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PubMed2003

Effects of hypoxia and thyroid hormone on mRNA levels and activity of phosphoglycerate mutase in rabbit tissues.

AIM: In the present work, we studied the effects of hypoxia and triiodothyronine (T(3)) on phosphoglycerate mutase (PGAM) activity and expression in rabbit liver, brain, and skeletal muscle under in vivo conditions. METHODS: Hypoxia was induced in a methacrylate cage with a mixture of 90% nitrogen and 10% oxygen. Hyperthyroidism was induced daily by T(3) injection (250 microg/kg). RESULTS: Hypoxia increases the PGAM activity in liver and brain, tissues which possess type PGAM-BB isozyme, but does not affect the PGAM activity in muscle which possesses type PGAM-MM isozyme. T(3) administration increases the PGAM activity in muscle and liver, but does not affect the enzyme activity in the brain. In all cases, the activity changes in parallel with those of PGAM mRNA levels. CONCLUSION: The tissue-specific effects of hypoxia and T(3) could be explained by the tissue-specific distribution of both PGAM isozyme and T(3) receptors.

Animals↗

PubMed1980

Relationship of 2,3-diphosphoglycerate and 2,3-diphosphoglycerate mutase in various mammals.

To investigate a possible mechanism involved in the regulation of 2,3-diphosphoglycerate (2,3-DPG) synthesis, 2,3-DPG mutase (DPGM) was measured in different mammals presenting large differences in 2,3-DPG concentration between fetal, neonatal and adult life to see the activity of this enzyme, necessary for 2,3-DPG synthesis, was related to the levels of 2,3-DPG. The data demonstrated that the minimal levels of 2,3-DPG in the adult sheep are likely due to the very low levels of DPGM. Also these findings show that the increases in 2,3-DPG levels, found in the newborn sheep during the 1st week of life, in adult rabbit and guinea pig when compared with their fetuses, are not due to an increase in levels of the DPGM.

2,3-Diphosphoglycerate↗

PubMed1992

Cloning and expression of a mutant methylmalonyl coenzyme A mutase with altered cobalamin affinity that causes mut- methylmalonic aciduria.

Distinct genotypic and phenotypic forms of methylmalonyl CoA mutase (MCM) apoenzyme deficiency can be delineated by biochemical analysis of mutant fibroblasts. One form, designated mut-, expresses a phenotype in which residual enzyme activity is evident in cultured cells exposed to high concentrations of hydroxycobalamin. We describe cloning of an MCM cDNA from cells exhibiting a mut- phenotype and characterization of the mutant gene product overexpressed in primary muto human fibroblasts and Saccharomyces cerevisiae. Three novel base changes were observed. Recombinant clones containing one of these base changes (G717V) express four characteristics of the mut- phenotype: failure to constitute [14C]propionate incorporation activity in fibroblasts assayed under basal cell culture conditions, constitution of [14C]propionate incorporation activity in fibroblasts stimulated with 0.1-1.0 micrograms/ml hydroxycobalamin, interallelic complementation with alleles bearing an R93H mutation, and an apparent Km (adenosylcobalamin) 1,000-fold higher than normal. These results demonstrate that the G717V mutation produces the mut- phenotype and localizes determinants for adenosylcobalamin binding near the carboxyl terminus of MCM.

Amino Acid Sequence↗

PubMed2002

Setting of graded levels of a protein in yeast by a t-degron technique as applied to phosphoglycerate mutase.

BACKGROUND: Setting of graded levels of a protein for in vivo studies by controlled gene expression has inconveniences, and we here explore the use of the t-degron technique instead. RESULTS: In a yeast t-degron (ubiquitin-argDHFR(ts))- phosphoglycerate mutase (GPM1) fusion strain, increasing periods of exposure to the non-permissive temperature 37 degrees C, even in the presence of cycloheximide, gave decreasing function, as assessed at 23 degrees C in vivo by glucose metabolism and confirmed by immunoblot. CONCLUSION: An ideal system would set a range of lower levels of a protein, do so without compensating protein synthesis, and give stable activity for in vitro comparisons. Although the first two aims appear obtainable, the third was not in this example of the application, limiting its uses for some but not all purposes.

Cell Division↗

PubMed1982

Muscle phosphoglycerate mutase deficiency.

A 52-year-old man complained since adolescence of cramps and pigmenturia after 15 to 30 minutes of intense exercise. There was no family history of neuromuscular diseases, and strength was normal. The rise of venous lactate after forearm ischemic exercise was abnormally low. Histochemical and ultrastructural studies of a muscle biopsy showed mild increase of glycogen, which was confirmed by biochemical analysis. Studies of anaerobic glycolysis in vitro showed decrease lactate formation with glycogen and with all hexosephosphate glycolytic intermediates, suggesting a defect below the phosphofructokinase reaction. Muscle phosphoglycerate mutase (PGAM) activity was 5.7% of the lowest control, while all other enzymes of glycolysis had normal activities. Electrophoretic, heat lability, and mercury inhibition studies showed that the small residual activity of PGAM in the patient's muscle was represented by the brain (BB) isoenzyme, suggesting a genetic defect of the M subunit that predominates in normal muscle. The prevalence of the BB isoenzyme in other tissues, including muscle culture, may explain why symptoms were confined to muscle.

Adult↗

PubMed1983

Muscle phosphoglycerate mutase (PGAM) deficiency: a second case.

Muscle phosphoglycerate mutase (PGAM) activity was markedly decreased (6% of the normal mean) in a 17-year-old girl with recurrent myoglobinuria after intense exercise. Muscle biopsy showed increased PAS stain; glycogen concentration was twice normal. Studies of anaerobic glycolysis in vitro showed decreased lactate production with glycogen, and with all hexose phosphate glycolytic intermediates, which was corrected by addition of purified PGAM to the reaction mixtures. A defect of the M subunit of PGAM was documented by electrophoretic, heat lability, and mercury inhibition studies. Intermediate PGAM activities (39 and 50% of normal) were found in muscle biopsies from the patient's asymptomatic parents. These data confirm the clinical, morphologic, and biochemical features described in the first patient with PGAM deficiency and suggest autosomal-recessive transmission of the trait.

Adolescent↗

PubMed1985

Physiologic assessment of phosphoglycerate mutase deficiency: incremental exercise test.

A third case of phosphoglycerate mutase (PGAM) deficiency, a metabolic myopathy involving terminal glycolysis, was identified in a 24-year-old black man with episodic, exercise-induced myoglobinuria since age 13. To better understand the physiologic consequences of PGAM deficiency, incremental exercise testing was performed. Results were compared with those of two patients having myophosphorylase deficiency and five normals. In contrast to the patients with phosphorylase deficiency, the PGAM-deficient patient achieved near-normal levels of maximal exercise and produced a normal peak lactate after exercise. The mechanisms underlying the asymptomatic performance of such strenuous exercise in this case are uncertain, but the data suggest that unidentified factors are operative in precipitating attacks of myoglobinuria in patients with some metabolic myopathies. Despite similar clinical histories, patients with different glycolytic enzyme deficiencies can have striking differences in exercise tolerance.

Adult↗

PubMed1999

Molecular analysis and heterologous expression of the gene encoding methylmalonyl-coenzyme A mutase from rifamycin SV-producing strain Amycolatopsis mediterranei U32.

The conversion of succinyl-coenzyme A (CoA) into methylmalonyl-CoA, catalyzed by adenosylcobalamin-dependent methylmalonyl-CoA mutase (MCM), represents an important source of building blocks for rifamycin SV biosynthesis. The structural gene for MCM from rifamycin SV-producing strain Amycolatopsis mediterranei U32 was isolated by using a heterologous gene probe encoding the MCM of Streptomyces cinnamonesis. A 7.8-kbp fragment was sequenced and four complete open reading frames (ORFs) and two incomplete ORFs were found. Two central ORFs, ORF3 and ORF4, overlap by four nucleotides and were found to encode MCM small (602 residues) and large (721 residues) subunits, respectively. Comparison showed that the MCM gene of A. mediterranei U32 was quite similar to those from other sources. The functionally unknown ORF5, immediately downstream of the mutAB gene, was quite similar to the ORFs downstream of mutAB from S. cinnamonensis and Mycobacterium tuberculosis. Such a striking cross-species conservation of gene order suggested that ORF5 could also be involved in the metabolism of methylmalonyl-CoA. MCM gene was overexpressed in Escherichia coli under T7 promoter, and MCM activity could be detected in the recombinant E. coli clone harboring MCM gene after the addition of coenzyme B12. A purification procedure based on the B12 affinity column was established to purify the MCM from E. coli. The molecular weight of purified MCM from E. coli was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, which corresponds to that calculated from the MCM protein sequence and is also the same size as that of the enzyme purified directly from A. mediterranei U32. MCM gene was overexpressed in polyketide monensin producing S. cinnamonensis, and the total monensin production was increased by 32%.

Actinomycetales↗

PubMed2006

Structure and molecular mechanism of Bacillus anthracis cofactor-independent phosphoglycerate mutase: a crucial enzyme for spores and growing cells of Bacillus species.

Phosphoglycerate mutases (PGMs) catalyze the isomerization of 2- and 3-phosphoglycerates and are essential for glucose metabolism in most organisms. This study reports the production, structure, and molecular dynamics analysis of Bacillus anthracis cofactor-independent PGM (iPGM). The three-dimensional structure of B. anthracis PGM is composed of two structural and functional domains, the phosphatase and transferase. The structural relationship between these two domains is different than in the B. stearothermophilus iPGM structure determined previously. However, the structures of the two domains of B. anthracis iPGM show a high degree of similarity to those in B. stearothermophilus iPGM. The novel domain arrangement in B. anthracis iPGM and the dynamic property of these domains is directly linked to the mechanism of enzyme catalysis, in which substrate binding is proposed to result in close association of the two domains. The structure of B. anthracis iPGM and the molecular dynamics of this structure provide unique insight into the mechanism of iPGM catalysis, in particular the roles of changes in coordination geometry of the enzyme's two bivalent metal ions and the regulation of this enzyme's activity by changes in intracellular pH during spore formation and germination in Bacillus species.

Bacillus anthracis↗

PubMed1977

Methylmalonyl-CoA mutase activity of leukocytes in variants and heterozygotes of methylmalonic acidemia.

An assay method for the methylmalonyl-CoA mutase of leukocytes obtained from 3 ml of blood was established. The enzyme activity which was measured with or without the in vitro addition of 5'-deoxyadenosylcobalamin was found to be of value for the diagnosis of two variants of methylmalonic acidemia (vitamin B12 responsive and unresponsive), and also for the detection of heterozygotes with the vitamin B12 unresponsive type.

Amino Acid Metabolism, Inborn Errors↗

PubMed1996

Partial deficiency of phosphoglycerate mutase with diabetic polyneuropathy: the first Japanese patient.

We report here findings in a 51-year-old Japanese man with non-insulin-dependent diabetes mellitus who complained of exercise-induced cramps. Muscle biopsy showed scattered regenerating fibers, small angular fibers and increased PAS positive particles. Electron microscopic examination revealed an abnormal accumulation of glycogen particles in subsarcolenmmal areas and between myofibrils while chemical studies showed an increased glycogen concentration and decreased phosphoglycerate mutase (PGAM), 46.9% of the normal mean value. Thus, partial PGAM deficiency, insulin resistance and mild diabetic sensory-motor polyneuropathy can induce severe cramps.

Biopsy↗

PubMed1988

Characterization of phosphoglycerate mutase isoenzymes from free dissected facial processes.

To characterize the three phosphoglycerate mutase (PGM) isoenzymes present in rat facial processes (types MM, BB, and MB), their sensitivity to reagents of the sulfhydryl groups and to heat treatment has been studied. Type BB PGM was not affected by the -SH group reagents; type MB PGM was inhibited about 50%, and type MM PGM was fully inhibited. Type MB PGM showed a greater heat lability than type MM PGM. There was a developmental change from type BB PGM from the 9th embryonic day to isoenzymes MB and MM on the 15th embryonic day. Isoenzyme development was first seen in mandibular processes, followed by maxillary, lateral nasal, and medial nasal processes.

Animals↗

PubMed2002

Purification and characterization of methylmalonyl-CoA mutase from a methanol-utilizing bacterium, Methylobacterium extorquens NR-1.

High activity (about 50 mU x mg protein(-1)) of methylmalonyl-CoA mutase (82-95% apo-enzyme) was constantly found during the cell growth of a methanol-utilizing bacterium, Methylobacterium extorquens NR-1. The apo-enzyme was purified to homogeneity and characterized. The purified enzyme was colorless. An apparent Mr of M. extorquens NR-1 enzyme was calculated to be 150,000 +/- 5,000 by Superdex 200 HR gel filtration. SDS-polyacrylamide gel electrophoresis of the purified enzyme gave two protein bands with an apparent Mr of 85.000 +/- 2,000 and 70,000 +/- 2,000, indicating that the M. extorquens NR-1 enzyme is composed of two nonidentical subunits. NH2-terminal amino acid sequences of the small and large subunits of M. extorquens NR-1 enzyme showed no significant homology to those of the enzyme from other species. Some enzymological properties of the M. extorquens NR-1 enzyme were studied.

Amino Acid Sequence↗

PubMed1999

p53 is a transcriptional activator of the muscle-specific phosphoglycerate mutase gene and contributes in vivo to the control of its cardiac expression.

The role that the p53 tumor suppressor gene product plays in cellular differentiation remains controversial. However, recent evidence indicates that p53 is required for proper embryogenesis. We have studied the effect of p53 on the expression mediated by the promoter of the rat muscle-specific phosphoglycerate mutase gene (M-PGAM), a marker for cardiac and skeletal muscle differentiation. Experiments involving transient transfection, mobility shift assay, and site-directed mutagenesis demonstrated that p53 specifically binds and transactivates the M-PGAM promoter. The p53-related proteins p51A and p73L also transactivated M-PGAM. Moreover, stable expression of a p53 dominant mutant in C2C12 cells blocked the induction of M-PGAM expression during the myoblast to myotube transition and the ability of p53, p51A, and p73L to transactivate the M-PGAM promoter. In addition, impaired expression of M-PGAM was observed in a subset of p53-null animals in heart and muscle tissues of anterior-ventral location. These results demonstrate that p53 is a transcriptional activator of M-PGAM that contributes in vivo to the control of its cardiac expression. These data support previous findings indicating a role for p53 in cellular differentiation.

Animals↗

PubMed1999

Co-ordinate variations in methylmalonyl-CoA mutase and methionine synthase, and the cobalamin cofactors in human glioma cells during nitrous oxide exposure and the subsequent recovery phase.

We investigated the co-ordinate variations of the two cobalamin (Cbl)-dependent enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MCM), and measured the levels of their respective cofactors, methylcobalamin (CH3Cbl) and adenosylcobalamin (AdoCbl) in cultured human glioma cells during nitrous oxide exposure and during a subsequent recovery period of culture in a nitrous oxide-free atmosphere (air). In agreement with published data, MS as the primary target of nitrous oxide was inactivated rapidly (initial rate of 0.06 h(-1)), followed by reduction of CH3Cbl (to <20%). Both enzyme activity and cofactor levels recovered rapidly when the cells were subsequently cultured in air, but the recovery was completely blocked by the protein-synthesis inhibitor, cycloheximide. During MS inactivation, there was a reduction of cellular AdoCbl and holo-MCM activity (measured in the absence of exogenous AdoCbl) to about 50% of pre-treatment levels. When the cells were transferred to air, both AdoCbl and holo-MCM activity recovered, albeit more slowly than the MS system. Notably, the regain of the holo-MCM and AdoCbl was enhanced rather than inhibited by cycloheximide. These findings confirm irreversible damage of MS by nitrous oxide; hence, synthesis of the enzyme is required to restore its activity. In contrast, restoration of holo-MCM activity is only dependent on repletion of the AdoCbl cofactor. We also observed a synchronous fluctuation in AdoCbl and the much larger hydroxycobalamin pool during the inactivation and recovery phase, suggesting that the loss and repletion of AdoCbl reflect changes in intracellular Cbl homoeostasis. Our data demonstrate that the nitrous oxide-induced changes in MS and CH3Cbl are associated with reversible changes in both MCM holoactivity and the AdoCbl level, suggesting co-ordinate distribution of Cbl cofactors during depletion and repletion.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1999

Muscle phosphoglycerate mutase deficiency with tubular aggregates: effect of dantrolene.

A patient with muscle phosphoglycerate mutase deficiency (PGAMD) and exercise-induced muscle cramps had tubular aggregates in muscle and increased muscle Ca2+-adenosine triphosphatase and calcium content. Two ischemic forearm exercise tests induced contractures in the patient. On dantrolene treatment, the patient became asymptomatic, and the ischemic test was performed without contracture. These findings suggest that cramps in muscle PGAMD are caused by a high calcium release from the sarcoplasmic reticulum relative to calcium re-uptake capacity.

Adult↗

PubMed2000

[Effect of artemether on phosphoglucomutase, aldolase, phosphoglycerate mutase and enolase of Schistosoma japonicum harbored in mice].

OBJECTIVE: To study the effect of artemether (Art) on phosphoglucomutase (GPM), aldolase (ALD), phosphoglycerate mutase (PGM) and enolase (ENO) of Schistosoma japonicum harbored in mice. METHODS: Mice infected with S. japonicum cercariae for 4-5 wk were treated ig with Art 100 mg/kg or 300 mg/kg and killed 24 h or 48 h after treatment for collection of worms. The activities of GPM, ALD, PGM and ENO in female and male worms were measured by the formation of NADPH or consumption of NADH. RESULTS: After the worms were exposed in vivo to Art 100 mg/kg for 24 h, the GPM, ALD, PGM and ENO activities in female worms were significantly decreased by 15%, 19%, 50% and 46%, respectively, while in male worms only the PGM and ENO activities were markedly decreased by 22% and 32%, respectively. Following exposure of the worms to Art 100 mg/kg for 48 h, the GPM and ALD activities in male worms were also significantly reduced by 21% and 18%, respectively, while the activities of GPM, ALD, PGM and ENO in female worms and those of PGM and ENO in male worms declined progressively with time. After the worms were exposed in vivo to Art 300 mg/kg for 24-48 h, all the activities of the above-mentioned enzymes in female and male worms declined significantly in a time-related pattern. CONCLUSION: Art showed an apparently inhibitory effect on GPM, ALD, PGM and ENO in female schistosomes.

Animals↗

PubMed1992

Compound heterozygosity in a complete erythrocyte bisphosphoglycerate mutase deficiency.

Erythrocyte bisphosphoglycerate mutase (BPGM) deficiency is a rare disease associated with a decrease in 2,3-diphosphoglycerate concentration. A complete BPGM deficiency was described in 1978 by Rosa et al (J Clin Invest 62:907, 1978) and was shown to be associated with 30% to 50% of an inactive enzyme detectable by specific antibodies and resulting from an 89 Arg-->Cys substitution. The propositus' three sisters exhibited the same phenotype, while his two children had an intermediate phenotype. Samples from the family were examined using polymerase chain reaction and allele-specific oligonucleotide hybridization and sequencing techniques. Amplification of erythrocyte total RNA from the propositus' sister around the 89 mutation indicated the presence of two forms of messenger RNAs, a major form with the 89 Arg-->Cys mutation and a minor form with a normal sequence. Sequence studies of the propositus' DNA samples indicated heterozygosity at locus 89 and another heterozygosity with the deletion of nucleotide C 205 or C 206. Therefore, the total BPGM deficiency results from a genetic compound with one allele coding for an inactive enzyme (mutation BPGM Créteil I) and the other bearing a frameshift mutation (mutation BPGM Créteil II). Examination of the propositus' two children indicated that they both inherited the BPGM Créteil I mutation.

Arginine↗