equipped with a 10-mm-diameter rotor knife. The homogenate was centrifuged for 10 minutes at 600g, 4°C. The pellet was discarded and the supernatant was centrifuged for 10 minutes at 10 000g to pellet mitochondria and lysosomes. The supernatant (crude cytosol) was further centrifuged for 30 minutes at 100 000g to obtain particulate-free cytosol (S100). The 10 000g-pellet from the previous centrifugation was resuspended in 10 mL of MSE buffer and centrifuged for 10 minutes at 8000g. This wash step was repeated once. The final pellet was resuspended in 3 mL of MSE buffer and was further purified by hybrid Percoll/metrizamide discontinuous gradient purification consisting of 5 mL of 6% Percoll, 2 mL of 17% metrizamide, and 2 mL of 35% metrizamide, all prepared in 0.25 mol/L sucrose and set up in 13-mL tubes.17 Three milliliters of the sample was overlaid on top of the gradient and centrifuged for 20 minutes at 50 000g, 4°C, using a Beckman SW41 rotor. The mitochondrial fraction at the interface between 17% and 35% metrizamide was collected and diluted at least 10-fold with MSE buffer, followed by centrifugation for 10 minutes at 10 000g to remove metrizamide. The pellet was resuspended in 20 mL of MSE buffer and centrifuged again. The final pellet was resuspended in 3 mL of MSE buffer and aliquots were stored at −80°C. Protein concentration was determined using the Bradford assay, and for all experiments, equal amounts of mitochondrial protein were loaded on the gels. Cytosol concentrations were adjusted to be equal in all conditions before incubating with mitochondria.
Suborganellar Fractionation of Mitochondria
We used a modification of the method of Comte and Gautheron18 to fractionate mitochondria (see the online data supplement available at http://www.circresaha.org).
Metabolic Inhibition of Adult Cardiomyocytes
The isolation of adult rabbit cardiomyocytes and metabolic inhibition were as previously described.19 Cardiomyocytes were disrupted by nitrogen cavitation.20 The mitochondria and cytosol were isolated as above except that no gradient purification was used.11
Labeling, Purification, and Identification of Mitochondrial Phosphoprotein
For phosphorylation reactions, 100 μg of purified mitochondria was incubated in MSE buffer supplemented with 25 mmol/L HEPES-KOH [pH 7.5], 10 mmol/L magnesium acetate, 10 μmol/L ATP (cold), and 10 μCi [γ-32P]ATP for 30 minutes at 30°C with or without 250 μg of cytosol. The reaction mixture was subsequently centrifuged for 5 minutes at 10 000g. The mitochondrial pellet was resuspended in 500 μL of MC buffer and washed twice. The mitochondrial proteins were resolved on a 12% polyacrylamide gel, transferred to nitrocellulose, and detected by autoradiography.
For protein kinase inhibition experiments, cytosols were incubated with the inhibitors at the indicated concentrations, then mitochondria were added and the reaction was initiated with the addition of ATP. Serine/threonine kinase inhibitors (Calbiochem, catalogue No. 539572) included bisindolylmaleimide I, 10 nmol/L; H-89, 48 μmol/L; protein kinase G inhibitor, 86 μmol/L; ML-7, 0.3 μmol/L; KN-93, 0.37 μmol/L; and staurosporine, 10 nmol/L. Tyrosine kinase inhibitors (Calbiochem, catalogue No. 657021) included genistein, 25 μmol/L; PP2, 5 nmol/L; AG490, 15 μmol/L; AG1296, 1 μmol/L; and AG1478, 3 nmol/L.
2D Gel Electrophoresis, Mass Spectrometry, and Phosphoamino Acid Analysis
Details can be found in the online data supplement.
Column Chromatography Purification of EF-Tu and Detection of p46
Mitochondria were labeled with [γ-32P]ATP and fractionated as above to obtain the matrix fraction. The mitochondrial matrix components were resolved by anion exchange chromatography on a 1-mL DEAE-Sepharose Fast-Flow column (Pharmacia) and eluted with a stepwise salt gradient (40 to 500 mmol/L KCl, in steps of 10 mmol/L, in a buffer containing 20 mmol/L Tris×HCl, pH 7.4). Fractions (0.5 mL) were collected, concentrated, and buffer-exchanged to 40 mmol/L KCl in 20 mmol/L Tris×HCl [pH 7.4] by use of a centrifugal filter concentrator (Ultrafree-4, Millipore). Protein fractions were resolved by 12% SDS-PAGE. Immunoblot analysis for EF-Tu was performed as described.21
Infarct Size Measurement
The measurement of infarct size was essentially identical to that detailed by Downey16 except the method of quantitation. After the TTC reaction, the heart slices were scanned into TIFF files and analyzed with Adobe Photoshop 5.5. The images were digitally manipulated in an identical manner to ensure equivalent outcome. The brightness and contrast were adjusted so that in the histogram essentially only red and white colors remained on the spectrum, corresponding to noninfarcted and infarcted regions, respectively. The histogram counts of red and white were recorded. The percent infarction was calculated as white counts divided by the sum of red plus white counts. To examine the effect of chloramphenicol (CAP) on infarction, 100 μg/mL CAP was included in the buffer throughout the entire procedure: 15 minutes of stabilization, 30 minutes of global ischemia, and 2 hours of reperfusion.16 As a control for the possible interference of CAP with TTC staining, CAP was added to the last 15 minutes of the 2-hour reperfusion.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
The phosphorylation pattern of mitochondrial proteins was analyzed in hearts subjected to global ischemia for 30 minutes followed by reperfusion for 15 minutes and compared with control perfused hearts and those subjected to preconditioning before ischemia and reperfusion. Because [γ-32P]orthophosphate labeling of whole hearts was impractical, mitochondria were prepared from hearts and incubated with [γ-32P]ATP. We observed phosphorylation of a number of mitochondrial proteins, but mitochondria from ischemic hearts consistently demonstrated greater phosphorylation (1.4-fold, P<0.01) of a single protein of 46 kDa (see Table and Figure 1A). The extent of phosphorylation of this protein (designated p46) from preconditioned hearts was not significantly different from control hearts. These results indicate that phosphorylation of p46 is inversely regulated by ischemia/reperfusion and preconditioning.
To determine if phosphorylation of p46 was regulated by cytosolic factors, cytosol was prepared from isolated control perfused hearts and from hearts subjected to global ischemia and reperfusion. These cytosols were then incubated with freshly isolated mitochondria from a normal rabbit heart, in the presence of [γ-32P]ATP. Phosphorylation of p46 in the presence of control cytosol was quite low but increased when normal mitochondria were incubated in the presence of cytosol from ischemic hearts (Figure 1B). Mitochondria incubated in the absence of any cytosol also demonstrated phosphorylation of p46, suggesting that a factor present in normal cytosol suppressed phosphorylation of p46 by an endogenous kinase.
To verify that this phosphorylation activity is associated with cardiomyocytes and not due to other cell types in the heart, we prepared cytosol from isolated adult rabbit cardiomyocytes incubated in the presence or absence of metabolic inhibitors (2-deoxyglucose and KCN).11 Cytosol from the control cardiomyocytes again suppressed the phosphorylation of p46. However, cytosol from the metabolically inhibited cardiomyocytes, either during metabolic inhibition or 10 minutes after recovery, was unable to inhibit the phosphorylation of the 46-kDa mitochondrial protein (Figure 1B).
Previously, we showed that JNK immunoreactivity and kinase activity translocated to mitochondria after ischemia/reperfusion but not after ischemia alone.11 Although p46 phosphorylation could be detected in the absence of metabolic recovery, we wondered whether JNK mediated phosphorylation of p46. To examine this possibility, cytosol was immunodepleted of JNK or p38 mitogen-activated protein kinase (MAPK). Mitochondria were then incubated with immunodepleted control or ischemic cytosol in the presence of [γ-32P]ATP. As indicated in Figure 2A, immunodepletion of JNK or p38 MAPK did not attenuate the increase in phosphorylation mediated by ischemic cytosols, indicating that neither of these protein kinases participates in the phosphorylation.
To gain additional information about the possible signal transduction pathway involved, we incubated the mitochondria with control or ischemic cytosols in the presence of a variety of kinase inhibitors and assessed the effect on phosphorylation of p46 in the mitochondria. We first tested a panel of serine/threonine kinase inhibitors, which included the broad-spectrum inhibitor staurosporine, and inhibitors of protein kinase C (PKC), PKA, Ca2+/calmodulin (CaM) kinase II, myosin light chain kinase, and PKG (see Materials and Methods for details). Mitochondria are recognized to have an associated protein kinase A that is anchored to the membrane by an A-kinase anchoring protein (AKAP). However, H-89, a PKA inhibitor, did not affect phosphorylation of p46 (data not shown), suggesting that PKA was not involved. None of the inhibitors in the serine/threonine kinase panel suppressed phosphorylation of p46. We then tested tyrosine kinase inhibitors including genistein, a broad-range inhibitor effective against EGFR and src; PP2, an inhibitor of p56 (lck), p59 (fynI), and Hck; and AG490, an inhibitor of JAK2. The only inhibitor that reduced phosphorylation was genistein (n=3) (Figure 2B). To distinguish between a tyrosine kinase in cytosol and one endogenous to the mitochondria, we incubated mitochondria from an ischemic heart with genistein and found that again genistein was able to inhibit the phosphorylation of p46 (Figure 2C).
To identify p46, the inner-membrane proteins of control and ischemic cytosol-treated mitochondria were analyzed by 2D SDS-PAGE gels under identical conditions. The isoelectric point (pI) of p46 appeared to be ≈6.5. In a preparative 2D gel for mass spectrometry analysis, three closely spaced Coomassie-stained spots on the 2D gel of inner-membrane proteins corresponding to radiolabeled p46 on the autoradiograph were selected for analysis by MALDI mass spectrometry (Figure 3A).
The obtained MALDI spectra from the three spots of interest revealed that all three spots represented the same protein. A total of 12 mass fingerprints were obtained and were subjected to a database search, but no protein could be successfully matched. Therefore, the sample obtained from the middle gel spot (Figure 3A) was further analyzed by nanoelectrospray mass spectrometry (MS). All peptide mass fingerprints that were observed by MALDI analysis were also found in the nanoelectrospray MS spectrum. All peptides observed as double-charged ions were analyzed in separate MS/MS experiments. Not all peptides fragmented in a way that a complete ion series could be obtained. Four of the analyzed peptides fragmented well enough to allow complete or partial sequencing. One peptide consisting of 12 amino acids was sequenced completely (Figure 3B), yielding AEAGDNI(L)GAI(L)VR. “L” in parentheses represents the possible alternative, because MS/MS is not able to distinguish residues I and L. The sequence I(L)I(L)DAVTYIPV was obtained from a second peptide of 13 amino acids, which was sequenced except for the last three residues. Two more peptides were partially sequenced yielding 6 and 4 amino acids, which are, respectively, EEI(L)DNA and YVSE.
The two longer-sequenced peptides were subjected to a BLAST search and were fully matched (100%) with the human and the bovine mitochondrial precursor of the elongation factor Tu (SwissProt accession numbers P49411 and P49410, respectively). Although the rabbit sequence is not in the database, the EF-Tu sequence is highly conserved between human and bovine (>94% identity). We obtained partial cDNA sequence for rabbit EF-Tumt (see online data supplement). For the partial sequence obtained, rabbit shares 92% homology with the human sequence and 95% homology with bovine at the amino acid level. One peptide sequence obtained by mass spectrometry fell within the partial cDNA sequence and matched exactly.
To verify that the phosphoprotein was indeed EF-Tumt, we performed immunoblot analysis of a sample of radiolabeled mitochondrial protein. Antibody to EF-Tumt21 recognized a series of spots that corresponded to the phosphoprotein (Figure 4A). To further confirm that the phosphoprotein represented EF-Tumt, mitochondrial extracts were partially purified on a DEAE-Sepharose anion exchange column following a protocol developed for the purification of EF-Tumt.21 Fractions were analyzed for the presence of EF-Tumt and for the p46 phosphoprotein. As shown in Figure 4B, the radiolabeled phosphoprotein (top panel) eluted in exactly the same fractions as EF-Tumt (bottom panel). Because EF-Tumt and EF-Tsmt elute as a complex on DEAE-Sepharose, this procedure depends on different biochemical properties than 2D gel electrophoresis.21 It is unlikely that another protein would copurify with EF-Tu in both schemes. Based on these studies, we conclude that the phosphoprotein is indeed EF-Tumt.
The foregoing observations indicated that EF-Tumt undergoes phosphorylation in vitro. To ascertain whether EF-Tumt was endogenously phosphorylated, mitochondria were isolated from heart in the presence of phosphatase inhibitors and subjected to 2D gel electrophoresis and immunodetection of EF-Tumt. As shown in Figure 4A (inset), the antibody against EF-Tumt detected two or possibly three spots that differed slightly by isoelectric point. The distribution of these multiple spots is comparable to that seen in the in vitro phosphorylation experiments and most likely represents the addition of acidic phosphate groups. The presence of three spots suggests the existence of at least two phosphorylation sites. This observation provides evidence that EF-Tumt is phosphorylated in vivo, even in control hearts. It also raises the possibility that the in vitro phosphorylation that is increased after ischemia could represent “back-phosphorylation” of available unphosphorylated sites, and that in control and preconditioned hearts, EF-Tumt could actually be highly phosphorylated (thereby leaving few sites available for the incorporation of radioactive phosphate in the in vitro reaction). Amino acid hydrolysis demonstrated that EF-Tumt is phosphorylated on serine (see online data supplement). This is surprising in light of our observation that genistein inhibited phosphorylation.
Phosphorylation of EF-Tu in prokaryotes inhibits protein translation. We hypothesized that the same might be true for eukaryotic EF-Tu. If so, then inhibition of mitochondrial protein synthesis would be expected to reproduce the effects of EF-Tumt phosphorylation. To test this possibility, we pretreated hearts with CAP, a potent inhibitor of mitochondrial protein synthesis, and examined the effects on infarct size after ischemia and reperfusion. As shown in Figure 5, CAP treatment reduced the infarct size from 64.1 (±4.1) to 43.1 (±6.4) (mean±SEM), suggesting that inhibition of mitochondrial protein synthesis may be cardioprotective. CAP administered in the last 15 minutes of the 2-hour reperfusion was not protective (infarct size 69.2±5.7).
A number of protein kinases are activated during ischemia, reperfusion, or preconditioning, and at least two of these are believed to translocate to the mitochondria. It is possible that either a small molecule or a cytosolic kinase could transmit a signal to the mitochondria to stimulate phosphorylation of EF-Tumt. In fact, we observed that cytosol from normal hearts suppresses phosphorylation of EF-Tumt, whereas the absence of any cytosol, or the presence of ischemic cytosol, favors phosphorylation. Because our experiments were performed in the presence of phosphatase inhibitors, this could represent a balance between kinases and phosphatases that are differentially regulated by cytosolic factors.
Because phosphorylation of EF-Tumt is increased in response to ischemia, we were interested in determining the effect of ischemic preconditioning on EF-Tumt phosphorylation. To measure the activity of the endogenous mitochondrial kinase with respect to EF-Tumt phosphorylation, we isolated mitochondria from control, ischemic, and preconditioned hearts. The purified mitochondria were incubated with [γ-32P]ATP in the absence of cytosol. Preconditioning diminished the amount of phosphorylation seen at 15 minutes of reperfusion (Figure 1). As previously noted, mitochondria incubated in buffer only demonstrated a basal level of phosphorylation of EF-Tumt, whereas cytosol from control or preconditioned hearts suppressed phosphorylation. Cytosol from ischemic hearts, or mitochondria prepared from ischemic hearts, stimulated phosphorylation of EF-Tumt. These observations suggest that the cytosol of normal, metabolically active cardiomyocytes is effective in regulating the phosphorylation of EF-Tumt, but that the cytosol of ischemic or metabolically inhibited cells has lost the ability to regulate this phosphorylation process. An alternative interpretation is that there is a factor present in cytosols from ischemic hearts that stimulates phosphorylation of EF-Tumt. The altered phosphorylation of EF-Tumt in ischemic versus control cytosols is unlikely to be due to dilution of [γ-32P]ATP by different concentrations of cytosolic ATP nor due to altered ATP transport through the adenine nucleotide translocator, as other phosphoprotein bands showed similar levels of phosphorylation under all conditions. This phosphorylation was increased in mitochondria from hearts subjected to ischemia and 15 minutes of reperfusion (Figure 1), but the level decreased to baseline if reperfusion was extended to 90 minutes (data not shown). These results indicate that phosphorylation of EF-Tumt is regulated by ischemia and by preconditioning. Members of the MAPK family have been implicated in ischemic injury11,22,23 and in preconditioning.24–26 However, immunodepletion of JNK and p38 did not affect the phosphorylation of EF-Tumt, suggesting that this may be regulated by other kinase pathways. The finding that genistein inhibited phosphorylation and that EF-Tumt is phosphorylated on serine suggests that the signal transduction pathway responsible for EF-Tumt phosphorylation involves both a tyrosine kinase and a serine/threonine kinase. Work is underway to identify the immediate upstream kinase.
What is the significance of EF-Tumt phosphorylation? Mitochondria contain an organelle-specific protein-synthesizing system that is essential for the synthesis of the 13 polypeptides encoded by the mitochondrial genome. All of the protein products of this system are components of the electron transport complexes and the F0F1 ATP synthase located in the inner membrane of mitochondria. Previous investigations have identified alterations in the function of electron transport complexes I and IV, which include mitochondrial encoded subunits, whereas complex II, which only contains nuclear-encoded subunits, is unaffected. We were surprised to find that the major target of phosphorylation was EF-Tumt. EF-Tu is a GTPase that serves to bind aa-tRNAs and bring them to the ribosome. In bacteria, phosphorylation of EF-Tu on threonine 382 has been shown to prevent ternary complex formation.13,27 This Thr residue is highly conserved in EF-Tu and is present in the mammalian mitochondrial factors whose sequences are currently available. This residue is, therefore, likely to be a Thr in rabbit EF-Tumt. The site of serine phosphorylation in rabbit EF-Tumt remains to be determined. In addition, the number of phosphorylation sites is undetermined. The MALDI spectra were identical for all three spots that differed by isoelectric points, which is consistent with two phosphorylation sites. However, it is also possible that glycosylation or minor proteolysis at the N- or C-terminus could give rise to a spot with a shifted pI without a detectable difference in molecular mass. EF-Tumt has not previously been reported to be phosphorylated and further studies will be required to determine the effect of this modification on its activity in protein synthesis. The fact that EF-Tumt phosphorylation is enhanced in ischemic hearts compared with control or preconditioned hearts leads us to believe that the phosphorylation carries some physiological significance. It is attractive to speculate that EF-Tumt phosphorylation leads to inactivation of mitochondrial protein synthesis with consequent loss of mitochondrial subunits essential for function of complexes I and IV. This may explain the cardioprotective effects of CAP treatment, particularly if complex I participates in superoxide production during reperfusion. In addition, inhibition of mitochondrial protein synthesis may be energy-sparing, allowing the utilization of limited ATP for more essential needs.
The abundance of EF-Tumt has been shown to increase in tumor cells28 and nearly disappears in pacing-induced heart failure, along with a number of other mitochondrial proteins.29 Escherichia coli EF-Tu has also been reported to function as a chaperone.30 It is possible that this activity is regulated by phosphorylation. EF-Tumt may play a role analogous to the heat shock proteins by participating in mitochondrial protein refolding after ischemic injury. Further work will be necessary to understand the functional consequences of EF-Tumt phosphorylation and to understand the basis of CAP-mediated cardioprotection.
This work was supported by NIH HL 60590 and HL 61518 and by a Pew Scholar’s Award (to R.A.G.). The mass spectrometric analysis was performed on a Voyager DE-Str and a Q-Star instrument in the University of California–San Francisco (UCSF) Mass Spectrometry Facility, which is supported in part by the National Center for Research Resources (RR01614). The authors thank Robert L. Engler, Bernard M. Babior, and members of their laboratories for generously sharing equipment and technical advice. We appreciate the technical assistance of Marc Heiser and Hai-Ling Li. We are grateful to the TSRI Protein Core Facility for 2D gel electrophoresis services. Oligonucleotide synthesis and DNA sequencing were performed in the Molecular and Experimental Medicine DNA Core Facility supported by the Sam and Rose Stein Endowment Fund.
Original received February 16, 2001; revision received July 13, 2001; accepted July 13, 2001.
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