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PubMed1983

Changes in the levels of glucose 1,6-diphosphate and cyclic GMP, and in the activities of phosphofructokinase and phosphoglucomutase induced by serotonin in muscle.

Injection of serotonin (5-hydroxytryptamine) induced a marked decrease in the level of glucose 1,6-diphosphate (Glc-1,6-P2) in the rat tibialis anterior muscle. Concomitant to the decrease in Glc-1,6-P2, the potent activator of phosphofructokinase and phosphoglucomutase, the activities of both these enzymes were markedly reduced by serotonin. The level of Glc-1,6-P2 and the activities of phosphofructokinase and phosphoglucomutase increased with age in the tibialis anterior muscle and the effect of serotonin was more pronounced in the older animals. Serotonin also induced a significant increase in the level of cyclic GMP in muscle. The serotonin-induced changes in the normal muscle mimic the changes in carbohydrate metabolism we found previously in muscular dystrophy.

Animals↗

PubMed1982

Effect of vanadate on the formation and stability of the phosphoenzyme forms of 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase and of phosphoglucomutase.

2,3-Bisphosphoglycerate-dependent phosphoglycerate mutase (2,3-bisphospho-D-glycerate:2-phospho-D-glycerate phosphotransferase, EC 2.7.5.3) and phosphoglucomutase (alpha-D-glucose-1,6-bisphosphate:alpha -D-glucose-1-phosphate phosphotransferase, EC 2.7.5.1), which are markedly inhibited by vanadate, possess a ping-pong mechanism involving an intermediate phosphoenzyme. The formation and the stability of these phosphoenzymes have been examined spectrophotometrically in the absence of vanadate. Vanadate does not inhibit the phosphorylation of either mutase by its cofactor. The instability of the phosphoenzyme form of phosphoglycerate mutase increases in the presence of vanadate, but the stability of the phosphorylated phosphoglucomutase is not affected.

2,3-Diphosphoglycerate↗

PubMed1985

Inhibition of phosphoglucomutase by fructose 2,6-bisphosphate.

Fructose 2,6-bisphosphate inhibits phosphoglucomutase. The inhibition is mixed with respect to glucose 1,6-bisphosphate and non-competitive with respect to glucose 1-phosphate. In contrast with fructose 1,6-bisphosphate and glycerate 1,3-bisphosphate, which also possess inhibitory effect, fructose 2,6-bisphosphate does not phosphorylate phosphoglucomutase. Fructose 2,6-bisphosphate preparations contain contaminants which can explain artefactual results previously reported.

Animals↗

PubMedDate not supplied

Determination of phosphoglucomutase phenotypes in hair bulbs.

The research was to determine a simple method of phosphoglucomutase phenotype identification in hair bulb. The agarose technique and electrophoresis on cellulose-acetate foil methods were chosen because a small quantity of the maternal available for examination. It was found out that 1 or 2 bulbs are sufficient to identify the PGM1 features if the electrophoresis method is applied and if more bulbs are available, the PGM3 characteristic can also be identified. The modified technique was used for staining the phosphoglucomutase phenotypes.

Hair↗

PubMed2003

Enhanced phosphorylation and enzymatic activity of phosphoglucomutase by the Btk29A tyrosine kinase in Drosophila.

The Drosophila Btk29A tyrosine kinase is suggested to be involved in diverse processes, although its target proteins are unknown. In the present study, we investigated substrates of Btk29A tyrosine kinase by expressing a catalytically activated form of Btk29A-P1 (Btk-EG) in Drosophila compound eyes. Expression in eye disks led to the development of the rough-eye phenotype and increased tyrosine phosphorylation of a 65-kDa protein. Partial amino acid sequence analysis of this protein showed that it was phosphoglucomutase. Phosphoglucomutase activity in heads from Btk-EG-expressing flies was higher than that in controls, suggesting that the levels of tyrosine phosphorylation and activity of the enzyme are associated with Btk29A tyrosine kinase activity.

Animals↗

PubMed1993

Structural changes at the metal ion binding site during the phosphoglucomutase reaction.

An electron density map of the reactive, Cd2+ form of crystalline phosphoglucomutase from X-ray diffraction studies shows that the enzymic phosphate donates a nonbridging oxygen to the ligand sphere of the bound metal ion, which appears to be tetracoordinate. 31P and 113Cd NMR spectroscopy are used to assess changes in the properties of bound Cd2+ produced by substrate/product and by substrate/product analog inhibitors. The approximately 50 ppm downfield shift of the 113Cd resonance on formation of the complex of dephosphoenzyme and glucose 1,6-bisphosphate is associated with the initial sugar-phosphate binding step and likely involves a change in the geometry of the coordinating ligands. This interpretation is supported by spectral studies involving various complexes of the active Co2+ and Ni(2+)-enzyme. In addition, there is a loss of the 31P-113Cd J coupling that characterizes the monophosphate complexes of the Cd2+ enzyme either during or immediately after the PO3- transfer step that produces the bisphosphate complex, indicating a further change at the metal binding site. The implications of these observations with respect to the PO3- transfer process in the phosphoglucomutase reaction are considered. The apparent plasticity of the ligand sphere of the active site metal ion in this system may allow a single metal ion to act as a chaperone for a nonbridging oxygen during PO3- transfer or to allow a change in metal ion coordination during catalysis. A general NMR line shape/chemical-exchange analysis for evaluating binding in protein-ligand systems when exchange is intermediate to fast on the NMR time scale is described. Its application to the present system involves multiple exchange sites that depend on a single binding rate, thereby adding further constraints to the analysis.

Binding Sites↗

PubMed1993

Comparison of vibrational frequencies of critical bonds in ground-state complexes and in a vanadate-based transition-state analog complex of muscle phosphoglucomutase. Mechanistic implications.

The symmetric stretching frequency of the P-O bonds of the enzymic phosphate group in muscle phosphoglucomutase was measured via 16O/18O Raman difference spectroscopy. This frequency, and its shift on isotopic substitution, is characteristic of a dianionic phosphate ester. The P-O stretching frequency is not detectably altered by the binding of the metal ion activators Mg2+, Zn2+, or Cd2+ nor by the subsequent binding of glucose phosphate. Hence, a binding-induced distortion/polarization of the enzymic phosphate group in the ground state, or enzyme-substrate complex, cannot serve as a rationale for the large value of kcat in the phosphoglucomutase reaction. By contrast, the stretching frequency of the V-O bonds within a vanadate group bound at the same site in the transition-state analog complex involving glucose 1-phosphate 6-vanadate is much lower than for a normal dianionic vanadate. This low V-O stretching frequency is best rationalized in terms of the extensive polarization of all three nonbridging oxygens of the vanadate ester dianion plus the formation of a weak, fifth bond to the vanadium atom. This distortion/polarization of the VO3(2-) group depends on the metal ion activator, since it is largely abolished, and the involvement of the fifth ligand eliminated, by substitution of Li+ for Mg2+ at the metal activation site.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Removal of salt from a salt-induced protein crystal without cross-linking. Preliminary examination of "desalted" crystals of phosphoglucomutase by X-ray crystallography at low temperature.

A model procedure for removing salt from relatively fragile salt-induced protein crystals is proposed. The procedure is based on physical principles and is validated by using millimeter-size crystals of rabbit muscle phosphoglucomutase grown from a 2.1 M solution of ammonium sulfate. Three types of operations are included in the procedure: initial transfer to salt solutions of reduced concentration; transfer to the organic-rich phase of an equilibrium biphasic mixture obtained with aqueous solutions of polyoxyethylene and the salt; and addition of various replacement cosolutes in aqueous solutions of polyoxyethylene to reduce osmotic stress on the crystal as the remaining salt is removed. A critical feature of the overall procedure is maintenance of near equilibrium throughout by using a large number of steps involving small changes in solute concentration. The conditions used in the actual transfer were adjusted to eliminate the fracturing of crystals by visually distinguishing between two opposing types of fracture patterns: those produced by osmotic crushing as opposed to osmotic expansion. Basic requirements for a successful procedure with other protein crystals are a high permeability toward small solutes and a relatively slow dissolution rate at salt concentrations for which biphasic mixtures can be obtained. Desalted crystals of phosphoglucomutase have no visible fractures, are stable in the final solution for at least a week, and exhibit no noticeable change in the resolution of their X-ray diffraction pattern. In fact, desalted crystals can be rapidly cooled to 160 K, whereas untreated crystals are almost completely disordered by the same cooling procedure. The component of the desalting mixture whose presence is crucial to the success of the cooling process is polyoxyethylene, which apparently impedes the formation of ice within the protein crystal. Diffraction data obtained with an area-detector diffractometer did not differ significantly, either in terms of quality or resolution range, between crystals in 2.3 M ammonium sulfate at room temperature and crystals at 160 K in which ammonium sulfate had been replaced by glycine. The successful use of the following replacement solutes, instead of glycine, also is documented: sucrose, glycerol, and a low molecular weight poly(ethylene glycol) (PEG-400).

Animals↗

PubMed1991

Formation of substrate and transition-state analogue complexes in crystals of phosphoglucomutase after removing the crystallization salt.

Crystals of phosphoglucomutase, grown in 2.1 M ammonium sulfate, "desalted", and suspended in a 30% polyoxyethylene-8000/1 M glycine solution as described in the accompanying paper [Ray, W. J., Jr., Puvathingal, J. M., Bolin, J. T., Minor, W., Liu, Y., & Muchmore, S. W. (1991) Biochemistry 30 (preceding paper in this issue)], were treated with glucose phosphates to form an equilibrium mixture of the catalytically active substrate/product complexes. However, this treatment extensively fractured the crystals, even when very dilute solutions of glucose phosphates were used. But formation of the desired complexes was achieved, without fracturing, by introducing the glucose phosphates at high salt concentration, where they do not bind significantly to the enzyme, and maintaining their presence during subsequent sulfate-removal steps, in order to obtain essentially uniform binding throughout the crystal at all times. Although this procedure produced unfractured crystals of the catalytically active complexes, an adjustment in water activity was required to prevent the crystals from slowly liquefying in the presence of the added glucose phosphates. After this adjustment, the quality of diffraction-grade crystals subjected to this treatment was not significantly altered. An even larger adjustment in water activity was required to stabilize crystals that had been largely converted into a mixture of vanadate-based transition-state analogue complexes [cf. Ray, W. J., Jr., & Puvathingal, J. M. (1990) Biochemistry 29, 2790-2801] by means of an analogous procedure. The rationale for, and the implications of, this adjustment of water activity are discussed. The phenomenon of lattice-based binding cooperativity also is discussed together with a possible role for such cooperativity in the fracturing of protein crystals during formation of ligand complexes and possible ways to circumvent such fracturing based on the annealing of crystals at fractional saturation. An assay for quantifying the extent of formation of the vanadate-based transition-state analogue complexes in crystals of phosphoglucomutase is described. A solution to problems associated with producing and maintaining a steady-state in treated crystals is discussed within the context of maximizing the fraction of the crystalline enzyme present as a complex with one such inhibitor, glucose alpha-1-phosphate-6-vanadate. One of these problems, achieving a substantial reduction in sulfate concentration, could not be successfully addressed by employing the desalting procedure used to produce the substrate/product complexes, because of reduced diffusional rates in the final solution.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystallization↗

PubMed1989

Time-dependent 31P saturation transfer in the phosphoglucomutase reaction. Characterization of the spin system for the Cd(II) enzyme and evaluation of rate constants for the transfer process.

Time-dependent 31P saturation-transfer studies were conducted with the Cd2+-activated form of muscle phosphoglucomutase to probe the origin of the 100-fold difference between its catalytic efficiency (in terms of kcat) and that of the more efficient Mg2+-activated enzyme. The present paper describes the equilibrium mixture of phosphoglucomutase and its substrate/product pair when the concentration of the Cd2+ enzyme approaches that of the substrate and how the nine-spin 31P NMR system provided by this mixture was treated. It shows that the presence of abortive complexes is not a significant factor in the reduced activity of the Cd2+ enzyme since the complex of the dephosphoenzyme and glucose 1,6-bisphosphate, which accounts for a large majority of the enzyme present at equilibrium, is catalytically competent. It also shows that rate constants for saturation transfer obtained at three different ratios of enzyme to free substrate are mutually compatible. These constants, which were measured at chemical equilibrium, can be used to provide a quantitative kinetic rationale for the reduced steady-state activity elicited by Cd2+ relative to Mg2+ [cf. Ray, W.J., Post, C.B., & Puvathingal, J.M. (1989) Biochemistry (following paper in this issue)]. They also provide minimal estimates of 350 and 150 s-1 for the rate constants describing (PO3-) transfer from the Cd2+ phosphoenzyme to the 6-position of bound glucose 1-phosphate and to the 1-position of bound glucose 6-phosphate, respectively. These minimal estimates are compared with analogous estimates for the Mg2+ and Li+ forms of the enzyme in the accompanying paper.

Binding Sites↗

PubMed1990

The oxyvanadium constellation in transition-state-analogue complexes of phosphoglucomutase and ribonuclease. Structural deductions from electron-transfer spectra.

The absorbance peak in the near ultraviolet electron-transfer spectrum of the oxyvanadium constellation in the "transition-state-analogue complexes" obtained by treating the dephospho form of phosphoglucomutase with inorganic vanadate in the presence of either glucose 1-phosphate or glucose 6-phosphate, as described in an accompanying paper [Ray, W. J., Jr., Burgner, J. W., II, & Post, C. B. (1990) Biochemistry (second of four papers in this issue)], is centered at a wavelength of 312 nm. The position of this peak amounts to a change in oscillator frequency of about -5000 cm-1 relative to that of tetrahedral VO4(3-). To provide a rationale for this spectral change, the near ultraviolet spectra of the di- and monoanions of inorganic vanadate and a number of derivatives of these anions are compared with that of vanadium (V) in the enzymic complexes, in terms of both what is observed experimentally and what is expected from crystal field theory. Comparisons in water and in largely anhydrous solvents show that water is not an essential element in the coordination sphere of inorganic vanadate or its mono- or diesters and hence that the coordination number of V(V) in such compounds likely is four. These comparisons also show that loss of solvating water from a 4-coordinate vanadate on binding cannot provide a rationale for the spectra of the enzymic complexes. Other comparisons show that neither the binding of metal ions nor protonation nor the binding of vanadate at a site with an unusually high or an unusually low dielectric constant can provide such a rationale. Further comparisons with vanadates known to be pentacoordinate strongly suggest that the coordination number of V(V) in the transition-state-analogue complexes of phosphoglucomutase does not exceed four. In fact, from the standpoint of crystal field theory the marked red shift observed in the electron-transfer absorbance spectrum of the oxyvanadium constellation in these complexes is more reasonably interpreted in terms of a decreased coordination at vanadium (V), viz., in terms of a weakened bonding between vanadium and one or more of its coordinating oxygens. This decreased coordination could be produced by a physical stretching of the vanadate ester linkage. By contrast, the near ultraviolet spectrum of the transition-state-analogue complex that ribonuclease forms with an adduct of uridine and vanadate [Lindquist, R. N., Lynn, J. L., & Lienhard, G. E. (1973) J. Am. Chem. Soc. 95, 8762] is similar to spectra of pentacoordinate model compounds of vanadium(V).(ABSTRACT TRUNCATED AT 400 WORDS)

Electrons↗

PubMed1990

Characterization of a vanadate-based transition-state-analogue complex of phosphoglucomutase by kinetic and equilibrium binding studies. Mechanistic implications.

The inhibitor complex produced by the binding of alpha-D-glucose 1-phosphate 6-vanadate to the dephospho form of muscle phosphoglucomutase exhibits an unusually small dissociation constant: about 15 fM for the Mg2+ enzyme at pH 7.4, when calculated in terms of the tetraanion. Such tight binding suggests that the enzyme/vanadate/glucose phosphate complex mimics a state that at least approaches the transition state for (PO3-) transfer in the normal enzymic reaction. This hypothesis also is supported by the observation that replacement of Mg2+, the normal metal ion activator, by Li+, a poor activator, substantially reduces the binding constant for the glucose phosphate/vanadate mixed diester. Other indicators that support this hypothesis are described. One is the derived equilibrium constant for replacement of a PO4(2-) group in bound glucose bisphosphate by VO4(2-): 3 x 10(6) when the replaced group is the phosphate at the (PO3-) transfer site of the Mg2+ enzyme--in contrast to about 10 for the same replacement (of PO4(2-) by VO4(2-)) in an aqueous solution of a phosphate ester. Another is the greatly decreased rate at which Mg2+ dissociates from the glucose phosphate/vanadate complex of the enzyme, relative to the rate at which it dissociates from the corresponding bisphosphate complex (rate ratio less than or equal to 3 x 10(-4)), presumably because Mg2+ binds more tightly to the glucose phosphate/vanadate complex than to the corresponding bisphosphate complex. This apparent increase in Mg2+ binding occurs in spite of what appears to be a reduced charge density at the bound vanadate grouping, relative to the bound phosphate grouping, and in spite of the somewhat weaker binding of Mg2+ by dianionic vanadate than by the phosphate dianion. Although a direct assessment of the binding constant for Mg2+ was not possible, the equilibrium constant for Mg2+/Li+ exchange could be evaluated for the complexes of dephospho enzyme with glucose bisphosphate or glucose 1-phosphate 6-vanadate. The results suggest that the glucose phosphate/vanadate complex of the Mg2+ enzyme mimics a state about halfway between the ground state and the transition state for (PO3-) transfer. This estimate also is in accord with the binding of glucose phosphate/vanadate relative to that expected for transition-state binding of glucose bisphosphate. A possible scenario for the (PO3-) transfer catalyzed by the Mg2+ form of phosphoglucomutase is discussed, on the basis of these observations, together with possible reasons why the bound vanadate group appears to mimic an intermediate state for (PO3-) transfer rather than the ground state for phosphate binding.

Animals↗

PubMed1973

The reactive serine residue in phosphoglucomutase of Micrococcus lysodeikiticus.

1. Phosphoglucomutase of Micrococcus lysodeikticus was labelled at the active site by exchange with (32)P-labelled substrates of high specific radioactivity. 2. Partial acid hydrolysis gave rise to radioactive peptides; serine phosphate was identified as one of the derivatives. 3. Comparison of the other (32)P-labelled peptides with the peptides obtained from the (32)P-labelled rabbit muscle phosphoglucomutase indicates that the sequence around the reactive serine residue is identical in both enzymes.

Autoradiography↗

PubMed1974

Preparation and properties of a phosphoenzyme from the phosphoglucomutase of Micrococcus lysodeikticus.

1. Phosphoglucomutase from Micrococcus lysodeikticus was incubated with (14)C- and (32)P-labelled glucose 1,6-diphosphate and separated from the cofactor on a Sephadex column. (32)P-labelled phosphate (0.7mol/mol of enzyme) was associated with the enzyme, but no (14)C label was. 2. The (32)P-labelled enzyme exchanged its label with the substrates. When the labelled enzyme was incubated in Tris buffer, pH8.3, at 30 degrees C the proportion of exchangeable label slowly fell indicating a half-life of the phosphoenzyme of about 50h. 3. When HClO(4) was added to the labelled phosphoenzyme all of the label was precipitated with the protein and none was released as P(i). On alkaline hydrolysis P(i) was released at a rate comparable with the rate of hydrolysis of the phosphoenzyme from rabbit muscle. 4. We conclude that the phosphoenzyme from Micrococcus lysodeikticus yields a relatively stable, catalytically active phosphoenzyme when treated with cofactor, and that there is no evidence for the formation of an enzyme-glucose 1,6-diphosphate complex. The properties of the phosphoenzyme, which resemble those of rabbit muscle phosphoglucomutase, suggest that the phosphate may be bound to serine.

Animals↗

PubMed1981

A stereochemical investigation of phosphoryl transfer catalysed by phosphoglucomutase by the use of alpha-D-glucose 1-[(S)-16O,17O,18O]phosphate.

Rabbit muscle phosphoglucomutase converts alpha-D-glucose 1-[(S)-16O,17O,18O]phosphate into D-glucose 6-[16O,17O,18O]phosphate, which is shown by 31P n.m.r. spectroscopy, after cyclization and methylation, to have the (S)-configuration at the phosphorus atom. Since phosphoglucomutase is known to catalyse phosphoryl transfer by way of a phospho-enzyme intermediate, and since individual phosphoryl-transfer steps appear in general to occur with inversion of configuration, this observation is most simply interpreted in terms of a double-displacement mechanism with two phosphoryl-transfer steps.

Animals↗

PubMed2002

Field experiments on the distributions of eggs of different phosphoglucomutase (PGM) genotypes in the yellow dung fly Scathophaga stercoraria (L.).

Female yellow dung flies can, in the laboratory, influence the probability that stored sperm from different males are used to fertilize eggs. This matches offspring phosphoglucomutase genotypes to the environmental conditions in which the larvae will grow, increasing larval growth success. We conducted field experiments in which dung topology or shading conditions were controlled. The proportions of the five common phosphoglucomutase alleles in eggs laid in north-facing slopes or in shaded conditions was related to their electrophoretic mobility. We suggest that females lay eggs of different genotypes, by appropriately choosing their fathers, in different places.

Alleles↗

PubMed2000

Antisense inhibition of plastidial phosphoglucomutase provides compelling evidence that potato tuber amyloplasts import carbon from the cytosol in the form of glucose-6-phosphate.

The aim of this work was to establish whether plastidial phosphoglucomutase is involved in the starch biosynthetic pathway of potato tubers and thereby to determine the form in which carbon is imported into the potato amyloplast. For this purpose, we cloned the plastidial isoform of potato PGM (StpPGM), and using an antisense approach generated transgenic potato plants that exhibited decreased expression of the StpPGM gene and contained significantly reduced total phosphoglucomutase activity. We confirmed that this loss in activity was due specifically to a reduction in plastidial PGM activity. Potato lines with decreased activities of plastidial PGM exhibited no major changes in either whole-plant or tuber morphology. However, tubers from these lines exhibited a dramatic (up to 40%) decrease in the accumulation of starch, and significant increases in the levels of sucrose and hexose phosphates. As tubers from these lines exhibited no changes in the maximal catalytic activities of other key enzymes of carbohydrate metabolism, we conclude that plastidial PGM forms part of the starch biosynthetic pathway of the potato tuber, and that glucose-6-phosphate is the major precursor taken up by amyloplasts in order to support starch synthesis.

Antisense Elements (Genetics)↗

PubMed1994

The addition of glucose-1-phosphate to the cytoplasmic glycoprotein phosphoglucomutase is modulated by intracellular calcium in PC12 cells and rat cortical synaptosomes.

In a number of different cell types, phosphorylation of a 63-kDa protein has been shown to increase rapidly in response to stimuli that lead to an increase in intracellular calcium. Here, a stimulus-sensitive protein at this molecular weight is identified in PC12 cells and rat cortical synaptosomes as phosphoglucomutase. In addition, the added phosphate is shown to be in an oligosaccharide terminating in phosphodiester-linked glucose. In synaptosomes, incorporated radioactivity, following incubation with [14C]glucose or the [beta-35S]phosphorothioate analogue of UDP-glucose, was found to increase within 5 s of stimulation and return to baseline within 25 s. Despite the many pathways utilizing glucose, this was the only detectable protein glycosylation observed in synaptosomes. These results indicate that cytoplasmic glycosylation is reversible and rapidly regulated, and suggest that phosphoglucomutase undergoes an alteration in function and/or topography in response to increases in intracellular calcium.

Animals↗