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European Journal of Biochemistry Apr 19781. The testis-specific isoenzyme of phosphoglycerate kinase (phosphoglycerate kinase B) has been isolated from ram testes using a procedure which separates it from... (Comparative Study)
Comparative Study
1. The testis-specific isoenzyme of phosphoglycerate kinase (phosphoglycerate kinase B) has been isolated from ram testes using a procedure which separates it from 'normal' phosphoglycerate kinase which is also present in testis tissue. The purification procedure is described. 2. The best preparations had no detectable impurity on electrophoresis, and had specific activities comparable with the same enzyme from other sources. 3. Kinetic studies indicated that the two isoenzymes have identical properties, within experimental error, for substrate affinity (for MgATP, 3-phosphoglycerate and MgADP), energy of activation and thermal denaturation. 4. The molecular weights of both isoenzymes were not distinguishably different from those previously reported, as measured by polyacrylamide/dodecylsulphate electrophoresis. The amino acid compositions showed only slight differences, and tryptic peptide maps showed that there was close homology of sequence. Starch gel electrophoresis at pH 6.5 indicates that the B isoenzyme has 1--2 more positive charges than the A. 5. Phosphoglycerate kinase A isolated from sheep muscle was shown, within experimental error, to be identical to the phosphoglycerate kinase A isolated from testis. 6. The results further substantiate the suggestion that the B isoenzyme is coded by a gene which was duplicated from the phosphoglycerate kinase A gene.
Topics: Amino Acids; Animals; Isoenzymes; Kinetics; Male; Molecular Weight; Phosphoglycerate Kinase; Sheep; Testis
PubMed: 639826
DOI: 10.1111/j.1432-1033.1978.tb12215.x -
British Journal of Haematology Sep 2003We report the case of a 3-year-old Japanese boy with phosphoglycerate kinase 1 (PGK1) deficiency (Online Mendelian Inheritance in Man entry 311800). The patient had... (Review)
Review
We report the case of a 3-year-old Japanese boy with phosphoglycerate kinase 1 (PGK1) deficiency (Online Mendelian Inheritance in Man entry 311800). The patient had anaemia and jaundice at birth, necessitating exchange transfusions for 2 d. After one red blood cell transfusion at age 2 months, his Hb level was 8-9 g/dl, his reticulocyte counts were 300-500 x 109/l, and his total bilirubin level was 25.65-42.75 micro mol/l. The patient suffered two episodes of respiratory infection-associated haemolytic crisis and rhabdomyolysis during early infancy. At age 3.0 years, his developmental milestones (developmental quotients measured using the Tsumori-Inage methods) score was 49% (normal 74-131%), and his height was below average by -2.0 standard deviations. The diagnosis of PGK1 deficiency was made based on his remarkably low (< 10% of normal) erythrocyte PGK enzyme activity level and the identification of a novel missense (1060G-->C) PGK1 gene mutation. This mutation results in the Ala-353Pro amino acid substitution, which has been designated PGK Kyoto. The patient developed the full clinical symptoms of PGK1 deficiency including haemolytic anaemia, myopathy, central nervous system disorder and growth retardation, which is unusual.
Topics: Anemia, Hemolytic, Congenital; Child, Preschool; Developmental Disabilities; Humans; Male; Models, Molecular; Mutation, Missense; Phosphoglycerate Kinase; Rhabdomyolysis
PubMed: 12956773
DOI: 10.1046/j.1365-2141.2003.04543.x -
The FEBS Journal Dec 2020Recently, we described the PAS domain-containing phosphoglycerate kinase (PGK) from Leishmania major (LmPAS-PGK) that shows acidic pH (5.5)-dependent optimum catalytic...
Recently, we described the PAS domain-containing phosphoglycerate kinase (PGK) from Leishmania major (LmPAS-PGK) that shows acidic pH (5.5)-dependent optimum catalytic activity. The PAS domain of LmPAS-PGK is expected to regulate PGK activity during catalysis, but the mechanism of regulation by PAS domain at the molecular level is uncharacterized. In this work, we have utilized the full-length, PAS domain-deleted, and mutant enzymes to measure the enzymatic activity in the presence of divalent cation at various pH values. Catalytic activity measurement indicates that Mg binding through PAS domain inhibits the PGK activity at pH 7.5, and this inhibition is withdrawn at pH 5.5. To identify the Mg binding residues of the PAS domain, we exploited a systematic mutational analysis of all (four) His residues in the PAS domain for potential divalent cation binding. Replacement of His-57 with alanine resulted in depression in the presence of Mg at pH 7.5, but H71A, H89A, and H111A showed similar characteristics with respect to the wild-type protein. Fluorescence and isothermal titration calorimetry studies revealed that H57 is responsible for Mg binding in the absence of substrates. Thus, the protonated form of His57 at acidic pH 5.5 destabilizes the Mg binding in the PAS domain, which is an essential requirement in the wild-type LmPAS-PGK for a conformational alteration in the sensor domain that, sequentially, activates the PGK domain, resulting in the synthesis of higher amounts of ATP.
Topics: Binding Sites; Catalysis; Hydrogen-Ion Concentration; Leishmania major; Magnesium; Mutant Proteins; Mutation; Phosphoglycerate Kinase; Protein Serine-Threonine Kinases
PubMed: 32196942
DOI: 10.1111/febs.15305 -
Methods in Enzymology 1975
Topics: Ammonium Sulfate; Catalysis; Chemical Phenomena; Chemistry; Chromatography, DEAE-Cellulose; Chromatography, Gel; Escherichia coli; Fractional Precipitation; Methods; Molecular Weight; Phosphoglycerate Kinase; Phosphoglycerate Mutase; Phosphotransferases; Spectrophotometry; Streptomycin; Ultracentrifugation
PubMed: 166274
DOI: 10.1016/0076-6879(75)42107-3 -
The EMBO Journal Feb 1995Phosphoglycerate kinase (PGK) from the hyperthermophilic bacterium Thermotoga maritima has been purified to homogeneity. A second larger enzyme with PGK activity and...
Phosphoglycerate kinase (PGK) from the hyperthermophilic bacterium Thermotoga maritima has been purified to homogeneity. A second larger enzyme with PGK activity and identical N-terminal sequence was also found. Surprisingly, this enzyme displayed triosephosphate isomerase (TIM) activity. No other TIM is detectable in T. maritima crude extracts. As shown by ultracentrifugal analysis, PGK is a 43 kDa monomer, whereas the bifunctional PGK-TIM fusion protein is a homotetramer of 240-285 kDa. SDS-PAGE indicated a subunit size of 70 kDa for the fusion protein. Both enzymes show high thermostability. Measurements of the catalytic properties revealed no extraordinary results. pH optima, Km values and activation energies were found to be in the range observed for other PGKs and TIMs investigated so far. The corresponding pgk and tpi genes are part of the apparent gap operon of T. maritima. This gene segment contains two overlapping reading frames, where the 43 kDa PGK is encoded by the upstream open reading frame, the pgk gene. On the other hand, the 70 kDa PGK-TIM fusion protein is encoded jointly by the pgk gene and the overlapping downstream open reading frame of the tpi gene. A programmed frameshift may be responsible for this fusion. A comparison of the amino acid sequence of both the PGK and the TIM parts of the fusion protein with those of known PGKs and TIMs reveals high similarity to the corresponding enzymes from different procaryotic and eucaryotic organisms.
Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Enzyme Stability; Escherichia coli; Genes, Bacterial; Gram-Negative Anaerobic Bacteria; Hot Temperature; Hydrogen-Ion Concentration; Molecular Sequence Data; Molecular Weight; Multienzyme Complexes; Osmolar Concentration; Phosphoglycerate Kinase; Reading Frames; Recombinant Fusion Proteins; Recombinant Proteins; Sequence Analysis; Triose-Phosphate Isomerase
PubMed: 7859734
DOI: 10.1002/j.1460-2075.1995.tb07020.x -
Communications Biology Feb 2021Protein-metabolite interactions are of crucial importance for all cellular processes but remain understudied. Here, we applied a biochemical approach named PROMIS, to...
Protein-metabolite interactions are of crucial importance for all cellular processes but remain understudied. Here, we applied a biochemical approach named PROMIS, to address the complexity of the protein-small molecule interactome in the model yeast Saccharomyces cerevisiae. By doing so, we provide a unique dataset, which can be queried for interactions between 74 small molecules and 3982 proteins using a user-friendly interface available at https://promis.mpimp-golm.mpg.de/yeastpmi/ . By interpolating PROMIS with the list of predicted protein-metabolite interactions, we provided experimental validation for 225 binding events. Remarkably, of the 74 small molecules co-eluting with proteins, 36 were proteogenic dipeptides. Targeted analysis of a representative dipeptide, Ser-Leu, revealed numerous protein interactors comprising chaperones, proteasomal subunits, and metabolic enzymes. We could further demonstrate that Ser-Leu binding increases activity of a glycolytic enzyme phosphoglycerate kinase (Pgk1). Consistent with the binding analysis, Ser-Leu supplementation leads to the acute metabolic changes and delays timing of a diauxic shift. Supported by the dipeptide accumulation analysis our work attests to the role of Ser-Leu as a metabolic regulator at the interface of protein degradation and central metabolism.
Topics: Energy Metabolism; Glycolysis; Metabolome; Metabolomics; Phosphoglycerate Kinase; Protein Interaction Maps; Proteolysis; Proteome; Proteomics; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 33568709
DOI: 10.1038/s42003-021-01684-3 -
Biochemistry Jun 1976The "phosphoryl-enzyme" prepared from phosphoglycerate kinase and adenosine 5'-triphosphate in the presence of an adenosine 5'-diphosphate trap is shown to contain...
The "phosphoryl-enzyme" prepared from phosphoglycerate kinase and adenosine 5'-triphosphate in the presence of an adenosine 5'-diphosphate trap is shown to contain stoichiometric amounts of 3-phosphoglycerate. This "phosphoryl-enzyme" is chemically competent, but is probably just a tight complex between 1,3-bisphosphoglycerate and the enzyme. The two partial exchange reactions (between adenosine 5'-diphosphate, and adenosine 5'-triphosphate, and between 3-phosphoglycerate and 1,3-bisphosphoglycerate) can both be observed, but their rates are very much slower than the rate of overall catalysis. No substrate analogue was found that accelerated the partial exchange reactions. Catalysis of each of the two exchange reactions and of the kinase reaction coincides after isoelectric focusing of purified enzyme, but the amount of cosubstrate necessary to cause the observed partial exchange rates is so small that these reactions may well be artifactual. The balance of evidence does not support a ping-pong pathway via phosphoryl-enzyme, and the reaction may be a sequential one in which the phosphoryl group is transferred between substrates in a ternary complex. The results point to the dangers in the interpretation of experiments where very small amounts of contaminating cosubstrate can lead to large kinetic effects, and to the possibility of mistaken deductions about the identity of reaction intermediates.
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Binding Sites; Diphosphoglyceric Acids; Glyceric Acids; Horses; Hydroxamic Acids; Muscles; Phosphoglycerate Kinase; Protein Binding; Saccharomyces cerevisiae
PubMed: 779834
DOI: No ID Found -
Biochimica Et Biophysica Acta Dec 1962
Topics: Erythrocytes; Phosphoglycerate Kinase; Phosphotransferases
PubMed: 13960866
DOI: 10.1016/0006-3002(62)91057-0 -
Experimental Parasitology Dec 2015In the present study, a full-length cDNA encoding the Schistosoma japonicum 3-phosphoglycerate kinase (SjPGK) with an open reading frame of 1251 bp was isolated from...
In the present study, a full-length cDNA encoding the Schistosoma japonicum 3-phosphoglycerate kinase (SjPGK) with an open reading frame of 1251 bp was isolated from 42-day-old (42-d) schistosome cDNAs. Real-time quantitative reverse transcription PCR analysis revealed that SjPGK was expressed in all investigated developmental stages and at a higher transcript levels in 21- and 42-d worms. Moreover, the SjPGK mRNA level was significantly downregulated in 10-d schistosomula from Wistar rats (non-susceptible host). SjPGK was subcloned into pET28a(+) and expressed as both supernatant and inclusion bodies in Escherichia coli BL21 cells. The enzymatic activity of recombinant SjPGK protein (rSjPGK) was 125 U/mg. Kinetic analyses with respect to 3-phosphoglycerate (3-PGA) as substrate gave a Km of 2.69 mmol/L and a Vmax of 748 μmol/min/mg protein. rSjPGK was highly stable over a range of pH 8.0-9.0 and temperature of 30°C-40 °C under physiological conditions. Immunolocalization analysis showed that SjPGK was mainly distributed in the tegument and parenchyma of schistosomes. Western blotting showed that rSjPGK had good immunogenicity. We vaccinated BALB/c mice with rSjPGK combined with Seppic 206 adjuvant. However, there were no significant reductions in the numbers of worms of eggs in the liver, as compared to adjuvant or blank control groups in two independent vaccination tests. This study provides the basis for further investigations into the biological function of SjPGK, although it might not be suitable as a potential vaccine candidate against schistosomiasis.
Topics: Amino Acid Sequence; Animals; Cloning, Molecular; Gene Expression Regulation, Enzymologic; Glyceric Acids; Male; Mice; Mice, Inbred BALB C; Phosphoglycerate Kinase; Phylogeny; Rabbits; Random Allocation; Rats; Rats, Wistar; Schistosoma japonicum; Sequence Alignment; Vaccination
PubMed: 26299245
DOI: 10.1016/j.exppara.2015.08.016 -
The Journal of Physical Chemistry. B Mar 2016It is frequently assumed that fluorescent protein tags used in biological imaging experiments are minimally perturbing to their host protein. As in-cell experiments...
It is frequently assumed that fluorescent protein tags used in biological imaging experiments are minimally perturbing to their host protein. As in-cell experiments become more quantitative and measure rates and equilibrium constants, rather than just "on-off" activity or the presence of a protein, it becomes more important to understand such perturbations. One criterion for a protein modification to be a perturbation is additivity of two perturbations (a linear effect on the protein free energy). Here we show that adding fluorescent protein tags to a host protein in vitro has a large nonadditive effect on its folding free energy. We compare an unlabeled, three singly labeled, and a doubly labeled enzyme (phosphoglycerate kinase). We propose two mechanisms for nonadditivity. In the "quinary interaction" mechanism, two tags interact transiently with one another, relieving the host protein from unfavorable tag-protein interactions. In the "crowding" mechanism, adding two tags provides the minimal crowding necessary to overcome destabilizing interactions of individual tags with the host protein. Both of these mechanisms affect protein stability in cells; we show here that they must also be considered for tagged proteins used for reference in vitro.
Topics: Enzyme Stability; Fluorescence; Luminescent Proteins; Models, Molecular; Phosphoglycerate Kinase; Protein Folding; Saccharomyces cerevisiae; Thermodynamics
PubMed: 26923443
DOI: 10.1021/acs.jpcb.5b11915