Alpha Subunit Of Pyruvate Dehydrogenase Research Articles

Mitochondrial Encephalomyopathy Associated with Pyruvate Dehydrogenase Complex Deficiency: Eight Clinical Cases

Background. Defects in pyruvate dehydrogenase complex (PDC), involved in the glycolysis products integration into the cells' energy metabolism, are one of the reasons of mitochondrial pathology development. The diagnosis of this condition can be pretty complicated also due to the lack of description of such patients with encephalomyopathy associated with PDC deficiency in Russian population. Clinical Cases Description. We have performed the analysis of clinical manifestations polymorphism of progressive mitochondrial encephalomyopathy caused by pathogenic variants in nuclear X linked gene, PDHA1 (encodes alpha subunit of pyruvate dehydrogenase), in 8 boys aged from 1 to 8 years. The adverse perinatal period was mentioned in all cases. The major features of symptom complex by the time of hospital examination were psychomotor retardation, ataxy, myopathic manifestations. Dystonic attacks were observed in 2 sibs. All patients had changes on brain magnetic resonance imaging: in basal ganglia in 6 children and ventriculomegaly in 2 children. All children had lactic acidosis. Clinical examination has shown that 4 patients had severe damage of nervous system, other 4 patients had moderate damage. Missense mutations in the PDHA1 gene were revealed in 6 children, insertions and duplications including 6 and 16 base pairs — in 2 children. The moderate positive dynamics was noticed as a result of complex treatment of children: stabilization of the overall condition, no metabolic crises, decrease in frequency of dystonic attacks. Conclusion. The clinical polymorphism of mitochondrial encephalomyopathy associated with PDC deficiency is described. The differences in manifestations of severe and moderate forms of disease are shown. The presented description may be useful for medico-genetic counseling and providing medico-genetic care for families.

Hexokinase II promotes the Warburg effect by phosphorylating alpha subunit of pyruvate dehydrogenase.

ObjectiveTumor cells rely heavily on glycolysis regardless of oxygen tension, a phenomenon called the Warburg effect. Hexokinase II (HKII) catalyzes the first irreversible step of glycolysis and is often overexpressed in tumor cells. Mitochondrial HKII couples glycolysis and oxidative phosphorylation while maintaining mitochondrial membrane integrity. In this study, we investigated the role of HKII in promoting the Warburg effect in cancer cells.MethodsHKII-mediated phosphorylation of the alpha subunit of pyruvate dehydrogenase (PDHA1) was tested in HEK293T cells and clear cell renal cell carcinoma (ccRCC) specimens using gene knockdown, western blotting, immunohistochemistry, and immunofluorescence.ResultsIt was determined that HKII could not only transform glucose into glucose-6-phosphate, but also transfer the phosphate group of ATP onto PDHA1. In addition, it was found that HKII increased the phosphorylation of Ser293 on PDHA1, decreasing pyruvate dehydrogenase (PDH) complex activity and thus rerouting the metabolic pathway and promoting the Warburg effect. The overexpression of HKII correlated with the phosphorylation of PDHA1 and disease progression in ccRCC.ConclusionsThe data presented here suggest that HKII is an important biomarker in the evaluation and treatment of cancer.

Phosphorylation status of pyruvate dehydrogenase distinguishes metabolic phenotypes of cultured rat brain astrocytes and neurons

Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria, and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons versus astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture, but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDH alpha). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorylated PDH alpha. Dephosphorylation of astrocytic PDH alpha restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed.

Monitoring phosphorylation of the pyruvate dehydrogenase complex

The pyruvate dehydrogenase multienzyme complex (PDC) is a key regulatory point in cellular metabolism linking glycolysis to the citric acid cycle and lipogenesis. Reversible phosphorylation of the pyruvate dehydrogenase enzyme is a critical regulatory mechanism and an important point for monitoring metabolic activity. To directly determine the regulation of the PDC by phosphorylation, we developed a complete set of phospho-antibodies against the three known phosphorylation sites on the E1 alpha subunit of pyruvate dehydrogenase (PDHE1α). We demonstrate phospho-site specificity of each antibody in a variety of cultured cells and tissue extracts. In addition, we show sensitivity of these antibodies to PDH activity using the pyruvate dehydrogenase kinase-specific inhibitor dichloroacetate. We go on to use these antibodies to assess PDH phosphorylation in a patient suffering from Leigh’s syndrome. Finally, we observe changes in individual phosphorylation states following a small molecule screen, demonstrating that these reagents should be useful for monitoring phosphorylation of PDHE1α and, therefore, overall metabolism in the disease state as well as in response to a myriad of physiological and pharmacological stimuli.

Import, processing, and assembly of the alpha- and beta-subunits of chloroplast pyruvate dehydrogenase.

Sequence comparisons were used to identify cDNAs potentially encoding the alpha- and beta-subunits of chloroplast pyruvate dehydrogenase. Coupled in-vitro transcription plus translation was used to synthesize radiolabeled pyruvate dehydrogenase alpha- and beta-subunit precursor proteins. When the precursors were incubated with intact pea (Pisum sativum L.) seedling chloroplasts in the presence of ATP, they were imported and proteolytically processed. In contrast, there was no import or processing of the precursors by isolated, intact pea seedling mitochondria. Monospecific antibodies to the recombinant pyruvate dehydrogenase alpha-subunit were additionally able to co-precipitate radiolabeled pyruvate dehydrogenase beta-subunit, indicating association between subunits after import and processing. Furthermore, size-exclusion chromatography was used to identify an alphabeta heterodimer that is an intermediate in the assembly of the native alpha2beta2 heterotetrameric enzyme.

Regulation of mammalian pyruvate dehydrogenase alpha subunit gene expression by glucose in HepG2 cells.

We report the effect of glucose on the expression of the gene encoding the pyruvate dehydrogenase (E1) alpha subunit (E1alpha) in human hepatoma (HepG2) cells. Total pyruvate dehydrogenase complex activity as well as the levels of protein and mRNA of the E1alpha subunit were significantly increased in HepG2 cells cultured in medium containing 16.7 mM glucose compared with 1.0 mM glucose for a period of 4 weeks. The level of E1alpha mRNA was elevated approx. 2-fold in HepG2 cells cultured for 24 h in medium containing 16.7 mM glucose compared with 1 mM glucose. This effect was specific to glucose and independent of insulin. Nuclear run-on assays and promoter analysis indicate that the glucose-induced increases in the levels of E1alpha mRNA in HepG2 cells are due to increased transcription of the human E1alpha (PDHA1) gene. Mutational analysis of the E1alpha promoter region has identified two regions, from -78 to -73 bp (CCCCTG) and from -8 to -3 bp (GCGGTG), that are responsible for the effect of glucose on promoter activity; the former exhibits a larger effect. These two sequences represent new variations of the carbohydrate-response element that has been identified in other genes. The stimulation of E1alpha promoter activity by glucose was abolished by okadaic acid at 100 nM but not at 5 nM, suggesting that glucose-mediated regulation of pyruvate dehydrogenase complex E1alpha gene transcription involves a phosphorylation/dephosphorylation mechanism, possibly involving protein phosphatase-1.

Molecular Analysis of Two Pyruvate Dehydrogenase Kinases from Maize

Two maize cDNAs were isolated and sequenced that had open reading frames with approximately 37% amino acid identity to mammalian pyruvate dehydrogenase kinases. Both maize kinase sequences contain the five domains with conserved signature residues typical of procaryotic two-component histidine kinases. Sequence comparisons identified six other highly conserved motifs that are proposed to be specific to pyruvate dehydrogenase kinases. In addition, specific Trp and Cys residues are also invariant in these sequences. The maize cDNAs are 1332 (PDK1) and 1602 (PDK2) nucleotides in length, encoding polypeptides with calculated molecular masses of 38,867 and 41,327 Da that share 77% amino acid identity. Reverse transcriptase-polymerase chain reaction analysis with oligonucleotide-specific primers revealed a differential expression pattern for the two isoforms. PDK1 and PDK2 were expressed in Escherichia coli with N-terminal His6 tags to facilitate purification. The recombinant proteins migrated at 44 and 48 kDa, respectively, during SDS-polyacrylamide gel electrophoresis. Anti-PDK1 antibodies immunoprecipitated 75% of pyruvate dehydrogenase kinase activity from a maize mitochondrial matrix fraction, and recognized a matrix protein of 43 kDa. Recombinant PDK2, expressed as a fusion with the maltose-binding protein, inactivated kinase-depleted maize pyruvate dehydrogenase complex when incubated with MgATP, coincident with incorporation of 32P from [gamma-32P]ATP into the alpha subunit of pyruvate dehydrogenase.

Mutations in the X-linked E1 alpha subunit of pyruvate dehydrogenase leading to deficiency of the pyruvate dehydrogenase complex.

Human PDH complex deficiency is an extremely heterogeneous disease in its presentation and clinical course. In an investigation at the level of the gene into ten cases of PDH complex (E1) deficiency, we found that all had mutations in the coding sequence of the X-linked E1 alpha gene while the E1 beta coding sequence was normal. Six of these patients (three males, three females) had missense mutations resulting in a changed amino acid residue in the E1 alpha subunit at positions amino acid 148 (in two siblings), 170, 202, 234 and 263 of the mature protein. Two of the females had one normal E1 alpha gene and one with a deletion at the sites of tandem repeats of AGTAAGA and TAT respectively. The two remaining females also had one normal E1 alpha gene and one with an insertion. Both insertions, one of 2 bp and one of 4 bp, occurred in DNA hotspots normally associated with deletions. Only two of these ten mutations have been reported in other patients previously. In the five cases (including the two siblings) where parent DNA was available, only in one case could the same mutation be found in the patient as well as the maternal genomic DNA.

Characterization of cDNA clones for the alpha subunit of pyruvate dehydrogenase from Ascaris suum

The pyruvate dehydrogenase complex of the adult parasitic nematode Ascaris suum functions in the reducing environment present in their anaerobic mitochondria. These organelles use fumarate and enoyl CoAs as terminal electron acceptors instead of oxygen. A λgt11 cDNA library was constructed from RNA isolated from adult ascarid muscle. Partial clones for the pyruvate dehydrogenase α subunit were isolated by screening the λgt11 library with a specific antiserum. Full-length clones (type I) were identified in a cDNA library prepared from RNA isolated from early embryos. During the hybridization screening, a second type of cDNA clone (type II) was identified. The nucleotide sequences of both clones are presented. The predicted amino acid sequences of the mature proteins are 91% identical to one another and about 55% identical to the predicted sequences of the α subunit of human pyruvate dehydrogenase. Northern blots were used to examine the expression of both mRNAs in various larval stages and in tissues of the adult. Type I sequences are found mainly in adult muscle. Type II sequences are abundant in third-stage larvae as well as in adult muscle.

Novel regulatory enhancer in the nuclear gene of the human mitochondrial ATP synthase beta-subunit.

The system coordinating expressions of nuclear coded mitochondrial proteins was investigated by examination of the 5'-flanking region of the human mitochondrial ATP synthase beta-subunit gene. The promoter activity was measured by a transient expression of a chloramphenicol acetyltransferase (CAT) gene connected with various 5'-deletion mutants of the 5'-flanking region. In this experiment, at least two regions enhanced this promoter activity and at least one region repressed it. In one of the enhancing regions, a consensus sequence was found for the genes of other mitochondrial proteins such as those for cytochrome c1 (Suzuki, H., Hosokawa, Y., Nishikimi, M., and Ozawa, T. (1989) J. Biol. Chem. 264, 1368-1374) and the pyruvate dehydrogenase alpha-subunit (Maragos, C., Hutchison, W. M., Hayasaka, K., Brown, G. K., and Dahl, H.-H. M. (1989) J. Biol. Chem. 264, 12294-12298; Ohta, S., Endo, H., Matsuda, K., and Kagawa, Y. (1989) Ann. N. Y. Acad. Sci. 573, 458-460). The characteristics of this enhancing element were examined by introducing a synthetic oligonucleotide element into the CAT plasmid with a deleted enhancing element. The resulting plasmid showed full recovery of promoter activity, and this activity was independent of the orientation or location of the insert. Therefore, this is an enhancer that may be common to the nuclear genes of some mitochondrial proteins involved in energy transduction.

Nucleotide and deduced amino acid sequence of the alpha subunit of yeast pyruvate dehydrogenase

A 413-base cDNA insert encoding a portion of the alpha subunit of pyruvate dehydrogenase (E 1α; EC 1.2.4.1) from Saccharomyces cerevisiae was isolated from a λgtll cDNA library by immunoscreening and by hybridization with an oligonucleotide probe which corresponded to the amino acid sequence around the phosphorylation site of E 1α. This cDNA was subcloned, sequenced and used as a probe to isolate two additional cDNA inserts which were subcloned and sequenced. These overlapping clones comprised the carboxyl-terminal part of E 1α. To identify the missing nucleotide sequence, the polymerase chain reaction was used to amplify yeast genomic DNA with synthetic oligonucleotide primers based on the amino-terminal sequence of E 1α and the 5′ end of one of the cDNA clones. Three DNA fragments were isolated and sequenced. The composite nucleotide sequence has an open reading frame of 1260 nucleotides encoding a putative presequence of 33 amino acids and a mature protein of 387 amino acids ( M r = 42,703). Hybridization analysis showed that the size of the mRNA is about I.4 kilobases.

In situ protein phosphorylation in hippocampal tissue slices

We have studied the subcellular distribution of phosphoproteins in intact hippocampal slices and examined factors that regulate their phosphorylation and dephosphorylation in situ. The presence of Ca2+ in slice equilibration and prelabeling buffers and high-K+-induced depolarization markedly increased 32Pi incorporation into endogenous proteins. Ca2+-stimulatory effects were significantly reduced by Ca2+-channel blockers and the calmodulin antagonist W-13. Certain proteins were dephosphorylated in situ, and their dephosphorylation was dependent on both Ca2+ and depolarization. A number of proteins phosphorylated in situ was similar to those previously characterized in synaptic fractions phosphorylated in vitro. Many phosphoproteins were identified on the basis of molecular weight, isoelectric point, immunoreactivity, and phosphopeptide mapping; these included the 87 kDa substrate of protein kinase C, synapsin I, the 50 and 60 kDa subunits of Ca2+/calmodulin-dependent protein kinase II (CKII), tubulin, B-50, the alpha-subunit of pyruvate dehydrogenase and myelin basic proteins. CKII phosphorylation in situ appeared similar but not identical to its in vitro counterpart. Phosphopeptide mapping analysis of in situ labeled substrate proteins indicated that cAMP-, Ca2+/calmodulin-, and Ca2+/phospholipid-dependent protein kinases were all active in slice preparations under basal conditions. Increased 32Pi labeling of hippocampal proteins following tissue depolarization appeared to be associated with increased activity of endogenous protein kinases since depolarization did not result in 32Pi-labeling of any new phosphoproteins.

Characterization of cDNAs encoding human pyruvate dehydrogenase alpha subunit.

A cDNA clone (1423 base pairs) comprising the entire coding region of the precursor form of the alpha subunit of pyruvate dehydrogenase (E1 alpha) has been isolated from a human liver cDNA library in phage lambda gt11. The first 29 amino acids deduced from the open reading frame correspond to a typical mitochondrial targeting leader sequence. The remaining 361 amino acids, starting at the N terminus with phenylalanine, represent the mature mitochondrial E1 alpha peptide. The cDNA has 43 base pairs in the 5' untranslated region and 210 base pairs in the 3' untranslated region, including a polyadenylylation signal and a short poly(A) tract. The nucleotide sequence of human liver E1 alpha cDNA was confirmed by the nucleotide sequences of three overlapping fragments generated from human liver and fibroblast RNA by reverse transcription and DNA amplification by the polymerase chain reaction. This consensus nucleotide sequence of human liver E1 alpha cDNA resolves existing discrepancies among three previously reported human E1 alpha cDNAs and provides the unambiguous reference sequence needed for the characterization of genetic mutations in pyruvate dehydrogenase-deficient patients.

Heterogeneous expression of protein and mRNA in pyruvate dehydrogenase deficiency.

Deficiency of pyruvate dehydrogenase [pyruvate:lipoamide 2-oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1], the first component of the pyruvate dehydrogenase complex, is associated with lactic acidosis and central nervous system dysfunction. Using both specific antibodies to pyruvate dehydrogenase and cDNAs coding for its two alpha and beta subunits, we characterized pyruvate dehydrogenase deficiency in 11 patients. Three different patterns were found on immunologic and RNA blot analyses. (i) Seven patients had immunologically detectable crossreactive material for the alpha and beta proteins of pyruvate dehydrogenase. (ii) Two patients had no detectable crossreactive protein for either the alpha or beta subunit but had normal amounts of mRNA for both alpha and beta subunits. (iii) The remaining two patients also had no detectable crossreactive protein but had diminished amounts of mRNA for the alpha subunit of pyruvate dehydrogenase only. These results indicate that loss of pyruvate dehydrogenase activity may be associated with either absent or catalytically inactive proteins, and in those cases in which this enzyme is absent, mRNA for one of the subunits may also be missing. When mRNA for one of the subunits is lacking, both protein subunits are absent, suggesting that a mutation affecting the expression of one of the subunit proteins causes the remaining uncomplexed subunit to be unstable. The results show that several different mutations account for the molecular heterogeneity of pyruvate dehydrogenase deficiency.

Molecular cloning and expression of the major protein kinase C substrate of platelets.

In platelets, agonists that stimulate phosphoinositide turnover cause the rapid phosphorylation of a protein of apparent relative molecular mass (Mr) 40-47,000, called P47, by protein kinase C (PKC). Diverse identities have been ascribed to P47 including lipocortin, inositol 1,4,5-trisphosphate 5-phosphomonoesterase, pyruvate dehydrogenase alpha subunit and an actin regulatory protein. We have isolated human P47 clones by immunological screening of a lambda gt11 complementary DNA library from HL-60 cells, a human promyelocytic leukaemia cell line. P47 recombinants thus identified hybridized to a 3.0 kilobase (kb) messenger RNA in mature white blood cell lines; the same mRNA was induced in HL-60 cells during differentiation. A 1,050 base pair (bp) open reading frame that could encode a protein of Mr40,087 was confirmed by comparison with peptide sequences from platelet P47, and by expression of the putative recombinant P47 in E. coli and in vitro. The P47 sequence appears to have been conserved throughout vertebrate evolution, and is not similar to any other known sequence including human lipocortin and the alpha subunit of pyruvate dehydrogenase. The P47 protein contains a potential Ca2+-binding 'EF-hand' structure and a region that strongly resembles known PKC phosphorylation sites.

Mitochondrial protein phosphorylation and cardiomyopathy in genetically diabetic mice: The effect of estrone treatment

The alpha-subunit of pyruvate dehydrogenase and succinyl-CoA synthetase are phosphorylated after incubation of cardiac mitochondria from genetically diabetic mice with [gamma-32P]ATP. There is significantly increased incorporation of 32P into pyruvate dehydrogenase from diabetic mice when compared to controls. The enhanced rate of pyruvate dehydrogenase phosphorylation correlates well with the previously reported defective oxidative metabolism and decreased activity of this enzyme from diabetic mice. The relationship between abnormal mitochondrial function and development of cardiomyopathy in the diabetic mice has been studied further by in vivo estrone treatment. The results indicate that ultrastructural alterations of myocardium are closely associated with the defective pyruvate oxidation (via phosphorylation of pyruvate dehydrogenase) and both processes can be prevented by 7-12 weeks estrone treatment.

Characterization of cyclic AMP-dependent phosphorylation of neuronal membrane proteins.

We examined the patterns of cyclic AMP-dependent protein phosphorylation in membranes prepared from rat cortical synaptosomes following gel electrophoresis and autoradiography. We determined the optimum pH (6.2), time (20 s), Mg2+ concentration (10 mM) and cyclic AMP concentration (5 microM) for the reaction. We also found that the detergents Triton X-100 and gramicidin S enhanced cyclic AMP-dependent protein phosphorylation. Inhibitors of the Na+, K+ ATPase (ouabain, NaF, vanadate) enhanced protein phosphorylation. This effect occurred in the presence but not in the absence of detergent. The addition of purified bovine brain cyclic AMP-dependent protein kinase catalytic subunit enhanced membrane protein phosphorylation. The addition of homogeneous neural (bovine brain) and non-neural (bovine skeletal muscle) cyclic AMP-dependent protein kinase type II regulatory subunit partially inhibited protein phosphorylation. Both neural and non-neural regulatory subunits behaved similarly. In addition to cyclic AMP-dependent phosphorylation, the alpha-subunit of pyruvate dehydrogenase (Mr = 41,000) is phosphorylated in a cyclic AMP-independent fashion. We also examined the phosphorylation pattern of membranes prepared from rat heart and found that the number of acceptor substrates was much less than that from the nervous system.

Brain protein phosphorylation [formula omitted]: Selective substrate action of insulin

Insulin action on [32P]-phosphate incorporation into brain membranes was determined. Hippocampal homogenate tissue was phosphorylated with [32P]-ATP, and insulin was introduced at various times before or after ATP addition. With 50μM Mg ++ in the medium, insulin selectively stimulated the phosphorylation of a 47kD phosphoprotein, Protein Fl. This effect required the prior presence of ATP. No effect of insulin on other phosphoproteins, or on [32P]-phosphate incorporation into TCA-precipitated material, was observed under these conditions. At 1mM Mg ++, insulin selectively decreased the phosphorylation of the alpha-subunit of pyruvate dehydrogenase. Insulin had no effect on other phosphoproteins, or on [32P]-phosphate incorporation into TCA-precipitated material under these conditions. The present study suggests a role for insulin in the modulation of brain protein phosphorylation. Since Protein Fl is phosphorylated by exogenous C kinase, and is likely the CNS-specific B-50 protein, these data also indicate a brain-specific function for insulin, possibly by action on a Ca ++/phospholipid protein kinase.

Long-term potentiation in the hippocampal slice: Possible involvement of pyruvate dehydrogenase

Tetanic stimulation of fibers in the hippocampal slice preparation produces long-term potentiation (LTP) and also decreases the in vitro incorporation of phosphate into the alpha subunit of pyruvate dehydrogenase (αPDH) 12. This paper describes 6 experiments that were undertaken to replicate this observation. Hippocampal slices were incubated in a specially designed chamber and stimulated with a tungsten wire electrode in the stratum radiatum for 1 s at 100 Hz. Two minutes after the tetanus, the stimulated slices were removed alternately with control (not tetanized) slices and each group was pooled for subcellular fractionation and labeling of the fractions with [ 32P]ATP. Proteins were separated by electrophoresis and relative 32P contents of 41K and 50K protein bands were studied. Tetanic stimulation of the stratum radiatum did not affect subsequent phosphorylation of a 50K M r protein that has been reported to be altered by perforant path activation 2. Stimulation also had no effect on pyruvate dehydrogenase enzyme activity or on the ratio of active (dephosphorylated) to inactive enzyme. In most cases tetanic stimulation produced no significant change in the in vitro phosphorylation of this enzyme. Only under one set of conditions, labeling with 250 μM [γ- 32P] ATP for 10 s, was a decrease in the in vivo labeling of αPDH shown to be statistically significant. These findings suggest that LTP is not necessarily accompanied by an initial change in PDH phosphorylation level or activity but may be associated with a decrease in the kinase activity directed toward this protein.

Apparent identity of alpha-subunit of pyruvate dehydrogenase and the protein phosphorylated in the presence of glutamate in P2-fractions of rat cerebral cortex.

Addition of L-glutamate or several citric acid cycle intermediates stimulated the phosphorylation of a protein with apparent molecular weight of 43,000 ( P43 ) in P2-fractions from rat cerebral cortex, and this phosphorylation was inhibited by dichloroacetic acid, a specific inhibitor of pyruvate dehydrogenase kinase. Comparison of several molecular properties of phosphorylated P43 and the phosphorylated alpha-subunit of pyruvate dehydrogenase indicated that both proteins are extracted by a similar procedure and have an identical apparent molecular weight and isoelectric point. Furthermore, digestion of both phosphorylated proteins by several different proteases in the presence of SDS yielded a similar pattern of phosphorylated peptides indicating that these proteins have a considerable sequence homology. Thus, various pieces of evidence indicate that P43 and the alpha-chain of pyruvate dehydrogenase are very similar if not identical. The possible implication of a glutamate stimulated phosphorylation of pyruvate dehydrogenase for long term potentiation and epilepsy is discussed.