Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Ligand binding can affect the thermal stability of PMM2. Heat-induced melting profile of wild-type PMM2 (A and C) and F119L-PMM2 (B and D) were recorded by thermal shift assay and by circular dichroism. For thermal shift assay, the proteins (0.2 mg/mL) were equilibrated in buffer (HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5) containing Sypro Orange2.5X and the appropriate ligands: MgCl 2 5 mmol/L, EDTA 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L, Glc-6-P 0.5 mmol/L + MgCl 2 5 mmol/L + vanadate 0.5 mmol/L, Glu-1,6-P 0.5 mmol/L + MgCl 2 5 mmol/L. The samples were distributed in 96-well PCR plates, the plates were sealed, and heated from 25 to 80° at 0.5°C/min. The experiment was run on an iCycler iQ Real Time PCR Detection System. An excitation wavelength of 490 nm and an emission wavelength of 575 nm were used to collect the data. When the melting profile was obtained by circular dichroism the proteins (0.2 mg/mL) were equilibrated in the same buffer in the presence of MgCl 2 1 mmol/L or EDTA 5 mmol/L. The signal at 222 nm was recorded while temperature was increased at 0.5°C/min from 20°C. The raw data were corrected by taking into account the slopes of the pre- and post-transition baselines, then they were normalized.

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Ligand Binding Assay, Thermal Shift Assay, Gas Chromatography, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Specific activity of PMM2 depends on glucose-1,6-bisphosphate concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glu-1-P (0.04 or 0.60 mmol/L), and yeast glucose 6-phosphate dehydrogenase 10 μg/mL, while Glc-1,6-P 2 was changed in the range 0–80 μmol/L. Enzymes concentrations were 107 nmol/L for wt-PMM2 and 73 nmol/L for F119L-PMM2. The hyperbolic dependence of velocity on the activator concentration was fitted using Michaelis and Menten equation to evaluate EC50.

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Activity Assay, Concentration Assay, Gas Chromatography

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Long-term stability of F119L-PMM2. F119L-PMM2 (0.027 mmol/L of monomer equivalents) was equilibrated in HEPES 50 mmol/L pH 7.1 containing NaCl 150 mmol/L. Aliquots containing 1.6 μg of protein were taken at known incubation time and diluted immediately to assay the residual activity with Glc-1-P under standard conditions. (A) Results obtained at 37°C in the presence of EDTA 0.1 mmol/L or MgCl 2 5 mmol/L. (B) Results obtained at 44°C in the presence of MgCl 2 5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L, MgCl 2 5 mmol/L plus Glc-1-P 0.5 mmol/L and vanadate 0.5 mmol/L or MgCl 2 5 mmol/L plus Glu-1,6-P 2 0.5 mmol/L

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Incubation, Activity Assay, Gas Chromatography

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Ligand binding can increase the resistance to proteases of PMM2. Purified wild-type PMM2 and F119L-PMM2 (A) were incubated (0.5 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w) for 1 or 2 h at 37°C before they were analyzed (5 μg of each sample) by SDS-PAGE. Purified F119L-PMM2 (B) was incubated (0.2 mg/mL) with thermolysin in HEPES 20 mmol/L, NaCl 150 mmol/L, MgCl 2 0.1 mmol/L, pH 7.5 at the indicated protease substrate ratio (w/w), in the presence of no ligands, Glc-1,6-P 2 0.5 mmol/L or Glu-6-P 0.5 mmol/L plus vanadate 0.5 mmol/L, for 2 h at 37°C before they were analyzed (2 μg of each sample) by SDS-PAGE. The protein bands were visualized by Coomassie blue staining and the intensity of the bands quantified. The not-digested protein was quantified and expressed as percentage of the starting material (no protease panel A; time 0 panel B).

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Ligand Binding Assay, Purification, Incubation, SDS Page, Gas Chromatography, Staining

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Ligand binding affects the quaternary structure of PMM2. Wild-type PMM2 (0.010 mg) and F119L-PMM2 (0.0065 mg) were subjected to size exclusion chromatography on BioSep-SEC-S3000 column equilibrated in HEPES 20 mmol/L pH 7.5, NaCl 150 mmol/L, MgCl 2 5 mmol/L (long dashed line for the wild-type PMM2 or short dashed line for F119L-PMM2) or in the same buffer containing Glc-6-P 0.5 mmol/L and vanadate 0.1 mmol/L (continuous line for wild-type PMM2 or dotted line for F119L-PMM2). The chromatography was run at room temperature at 0.5 mL/min.

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Ligand Binding Assay, Size-exclusion Chromatography, Gas Chromatography, Chromatography

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Specific activity of F119L-PMM2 depends on enzyme concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L, and yeast glucose 6-phosphate dehydrogenase 10 μg/mL. The reaction mixture also contained BSA at 0.5 mg/mL. Three sets of experiments were carried out in the presence of 0.04, 0.16, or 0.6 mmol/L Glc-1-P and the F119L-PMM2 concentration changed in the range 10–240 nmol/L (monomer equivalents).

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Activity Assay, Concentration Assay, Gas Chromatography

Journal: Molecular Genetics & Genomic Medicine

Article Title: Biochemical phenotype of a common disease-causing mutation and a possible therapeutic approach for the phosphomannomutase 2-associated disorder of glycosylation

doi: 10.1002/mgg3.3

Figure Lengend Snippet: Specific activity of wt-PMM2 changes as a function of protein concentration. The assay was performed at 32°C in a reaction mixture containing HEPES 20 mmol/L, pH 7.5, MgCl 2 5 mmol/L, NaCl 150 mmol/L, NADP+ 0.25 mmol/L, Glc-1,6-P 2 0.030 mmol/L and yeast glucose 6-phosphate dehydrogenase 0.010 mg/mL. The reaction mixture also contained Glc-1-P 0.020 mmol/L and BSA 0.5 mg/mL. The wt-PMM2 concentration changed in the range 2–110 nmol/L (monomer equivalents).

Article Snippet: The proteins (0.2 mg/mL) were equilibrated in the presence of HEPES 20 mmol/L, NaCl 150 mmol/L, pH 7.5, Sypro Orange 2.5× (Invitrogen Molecular Probes, http://lifetechnologies.com ).

Techniques: Activity Assay, Protein Concentration, Gas Chromatography, Concentration Assay

Journal: PLoS ONE

Article Title: Glutamate Uptake Triggers Transporter-Mediated GABA Release from Astrocytes

doi: 10.1371/journal.pone.0007153

Figure Lengend Snippet: Glu-induced GABA release is not mediated by vesicular release. (A) Elevation of [GABA] o in acute rat hippocampal slices in control conditions (n = 6) and in the presence of glutamate receptor antagonists (n = 7; P = 0.014; 128.5±13.4% of pre-stimulus control). (B) Cytosolic GABA level as determined by [ 3 H]GABA uptake in the presence of 100 µM NNC-711 and Glu receptor antagonists in rat cerebrocortical NPMV fractions (n = 4; P = 0.82) (C) Elevation of [GABA] o in acute rat hippocampal slices in control conditions (n = 6) and in low-[Ca 2+ ]/high-[Mg 2+ ] buffer (n = 5; P = 0.91; 148.3±15.5% of pre-stimulus control). (D) Cytosolic GABA level as determined by [ 3 H]GABA uptake in the presence of 100 µM NNC-711 in low-[Ca 2+ ] buffer in rat cerebrocortical NPMV fractions (n = 3; P = 0.49). Low-[Ca 2+ ] buffer contained 145 mM NaCl, 5 mM KCl, 20 mM MgCl 2 , 10 mM glucose and 20 mM HEPES (pH 7.5).

Article Snippet: Uptake studies were performed as follows: aliquots of NPMV fractions in HEPES buffer were preincubated with test compounds in the presence of NNC-711 (100 µM, Tocris) at 30°C for 10 min.

Techniques: