Phosphoglycerate Mutase Activity Research Articles

GLYCOLYTIC ENZYMES AS VACCINES AGAINST SCHISTOSOMIASIS: TESTING SCHISTOSOMA MANSONI PHOSPHOGLYCERATE MUTASE IN MICE.

Schistosomiasis is a globally burdensome parasitic disease caused by flatworms (blood flukes) in the genus Schistosoma. The current standard treatment for schistosomiasis is the drug praziquantel, but there is an urgent need to advance novel interventions such as vaccines. Several glycolytic enzymes have been evaluated as vaccine targets for schistosomiasis, and data from these studies are reviewed here. Although these parasites are canonically considered to be intracellular, proteomic analysis has revealed that many schistosome glycolytic enzymes are additionally found at the host-interactive surface. We have recently found that the intravascular stage of Schistosoma mansoni (Sm) expresses the glycolytic enzyme phosphoglycerate mutase (PGM) on the tegumental surface. Live parasites display PGM activity, and suppression of PGM gene expression by RNA interference diminishes surface enzyme activity. Recombinant SmPGM (rSmPGM) can cleave its glycolytic substrate, 3-phosphoglycerate and can both bind to plasminogen and promote its conversion to an active form (plasmin) in vitro, suggesting a moonlighting role for this enzyme in regulating thrombosis in vivo. We found that antibodies in sera from chronically infected mice recognize rSmPGM. We also tested the protective efficacy of rSmPGM as a vaccine in the murine model. Although immunization generates high titers of anti-SmPGM antibodies (against both recombinant and native SmPGM), no significant differences in worm numbers were found between vaccinated and control animals.

Role of Bacillus subtilis Spore Core Water Content and pH in the Accumulation and Utilization of Spores' Large 3-Phosphoglyceric Acid Depot, and the Crucial Role of This Depot in Generating ATP Early during Spore Germination.

The development of Bacillus spore cores involves the accumulation of 3-phosphoglycerate (3PGA) during sporulation, following core acidification to ~6.4, and before decreases in core water content occur due to Ca-dipicolinc acid (CaDPA) uptake. This core acidification inhibits phosphoglycerate mutase (PGM) at pH 6.4, allowing 3PGA accumulation, although PGM is active at pH 7.4. Spores' 3PGA is stable for months at 4 °C and weeks at 37 °C. However, in wild-type spore germination, increases in core pH to 7.5-8 and in core water content upon CaDPA release and cortex peptidoglycan hydrolysis allow for rapid 3PGA catabolism, generating ATP; indeed, the earliest ATP generated following germination is from 3PGA catabolism. The current work found no 3PGA in those Bacillus subtilis spores that do not accumulate CaDPA during sporulation and have a core pH of ~7.4. The ATP production in the germination of 3PGA-less spores in a poor medium was minimal, and the germinated spores were >99% dead. However, the 3PGA-replete spores that germinated in the poor medium accumulated >30 times more ATP, and >70% of the germinated spores were found to be alive. These findings indicate why 3PGA accumulation during sporulation (and utilization during germination) in all the Firmicute spores studied can be crucial for spore revival due to the generation of essential ATP. The latter finding further suggests that targeting PGM activity during germination could be a novel way to minimize the damaging effects of spores.

Structural analysis of EhPSP in complex with 3-phosphoglyceric acid from Entamoeba histolytica reveals a basis for its lack of phosphoglycerate mutase activity

Entamoeba histolytica phosphoserine phosphatase (EhPSP), a regulatory enzyme in the serine biosynthetic pathway, is also a structural homolog of cofactor-dependent phosphoglycerate mutase (dPGM). However, despite sharing many of its catalytic residues with dPGM, EhPSP displays no significant mutase activity. In the current work, we determined a crystal structure of EhPSP in complex with 3-PGA to 2.5 Å resolution and observed striking differences between the orientation of 3-PGA bound to EhPSP and that to its other homologous structures. We also performed computational modeling and simulations of the intermediate 2,3-bisphosphoglyceric acid into the active site of EhPSP to better understand its mechanistic details. Based on these results and those of a similar study with the dPGMs from E. coli and B. pseudomallei, the affinity of EhPSP for 2,3-BPG was concluded to be lower than those of the other proteins. Moreover, a different set of 2,3-BPG interacting residues was observed in EhPSP compared to dPGMs, with all of the crucial interacting residues of dPGMs either missing or substituted with weakly interacting residues. This study has expanded our understanding, at the structural level, of the inability of EhPSP to catalyze the mutase reaction and has strengthened earlier conclusions indicating it to be a true phosphatase.

Phosphoglycerate Mutase Cooperates with Chk1 Kinase to Regulate Glycolysis.

SummaryDysregulated glycolysis, including the cancerous Warburg effect, is closely involved in pathological mechanisms of diseased states. Among glycolytic enzymes, phosphoglycerate mutase (PGAM) has been known to exert certain physiological impact in vitro, whereas its regulatory role on glycolysis remains unclear. Here, we identified that PGAM plays a key role in regulating glycolysis in cancer cells but not in standard cells. Cancer-prone phenotype by PGAM overexpression in vivo was associated with upregulated glycolytic features. PGAM interacts and cooperates with Chk1 to regulate the enhanced glycolysis in cancer cells, especially under oncogenic Ras expressing conditions. Genetic or chemical interference of the PGAM-Chk1 interaction, with intact PGAM activity, abrogated the maintenance of cancerous enhanced glycolysis. Thus, the nonenzymatic function of PGAM is essential for the Warburg effect that accompanies cancerous proliferation.

Phosphoglycolate has profound metabolic effects but most likely no role in a metabolic DNA response in cancer cell lines.

Repair of a certain type of oxidative DNA damage leads to the release of phosphoglycolate, which is an inhibitor of triose phosphate isomerase and is predicted to indirectly inhibit phosphoglycerate mutase activity. Thus, we hypothesized that phosphoglycolate might play a role in a metabolic DNA damage response. Here, we determined how phosphoglycolate is formed in cells, elucidated its effects on cellular metabolism and tested whether DNA damage repair might release sufficient phosphoglycolate to provoke metabolic effects. Phosphoglycolate concentrations were below 5 µM in wild-type U2OS and HCT116 cells and remained unchanged when we inactivated phosphoglycolate phosphatase (PGP), the enzyme that is believed to dephosphorylate phosphoglycolate. Treatment of PGP knockout cell lines with glycolate caused an up to 500-fold increase in phosphoglycolate concentrations, which resulted largely from a side activity of pyruvate kinase. This increase was much higher than in glycolate-treated wild-type cells and was accompanied by metabolite changes consistent with an inhibition of phosphoglycerate mutase, most likely due to the removal of the priming phosphorylation of this enzyme. Surprisingly, we found that phosphoglycolate also inhibits succinate dehydrogenase with a Ki value of <10 µM. Thus, phosphoglycolate can lead to profound metabolic disturbances. In contrast, phosphoglycolate concentrations were not significantly changed when we treated PGP knockout cells with Bleomycin or ionizing radiation, which are known to lead to the release of phosphoglycolate by causing DNA damage. Thus, phosphoglycolate concentrations due to DNA damage are too low to cause major metabolic changes in HCT116 and U2OS cells.

Deacetylation of metabolic enzymes by Sirt2 modulates pyruvate homeostasis to extend insect lifespan.

Diapause in insects is akin to dauer in Caenorhabditis elegans and hibernation in vertebrates. Diapause causes a profound extension of lifespan by low metabolic activity. However, the detailed regulatory mechanisms for low metabolic activity remain unknown. Here, we showed that low pyruvate levels are present in the brains of diapause-destined pupae of the cotton bollworm Helicoverpa armigera, and three enzymes pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK), and phosphoglycerate mutase (PGAM) are closely correlated with pyruvate homeostasis. Notably, Sirt2 can deacetylate the three enzymes to increase their activity in vitro. Thus, low Sirt2 expression in the brains of diapause individuals decreases PK and PEPCK protein levels as well as PGAM activity, resulting in low pyruvate levels and low tricarboxylic acid cycle activity and eventually inducing diapause initiation by low metabolic activity. These findings suggest that pyruvate is a checkpoint for development or lifespan extension, and Sirt2 is a negative regulator to extend lifespan in insects.

Multiplex analyses of the changes of aromatic compounds during the development of peach fruit using GC–MS and iTRAQ proteomic techniques

Abstract This study aimed to combine GC–MS and iTRAQ techniques to investigate the changes in aromatic compounds and protein profiles of peach fruit peel (Prunus persica cv. Hujiingmilu) at its different developmental stages. The fruits were collected 50, 90, 95, 100, and 105 days after flowering. GC–MS during development and ripening revealed more than 200 volatile compounds, and 18 of them were aroma-related. Based on the varying amount and pattern, γ-decalactone, δ-decalactone, hexyl acetate, and 1-hexanol were involved in peach aroma. Proteomic analysis with the iTRAQ technique identified 1848 unique proteins, out of which 842 were related to biological processes and 100 related with aromatic compounds and energy transfer during fruit development and ripening. The up-regulated linoleate 9(S)-lipoxygenase plays an important role in the increase of hexanal and benzaldehyde content during ripening. The activity of phosphoglycerate mutase, glyceraldehyde 3-phosphate dehydrogenase, and inorganic pyrophosphatase regulated the terpenoids production for linalool through the 3-phosphoglyceric acid/pyruvic acid pathway but not the mevalonic acid pathway. Caffeoyl-CoA-O-methyltransferase had linear interactions with 4-coumarate-CoA ligase and 3-hydroxyisobutyryl-CoA hydrolase, and it might be the critical pathway of aromatic compounds during the development of peach fruit. Overall, the fatty acid metabolism was the primary pathway for aromatic compound formation in peach fruit and the AOC, AOS, LOX, and OPR were the key functional proteins for the formation of lactones and esters representing the characteristic aroma of honey peach. This study combined the techniques of GC–MS and proteomics to delineate the relationship between the change of aromatic compounds and proteins and their possible biological processes with aroma formation in peach fruit during development and ripening.

Deacetylation of phosphoglycerate mutase in its distinct central region by SIRT2 down-regulates its enzymatic activity.

Substantially high rate of glycolysis, known as the Warburg effect, is a well-known feature of cancers, and emerging evidence suggests that it supports cancerous proliferation/tumor growth. Phosphoglycerate mutase (PGAM), a glycolytic enzyme, is commonly up-regulated in several cancers, and recent reports show its involvement in the Warburg effect. Here, a comprehensive analysis shows that PGAM is acetylated at lysines 100/106/113/138 in its central region, and a member of the Sirtuin family (class III deacetylase), SIRT2, is responsible for its deacetylation. Over-expression of SIRT2 or mutations at the acetylatable lysines of PGAM attenuates cancer cell proliferation with a concomitant decrease in PGAM activity. We also report that the acetyltransferase PCAF (p300/CBP-associated factor) interacts with PGAM and acetylates its C-terminus, but not the central region. As prior evidence suggests that SIRT2 functions as a tumor suppressor, our results would provide support for the mechanistic basis of this activity.

Glycolytic enzyme activities and gene expression in Cicer arietinum exposed to water-deficit stress.

The specific activities and transcript levels of glycolytic enzymes were examined in shoots of chickpea (Cicer arietinum L.) cultivars, Pusa362 (drought tolerant) and SBD377 (drought sensitive), subjected to water-deficit stress 30 days after sowing. Water-deficit stress resulted in decrease in relative water content, chlorophyll content, plant dry weight, and NADP/NADPH ratio and increase in NAD/NADH ratio in both the cultivars. A successive decline in the specific activities of fructose-1,6-bisphosphate aldolase (aldolase), 3-phosphoglycerate kinase (PGK), and NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) and elevation in the specific activities of phosphoglycerate mutase (PGM) and triosephosphate isomerase (TPI) was observed in both the cultivars under stress as compared to their respective control plants. The specific activities of hexokinase, fructose-6-phosphate kinase (PFK), and NAD-GAPDH were least affected. The transcript levels of PGK and NADP-GAPDH decreased and that of glucose-6-phosphate isomerase (GPI), PGM, and PFK increased in response to water-deficit stress while water-deficit stress had no effect on the steady-state transcript levels of hexokinase, aldolase, TPI, and NAD-GAPDH. The results suggest that under water-deficit stress, the activities and transcript levels of most of the glycolytic enzymes are not significantly affected, except the increased activity and transcript level of PGM and decreased activities and transcript levels of PGK and NADP-GAPDH. Further, the glycolytic enzymes do not show much variation between the tolerant and sensitive cultivars under water deficit.

Oxidative stress activates SIRT2 to deacetylate and stimulate phosphoglycerate mutase.

Glycolytic enzyme phosphoglycerate mutase (PGAM) plays an important role in coordinating energy production with generation of reducing power and the biosynthesis of nucleotide precursors and amino acids. Inhibition of PGAM by small RNAi or small molecule attenuates cell proliferation and tumor growth. PGAM activity is commonly upregulated in tumor cells, but how PGAM activity is regulated in vivo remains poorly understood. Here we report that PGAM is acetylated at lysine 100 (K100), an active site residue that is invariably conserved from bacteria, to yeast, plant, and mammals. K100 acetylation is detected in fly, mouse, and human cells and in multiple tissues and decreases PGAM2 activity. The cytosolic protein deacetylase sirtuin 2 (SIRT2) deacetylates and activates PGAM2. Increased levels of reactive oxygen species stimulate PGAM2 deacetylation and activity by promoting its interaction with SIRT2. Substitution of endogenous PGAM2 with an acetylation mimetic mutant K100Q reduces cellular NADPH production and inhibits cell proliferation and tumor growth. These results reveal a mechanism of PGAM2 regulation and NADPH homeostasis in response to oxidative stress that impacts cell proliferation and tumor growth.

Functional characterization of two members of histidine phosphatase superfamily in Mycobacterium tuberculosis

BackgroundFunctional characterization of genes in important pathogenic bacteria such as Mycobacterium tuberculosis is imperative. Rv2135c, which was originally annotated as conserved hypothetical, has been found to be associated with membrane protein fractions of H37Rv strain. The gene appears to contain histidine phosphatase motif common to both cofactor-dependent phosphoglycerate mutases and acid phosphatases in the histidine phosphatase superfamily. The functions of many of the members of this superfamily are annotated based only on similarity to known proteins using automatic annotation systems, which can be erroneous. In addition, the motif at the N-terminal of Rv2135c is ‘RHA’ unlike ‘RHG’ found in most members of histidine phosphatase superfamily. These necessitate the need for its experimental characterization. The crystal structure of Rv0489, another member of the histidine phosphatase superfamily in M. tuberculosis, has been previously reported. However, its biochemical characteristics remain unknown. In this study, Rv2135c and Rv0489 from M. tuberculosis were cloned and expressed in Escherichia coli with 6 histidine residues tagged at the C terminal.ResultsCharacterization of the purified recombinant proteins revealed that Rv0489 possesses phosphoglycerate mutase activity while Rv2135c does not. However Rv2135c has an acid phosphatase activity with optimal pH of 5.8. Kinetic parameters of Rv2135c and Rv0489 are studied, confirming that Rv0489 is a cofactor dependent phosphoglycerate mutase of M. tuberculosis. Additional characterization showed that Rv2135c exists as a tetramer while Rv0489 as a dimer in solution.ConclusionMost of the proteins orthologous to Rv2135c in other bacteria are annotated as phosphoglycerate mutases or hypothetical proteins. It is possible that they are actually phosphatases. Experimental characterization of a sufficiently large number of bacterial histidine phosphatases will increase the accuracy of the automatic annotation systems towards a better understanding of this important group of enzymes.

Structural Units Important for Activity of a Novel-type Phosphoserine Phosphatase from Hydrogenobacter thermophilus TK-6 Revealed by Crystal Structure Analysis

Novel-type serine-synthesizing enzymes, termed metal-independent phosphoserine phosphatases (iPSPs), were recently identified and characterized from Hydrogenobacter thermophilus, a chemolithoautotrophic bacterium belonging to the order Aquificales. iPSPs are cofactor-dependent phosphoglycerate mutase (dPGM)-like phosphatases that have significant amino acid sequence similarity to dPGMs but lack phosphoglycerate mutase activity. Genes coding dPGM-like phosphatases have been identified in a broad range of organisms; however, predicting the function of the corresponding proteins based on sequence information alone is difficult due to their diverse substrate preferences. Here, we determined the crystal structure of iPSP1 from H. thermophilus in the apo-form and in complex with its substrate L-phosphoserine to find structural units important for its phosphatase activity toward L-phosphoserine. Structural and biochemical characterization of iPSP1 revealed that the side chains of His(85) and C-terminal region characteristic of iPSP1 are responsible for the PSP activity. The importance of these structural units for PSP activity was confirmed by high PSP activity observed in two novel dPGM-like proteins from Cyanobacteria and Chloroflexus in which the two structural units were conserved. We anticipate that our present findings will facilitate understanding of the serine biosynthesis pathways of organisms that lack gene(s) encoding conventional PSPs, as the structural information revealed here will help to identify iPSP from sequence databases.

2-Carboxy-D-arabinitol 1-phosphate (CA1P) phosphatase: evidence for a wider role in plant Rubisco regulation

The genes for CA1Pase (2-carboxy-D-arabinitol-1-bisphosphate phosphatase) from French bean, wheat, Arabidopsis and tobacco were identified and cloned. The deduced protein sequence included an N-terminal motif identical with the PGM (phosphoglycerate mutase) active site sequence [LIVM]-x-R-H-G-[EQ]-x-x-[WN]. The corresponding gene from wheat coded for an enzyme with the properties published for CA1Pase. The expressed protein lacked PGM activity but rapidly dephosphorylated 2,3-DPG (2,3-diphosphoglycerate) to 2-phosphoglycerate. DTT (dithiothreitol) activation and GSSG inactivation of this enzyme was pH-sensitive, the greatest difference being apparent at pH 8. The presence of the expressed protein during in vitro measurement of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activity prevented a progressive decline in Rubisco turnover. This was due to the removal of an inhibitory bisphosphate that was present in the RuBP (ribulose-1,5-bisphosphate) preparation, and was found to be PDBP (D-glycero-2,3-pentodiulose-1,5-bisphosphate). The substrate specificity of the expressed protein indicates a role for CA1Pase in the removal of 'misfire' products of Rubisco.

Inhibitory effects of decavanadate on several enzymes and Leishmania tarentolae In Vitro

Multiple studies report apparent effects of vanadium on various systems in vivo and in vitro. Vanadium species may be possible deterrents for the growth of the Leishmania parasite, which causes the sometimes deadly diseases known as leishmaniasis. The current studies focus specifically on decavanadate V10O286- (V10), which has a potential to be a potent effector for disease treatment. The X-ray structure of a new solvate salt of V10, namely (NH4)6V10O28·5H2O, is also reported. Other vanadium complexes with imidazole carboxylate, anthranilate, or picolinate were also evaluated. The yellow-orange oxoanion, used as the (NH4)6V10O28·6H2O salt, was tested (at 1–100μM) directly with two strains of Leishmania tarentolae promastigotes in culture to evaluate the effect on cell viability. Vanadium coordination complexes are known effective inhibitors of phosphatases. Using the artificial phosphatase substrate para-nitrophenylphosphate in the presence of a bovine calf intestine alkaline phosphatase enzyme, V10 (from 5 to 100μM) was shown to be a mixed inhibitor for this enzyme and decreased the activity of the other two phosphatases tested. The effect of V10 and the other vanadium complexes on the activity of phosphoglycerate mutase B (PGAM), an important enzyme in glycolysis and gluconeogenesis, was also evaluated. At 10μM, V10 was the most potent inhibitor of PGAM, with an apparent reduction of about 50%. Taken together, we speculate that V10 could have a role in treating Leishmania diseases.

Decreased oxidative phosphorylation and PGAM deficiency in horses suffering from atypical myopathy associated with acquired MADD

Earlier research on ten horses suffering from the frequently fatal disorder atypical myopathy showed that MADD (multiple acyl-CoA dehydrogenase deficiency) is the biochemical derangement behind atypical myopathy. From five horses that died as a result of this disease and seven healthy control horses, urine and plasma were collected ante mortem and muscle biopsies were obtained immediately post-mortem (2 patients and 7 control horses), to analyse creatine, purine and carbohydrate metabolism as well as oxidative phosphorylation.In patients, the mean creatine concentration in urine was increased 17-fold and the concentration of uric acid approximately 4-fold, compared to controls.The highest degree of depletion of glycogen was observed in the patient with the most severe myopathy clinically. In this patient, glycolysis was more active than in the other patients and controls, which may explain this depletion. One patient demonstrated very low phosphoglycerate mutase (PGAM) activity, less than 10% of reference values.Most respiratory chain complex activity in patients was 20–30% lower than in control horses, complex II activity was 42% lower than normal, and one patient had severely decrease ATP-synthase activity, more than 60% lower than in control horses.General markers for myopathic damage are creatine kinase (CK) and lactic acid in plasma, and creatine and uric acid in urine. To obtain more information about the cause of the myopathy analysis of carbohydrate, lipid and protein metabolism as well as oxidative phosphorylation is advised.This study expands the diagnostic possibilities of equine myopathies.

PGAM5: A Novel Type of Protein Serine/Threonine Phosphatase That Exists in the Mitochondria

Phosphoglycerate mutase (PGAM) family member 5 (PGAM5) is a member of the PGAM family, the prototypical members of which are enzymes of intermediary metabolism that convert 3-phosphoglycerate to 2-phosphoglycerate in glycolysis. It has recently been found that PGAM5 lacks authentic PGAM activity but possesses protein phosphatase activity highly specific to serine and threonine residues. Depending on its phosphatase activity, PGAM5 activates the stress-activated c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways through their upstream regulator ASK1. Furthermore, PGAM5 is localized to the mitochondria through its N-terminal transmembrane domain and appears to be involved in the regulation of mitochondrial functions. Here we introduce this novel type of protein phosphatase and discuss its roles as a signaling intermediate.

Phosphoglycerate Mutase Is a Highly Efficient Enzyme without Flux Control in Lactococcus lactis

The glycolytic enzyme phosphoglycerate mutase (PGM), which catalyzes the conversion of 3-phosphoglycerate to 2-phosphoglycerate, was examined in Lactococcus lactis with respect to its function, kinetics and glycolytic flux control. A library of strains with PGM activities ranging between 15–465% of the wild-type level was constructed by replacing the native promoter of pgm with synthetic promoters of varying strengths. The specific growth rate and glucose flux were found to be maximal at the wild-type level at which PGM had no flux control. Low flux control of PGM was found on mixed acid fluxes at highly reduced PGM activities. At the wild-type level PGM operated very far from V_max. Consequently, in a strain with only 15% PGM activity, the catalytic rate of PGM was almost six times higher than in the wild-type. K_mof PGM for 3-phosphoglycerate was 1.0 mM and k_catwas 3,200 s^–1. The L. lactis PGM was dependent on 2,3-bisphosphoglyceric acid for activity, which showed that the enzyme is of the dPGM type in accordance with its predicted homology to dPGM enzymes from other organisms. In conclusion, PGM from L. lactis is a highly efficient catalyst, which partially explains why this enzyme has limited control in wild-type L. lactis.

Mitochondrial phosphoglycerate mutase 5 uses alternate catalytic activity as a protein serine/threonine phosphatase to activate ASK1

Phosphoglycerate mutase (PGAM) is an enzyme of intermediary metabolism that converts 3-phosphoglycerate to 2-phosphoglycerate in glycolysis. Here, we discovered PGAM5 that is anchored in the mitochondrial membrane lacks PGAM activity and instead associates with the MAP kinase kinase kinase ASK1 and acts as a specific protein Ser/Thr phosphatase that activates ASK1 by dephosphorylation of inhibitory sites. Mutation of an active site His-105 in PGAM5 abolished phosphatase activity with ASK1 and phospho-Thr peptides as substrates. The Drosophila and Caenorhabditis elegans orthologs of PGAM5 also exhibit specific Ser/Thr phosphatase activity and activate the corresponding Drosophila and C. elegans ASK1 kinases. PGAM5 is unrelated to the other known Ser/Thr phosphatases of the PPP, MPP, and FCP families, and our results suggest that this member of the PGAM family has crossed over from small molecules to protein substrates and been adapted to serve as a specialized activator of ASK1.

Phosphoglycerate mutase deficiency: case report of a manifesting heterozygote with a novel E154K mutation and very late onset

Phosphoglycerate mutase (PGAM) deficiency is a rare glycogen storage disease (glycogenosis type X) due to recessive mutations in the muscle-specific isoform PGAMM. We present a 67-year-old German patient with exercise-induced cramps and myalgia for 1 year but no attacks of rhabdomyolysis. CK was 2.5-folds increased and forearm exercise test was normal. Sections of the muscle biopsy stained with PAS showed moderate glycogen accumulation. Gomori-trichome and NADH showed subsarcolemmal depositions (0.01% of the fibres) suggesting tubular aggregates (Fig. 1). PGAM activity in muscle homogenate [4] was moderately reduced (141 U/g wet weight, normal 332 ± 88). Deficiencies of alpha-glucosidase, myophosphorylase, CPT and MAD were excluded biochemically. The PGAM-M gene was amplified [5] and directly sequenced. A novel G to A transition at nucleotide position 460, resulting in exchange of amino acid glutamic acid at codon 154 to lysine, was identified in heterozygous state. This was confirmed by reverse sequencing. RFLP analysis in 70 normal controls from the same ethnic origin did not show the mutation. There are several lines of evidence suggesting that the patient is a manifesting heterozygous carrier. The E154K mutation is considered pathogenic because: (1) the mutation was not identified in normal controls, (2) it affects a highly conserved amino acid (Fig. 2), (3) it results in changing of an acidic amino acid to a basic amino acid with larger side chain, (4) it was the only mutant change identified in the coding region and exon– intron boundaries of the PGAM-M gene. However, it cannot be excluded that the patient carries a large deletion or a deep intronic mutation on the second allele. The biochemical result with half the normal enzyme activity, however, seems consistent with just one mutant allele. Finally, glycogen storage and tubular aggregates in muscle (in very low degree, though) are typical findings of PGAM deficiency. These histological changes might be scarce, even in cases with two mutant alleles. We identified another case of PGAM deficiency (with the typical phenotype of exercise-induced myalgia with rhabdomyolysis starting at age 35 years) with moderate glycogen accumulation and very few fibres suggestive of tubular aggregates (0.02% of total fibres). This patient of African origin had very reduced PGAM activity (9.6 U/g wet weight, normal 332 ± 88) and carried the homozygous W78X stopmutation that was identified in several African patients with PGAM deficiency [3, 6]. The patient had incomplete raise of lactate in the forearm test, as described for PGAM deficiency [1, 3, 7]. The manifesting heterozygote patient, however, had normal forearm exercise test, suggesting that 50% residual activity is enough so that the metabolic impairment is undetectable in this test. In both of our patients electron microscopy was unable to identify fibres with tubular aggregates because they were very scarce and abnormal fibres were not seen in semi-thin sections because of the small piece of muscle that is analysed. P. R. Joshi M. Knape S. Zierz M. Deschauer Neurologische Universitatsklinik, Halle, Germany

Biochemical and molecular genetic identification of patients with phosphoglycerate mutase deficiency

Phosphoglycerate mutase (PGAM) deficiency is an autosomal recessive glycogenosis (type X) characterised by exercise induced cramps, myalgia, and myoglobinuria. Myohistological features are glycogen deposition and tubular aggregates. Biochemically there is low muscle PGAM activity (most patients below 10%) but few manifesting heterozygotes with 50% activity are reported. Molecular genetic investigations have revealed 4 mutations among 13 patients, so far. A nonsense mutation W78X is frequent in patients of African origin.