Interaction of inorganic vanadate with glucose-6-phosphate dehydrogenase. Nonenzymic formation of glucose 6-vanadate

Inorganic vanadate (Vi) activates catalysis by glucose-6-phosphate dehydrogenase of the oxidation of glucose by NADP+. As the concentration of Glu-6-P dehydrogenase is increased, the rate of the vanadate-activated glucose oxidation becomes less sensitive to increases in enzyme concentration. The rate of glucose oxidation in the absence of Vi increases linearly with Glu-6-P dehydrogenase concentration. These results are interpreted in terms of nonenzymic formation of glucose 6-vanadate. At high enzyme concentration, vanadate ester formation becomes partially rate-limiting, and extrapolation to infinite Glu-6-P dehydrogenase concentration allows determination of the second order rate constant for formation of the ester. In separate experiments designed to test the proposed mechanism, it was found that Vi, at concentrations at which it strongly activates catalysis by Glu-6-P dehydrogenase of glucose oxidation, has no effect on the rates of oxidation of glucose 6-phosphate or 6-deoxyglucose catalyzed by Glu-6-P dehydrogenase. Sulfate, which is known to activate glucose oxidation and to inhibit glucose 6-phosphate oxidation, strongly activates 6-deoxyglucose oxidation. These experiments show that the 6-hydroxyl group of glucose is essential for the observed activation by Vi and are also consistent with the formation of glucose 6-vanadate. Also, the rate of the sulfate-activated glucose oxidation increases linearly with Glu-6-P dehydrogenase concentration. These results are consistent with the proposed mechanism for sulfate activation which involves sulfate binding to the enzyme (Anderson, W. B., Horne, R. N., and Nordlie, R. C. (1968) Biochemistry 7, 3997-4004). The second order rate constant calculated for formation of glucose 6-vanadate at pH 7.0 is 2.4 M-1 s-1. The corresponding values for glucose 6-phosphate and glucose 6-arsenate formation are approximately 9 X 10(-11) M-1 s-1 and 6.3 X 10(-6) M-1 s-1 (Lagunas, R. (1980) Arch. Biochem. Biophys. 205, 67-75).     

Complementarity in the regulation of phosphoglucomutase, phosphofructokinase and hexokinase; the role of glucose 1,6-bisphosphate.

ATP and citrate, the well known inhibitors of phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11), were found to inhibit the activities of the multiple forms of phosphoglucomutase (alpha-D-glucose 1,6-bisphosphate: alpha-D-glucose 1-phosphate phosphotransferase, EC 2.7.5.1) from rat muscle and adipose tissue. This inhibition could be reversed by an increase in the glucose 1,6-bisphosphate (Glc-1,6-P2) concentration. Other known activators (deinhibitors) of phosphofructokinase, viz. cyclic AMP, AMP, ADP or Pi, had no direct deinhibitory action on the ATP or citrate inhibited multiple phosphoglucomutases. Cyclic AMP and AMP, could however lead indirectly to deinhibition of the phosphoglucomutases, by activating phosphofructokinase which catalyzes the ATP-dependent phosphorylation of glucose 1-phosphate to form Glc-1,6-P2, the la-ter then released the multiple phosphoglucomutases from ATP or citrate inhibition. The Glc-1,6-P2 was also found to exert a selective inhibitory effect on hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) type II, the predominant form in skeletal muscle. This selective inhibition by Glc-1,6-P2 was demonstrated on the multiple hexokinases which were resolved by cellogel electrophoresis or isolated by chromatography on DEAE-cellulose. Based on the in vitro studies it is suggested that during periods of highly active epinephrine-induced glycogenolysis in muscle, the Glc-1,6-P2, produced by the cyclic AMP-stimulated reaction of phosphofructokinase with glucose 1-phosphate, will release the phosphoglucomutases from ATP or citrate inhibition, and will depress the activity of muscle type II hexokinase.     

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.     

Mammalian phosphomannomutase PMM1 is the brain IMP-sensitive glucose-1,6-bisphosphatase

Glucose 1,6-bisphosphate (Glc-1,6-P(2)) concentration in brain is much higher than what is required for the functioning of phosphoglucomutase, suggesting that this compound has a role other than as a cofactor of phosphomutases. In cell-free systems, Glc-1,6-P(2) is formed from 1,3-bisphosphoglycerate and Glc-6-P by two related enzymes: PGM2L1 (phosphoglucomutase 2-like 1) and, to a lesser extent, PGM2 (phosphoglucomutase 2). It is hydrolyzed by the IMP-stimulated brain Glc-1,6-bisphosphatase of still unknown identity. Our aim was to test whether Glc-1,6-bisphosphatase corresponds to the phosphomannomutase PMM1, an enzyme of mysterious physiological function sharing several properties with Glc-1,6-bisphosphatase. We show that IMP, but not other nucleotides, stimulated by >100-fold (K(a) approximately 20 mum) the intrinsic Glc-1,6-bisphosphatase activity of recombinant PMM1 while inhibiting its phosphoglucomutase activity. No such effects were observed with PMM2, an enzyme paralogous to PMM1 that physiologically acts as a phosphomannomutase in mammals. Transfection of HEK293T cells with PGM2L1, but not the related enzyme PGM2, caused an approximately 20-fold increase in the concentration of Glc-1,6-P(2). Transfection with PMM1 caused a profound decrease (>5-fold) in Glc-1,6-P(2) in cells that were or were not cotransfected with PGM2L1. Furthermore, the concentration of Glc-1,6-P(2) in wild-type mouse brain decreased with time after ischemia, whereas it did not change in PMM1-deficient mouse brain. Taken together, these data show that PMM1 corresponds to the IMP-stimulated Glc-1,6-bisphosphatase and that this enzyme is responsible for the degradation of Glc-1,6-P(2) in brain. In addition, the role of PGM2L1 as the enzyme responsible for the synthesis of the elevated concentrations of Glc-1,6-P(2) in brain is established.     

Intracellular glucose 1-phosphate and glucose 6-phosphate levels modulate Ca2+ homeostasis in Saccharomyces cerevisiae

The enzyme phosphoglucomutase plays a key role in cellular metabolism by virtue of its ability to interconvert Glc-1-P and Glc-6-P. It was recently shown that a yeast strain lacking the major isoform of phosphoglucomutase (pgm2Delta) accumulates a high level of Glc-1-P and exhibits several phenotypes related to altered Ca(2+) homeostasis when d-galactose is utilized as the carbon source (Fu, L., Miseta, A., Hunton, D., Marchase, R. B., and Bedwell, D. M. (2000) J. Biol. Chem. 275, 5431-5440). These phenotypes include increased Ca(2+) uptake and accumulation and sensitivity to high environmental Ca(2+) levels. In the present study, we overproduced the enzyme UDP-Glc pyrophosphorylase to test whether the overproduction of a downstream metabolite produced from Glc-1-P can also mediate changes in Ca(2+) homeostasis. We found that overproduction of UDP-Glc did not cause any alterations in Ca(2+) uptake or accumulation. We also examined whether Glc-6-P can influence cellular Ca(2+) homeostasis. A yeast strain lacking the beta-subunit of phosphofructokinase (pfk2Delta) accumulates a high level of Glc-6-P (Huang, D., Wilson, W. A., and Roach, P. J. (1997) J. Biol. Chem. 272, 22495-22501). We found that this increase in Glc-6-P led to a 1.5-2-fold increase in total cellular Ca(2+). We also found that the pgm2Delta/pfk2Delta strain, which accumulated high levels of both Glc-6-P and Glc-1-P, no longer exhibited the Ca(2+)-related phenotypes associated with high Glc-1-P levels in the pgm2Delta mutant. These results provide strong evidence that cellular Ca(2+) homeostasis is coupled to the relative levels of Glc-6-P and Glc-1-P in yeast.     

Glucose-1-phosphate transport into protoplasts and chloroplasts from leaves of Arabidopsis

Almost all glucosyl transfer reactions rely on glucose-1-phosphate (Glc-1-P) that either immediately acts as glucosyl donor or as substrate for the synthesis of the more widely used Glc dinucleotides, ADPglucose or UDPglucose. In this communication, we have analyzed two Glc-1-P-related processes: the carbon flux from externally supplied Glc-1-P to starch by either mesophyll protoplasts or intact chloroplasts from Arabidopsis (Arabidopsis thaliana). When intact protoplasts or chloroplasts are incubated with [U-(14)C]Glc-1-P, starch is rapidly labeled. Incorporation into starch is unaffected by the addition of unlabeled Glc-6-P or Glc, indicating a selective flux from Glc-1-P to starch. However, illuminated protoplasts incorporate less (14)C into starch when unlabeled bicarbonate is supplied in addition to the (14)C-labeled Glc-1-P. Mesophyll protoplasts incubated with [U-(14)C]Glc-1-P incorporate (14)C into the plastidial pool of adenosine diphosphoglucose. Protoplasts prepared from leaves of mutants of Arabidopsis that lack either the plastidial phosphorylase or the phosphoglucomutase isozyme incorporate (14)C derived from external Glc-1-P into starch, but incorporation into starch is insignificant when protoplasts from a mutant possessing a highly reduced ADPglucose pyrophosphorylase activity are studied. Thus, the path of assimilatory starch biosynthesis initiated by extraplastidial Glc-1-P leads to the plastidial pool of adenosine diphosphoglucose, and at this intermediate it is fused with the Calvin cycle-driven route. Mutants lacking the plastidial phosphoglucomutase contain a small yet significant amount of transitory starch.     

The participation of glucose-1,6-diphosphate in the regulation of hexokinase and phosphoglucomutase activities in brains of young and adult rats

1. The level of glucose-1,6-diphosphate (Glc-1,6-P2), the powerful regulator of carbohydrate metabolism, was found to be strikingly decreased in brains of adult rats (5 months of age) as compared to young (10-14 days of age). 2. This age-related decrease in Glc-1,6-P2, the potent inhibitor of hexokinase and activator of phosphoglucomutase, was accompanied by a correlated increase in the activity of hexokinase and a reduction in phosphoglucomutase. 3. Evidence is provided showing that Glc-1,6-P2 participates in the regulation of these enzymes' activities with age. 4. The age-related changes in Glc-1,6-P2 and in the enzymes' activities in brain were opposite to those which we previously found in skeletal muscle. 5. These results suggest that Glc-1,6-P2 is involved in the regulation of carbohydrate metabolism during growth in both brain and muscle, as well as in the interrelationship between these two tissues.     

Studies on the Formation and the Degradation of Glucose 1,6-Bisphosphate in the Beef Liver Homogenate

Four kinds of the enzyme reactions have been reported for the synthesis of Glc-1,6-P₂. However, any activity of Glc-1-P dismutase and phosphoglucokinase was not observed in the beef liver homogenate. When the liver homogenate was incubated with Glc-1-P and Fru-1,6-P₂, a significant amount of Glc-1,6-P₂ was formed. The Glc-1,6-P₂ synthesis activity from Glc-1-P and Fru-1,6-P₂ was caused by the action of phosphoglucomutase present in the liver homogenate. The most remarkable activity for Glc-1,6-P₂ synthesis was observed when the homogenate was incubated with Glc-1-P and glycerate-1,3-P₂. The Glc-1,6-P₂ synthesis activity from Glc-1-P and glycerate-1,3-P₂ was separated from the major peak of phosphoglucomutase activity by DEAE-Sephadex chromatography. The peak of Glc-1,6-P₂ synthesis activity, however, still retained phosphoglucomutase activity.

Glc-1,6-P₂ phosphatase activity was mainly observed in the mitochondria and microsome fraction. The properties of Glc-1,6-P₂ phosphatase were differentiated from those of acid phosphatase and Glc-6-P phosphatase.     

Actin mediated release of ATP from a myosin-ATP complex.

The apparent second-order rate constant, ka-2, of actin binding to a myosin-ATP state (M*.ATP) and releasing ATP to the medium has been determined by two methods. The first was the measurement of the amount of ATP released when actin was added to the intermediate state, M*.ATP; the second was the measurement of oxygen exchange between ATP and HOH. A quantitative treatment of ATP in equilibrium HOH exchange is given to allow extraction of elementary rate constants from the data. Agreement between the two methods was good and at low ionic strength and 23 degrees C, ka-2 is 6 X 10(5) M-1 s-1 which is about one-third the value of the apparent second-order rate constant, ka4, of actin binding to the myosin product state (M**.ADP.Pi). The determination of ka-2 allows a lower limit of 6 s-1 to be placed upon the first-order rate of ATP release from AM.ATP. This is to be compared with a value of less than or equal to 1.5 X 10(-4) s-1 for the equivalent steps of the myosin scheme; thus actin enhances the rate by a factor of 4 X 10(4) or more. A greater proportion of the bound ATP is released to the medium as ATP with increasing actin concentration. This reflects the contribution to rate limitation at saturating actin concentration of steps between myosin states dissociated from actin.     

Multifunctional Properties of Beef Liver Phosphoglucomutase

Liver phosphoglucomutase was found to catalyze also the reaction of Glc-1,6-P₂ formation from Glc-1-P and Fru-1,6-P_z or Glc-1-P and glycerate-1,3-P₂. The specific activity of Glc-1,6-P₂ formation from Glc-1-P and Fru-1,6-P₂ was 1/9200 of that of the mutase activity. The activity of Glc-1,6-P₂ formation from Glc-1-P and glycerate-1,3-P₂ was 1/122,000 of the mutase activity. From the results of the kinetics and the thermal inactivation experiments, the reaction of the mutase and Glc-1,6-P₂ synthesis were strongly suggested to occur at the same active site of liver phosphoglucomutase.

Liver phosphoglucomutase exhibited the Glc-1,6-P₂ phosphatase activity only in the presence of xylose 1-phosphate. The specific activity of phosphatase was only 1/154,000 of that of the mutase activity.     

Increase in glucose 1,6-bisphosphate levels, activation of phosphofructokinase and phosphoglucomutase, and inhibition of glucose 1,6-bisphosphatase in muscle induced by trifluoperazine

Injection of trifluoperazine (TFP) to rats induced a significant rise in the level of glucose 1,6-bisphosphate (Glc-1,6-P2) in muscle. This increase in Glc-1,6-P2, the potent activator of phosphofructokinase and phosphoglucomutase, was accompanied by a marked activation of both enzymes, when assayed in the absence of exogenous Glc-1,6-P2 under conditions in which these enzymes are sensitive to regulation by endogenous Glc-1,6-P2. Glucose-1,6-bisphosphatase (the enzyme that degrades Glc-1,6-P2) was markedly inhibited following the injection of TFP, which may account for the rise in the Glc-1,6-P2 level. Previous results from this laboratory have revealed that muscle damage or weakness is characterized by a decrease in Glc-1,6-P2 levels, leading to a marked reduction in the activities of phosphoglucomutase and phosphofructokinase (the rate-limiting enzyme in glycolysis). The present results suggest that TFP treatment may have a beneficial effect on the depressed glycolysis in muscle weakness or damage.     

Kinetic determination of talin-actin binding

Smooth muscle talin prepared from chicken gizzard binds to skeletal muscle actin in vitro. The stoichiometry of 1:3 for talin:fluorescent labelled G-actin was confirmed by steady state titration and viscosity measurements under non-polymerizing conditions. The binding constant (Kd) of talin and G-actin was determined by continuous fluorescence titration and gave a value of approx 0.3 microM. The association rate constant of talin and fluorescent labelled G-actin of approx 7 x 10(6) M-1 x s-1 was ascertained by the stopped flow method; the dissociation rate constant was calculated at approx 2-3 s-1.     

Identification of the 64 kilodalton chloroplast stromal phosphoprotein as phosphoglucomutase

Phosphorylation of the 64 kilodalton stromal phosphoprotein by incubation of pea (Pisum sativum) chloroplast extracts with [γ-³²P]ATP decreased in the presence of Glc-6-P and Glc-1,6-P₂, but was stimulated by glucose. Two-dimensional gel electrophoresis following incubation of intact chloroplasts and stromal extracts with [γ-³²P]ATP, or incubation of stromal extracts and partially purified phosphoglucomutase (EC 2.7.5.1) with [³²P]Glc-1-P showed that the identical 64 kilodalton polypeptide was labeled. A 62 kilodalton polypeptide was phosphorylated by incubation of tobacco (Nicotiana sylvestris) stromal extracts with either [γ-³²P]ATP or [³²P]Glc-1-P. In contrast, an analogous polypeptide was not phosphorylated in extracts from a tobacco mutant deficient in plastid phosphoglucomutase activity. The results indicate that the 64 (or 62) kilodalton chloroplast stromal phosphoprotein is phosphoglucomutase.     

Resolution of phosphoglucomatase and the 62-kDA acceptor for the glucosylphosphotransferase

The radioactive, photoactivatable labeling probe [beta-32P]5-azidouridine 5'-diphosphoglucose has recently been shown to label a 62-kDa protein in crude homogenates and in partially purified enzyme preparations without photoactivation. Here, we report that a portion of this radioactivity is due to labeling of phosphoglucomutase by contaminating levels of [32P]alpha Glc-1-P initially present at less than 1% of the total 32P. This conclusion is based in part on the ability of excess unlabeled alpha Glc-1-P and Glc-6-P, but not UDP-Glc, to block the labeling. In addition, the labeled protein in liver homogenates had a tryptic peptide pattern similar to that of authentic phosphoglucomutase. These findings, however, raised a second question. Assays for the UDP-Glc: glycoprotein glucosyl phosphotransferase (Glc phosphotransferase) have utilized [beta-32P]UDP-Glc and have resulted in the labeling of a small number of acceptors, including one of approximately 62 kDa. Despite the fact that these assays had routinely been performed in the presence of 1 mM alpha Glc-1-P, the coincidence in molecular weights led to these further studies. We conclude that the acceptor of approximately 62 kDa is distinct from phosphoglucomutase. This conclusion is based on differences in the time courses of incorporation, the specificity of blocking agents, the presence of covalently linked glucose, the products of acid hydrolysis and of beta-elimination, and isoelectric points.     

Comparison of rate constants for (PO3-) transfer by the Mg(II), Cd(II), and Li(I) forms of phosphoglucomutase

Net rate constants that define the steady-state rate through a sequence of steps and the corresponding effective energy barriers for two (PO3-)-transfer steps in the phosphoglucomutase reaction were compared as a function of metal ion, M, where M = Mg2+ and Cd2+. These steps involve the reaction of either the 1-phosphate or the 6-phosphate of glucose 1,6-bisphosphate (Glc-P2) bound to the dephosphoenzyme (ED) to produce the phosphoenzyme (EP) and the free monophosphates, glucose 1-phosphate (Glc-1-P) or glucose 6-phosphate (Glc-6-P): EP.M + Glc-1-P----ED.M.Glc-P2----EP.M.Glc-6-P6. Before this comparison was made, net rate constants for the Cd2+ enzyme, obtained at high enzyme concentration via 31P NMR saturation-transfer studies [Post, C. B., Ray, W. J., Jr., & Gorenstein, D. G. (1989) Biochemistry (preceding paper in this issue)], were appropriately scaled by using the observed constants to calculate both the expected isotope-transfer rate at equilibrium and the steady-state rate under initial velocity conditions and comparing the calculated values with those measured in dilute solution. For the Mg2+ enzyme, narrow limits on possible values of the corresponding net rate constants were imposed on the basis of initial velocity rate constants for the forward and reverse directions plus values for the equilibrium distribution of central complexes, since direct measurement is not feasible. The effective energy barriers for both the Mg2+ and Cd2+ enzymes, calculated from the respective net rate constants, together with previously values for the equilibrium distribution of complexes in both enzymic systems [Ray, W. J., Jr., & Long, J. W. (1976) Biochemistry 15, 4018-4025], show that the 100-fold decrease in the kappa cat for the Cd2+ relative to the Mg2+ enzyme is caused by two factors: the increased stability of the intermediate bisphosphate complex and the decreased ability to cope with the phosphate ester involving the 1-hydroxyl group of the glucose ring. In fact, it is unlikely that the efficiency of (PO3-) transfer to the 6-hydroxyl group of bound Glc-1-P (thermodynamically favorable direction) is reduced by more than an order of magnitude in the Cd2+ enzyme. By contrast, the efficiency of the Li+ enzyme in the same (PO3-)-transfer step is less than 4 x 10(-8) that of the Mg2+ enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ligand binding by the chlorocruorin from Eudistylia vancouverii.

Carbon monoxide chlorocruorin from Eudistylia vancouverii shows three distinct first-order relaxations with rates of 2.9 x 10(9) s-1, 6.5 x 10(7) s-1, and 3.2 x 10(6) s-1 (geminate reactions) and three second-order relaxations with rates of 4.7 x 10(6) M-1 s-1, 7 x 10(5) M-1 s-1, and 7 x 10(4) M-1 s-1, when studied by flash photolysis. The amplitudes of the second-order reactions depend on the extent of photolysis. This may be due to relaxation from the liganded (R) to the unliganded (T) conformation following photolysis and suggests that the combination rates contribute to cooperativity. In a stopped-flow experiment only the slowest phase with a rate of 7 x 10(4) M-1 s-1 is observed. It is assigned to binding to the T-state protein. Fragments of the native protein containing 12 and 4 hemes react like the holoprotein suggesting that the tetramer is a major cooperative unit. Oxygen binding shows three geminate relaxations with rates of 2.5 x 10(10) s-1, 3.5 x 10(7) s-1, and 4.5 x 10(6) s-1, and two second-order rates of 1.5 x 10(7) M-1 s-1 and 1 x 10(6) M-1 s-1. The amplitudes of the second-order phases do not correlate with the extent of photolysis. The results with the two ligands are consistent with an allosteric transition fast enough to compete with a rebinding rate of 500 s-1 in the R to T direction (CO rebinding) but not fast enough to compete with oxygen rebinding. There is significant heterogeneity in the R-state kinetics, but the T-state reaction is homogeneous.     

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.     

Capsule biosynthesis and basic metabolism in Streptococcus pneumoniae are linked through the cellular phosphoglucomutase

Synthesis of the type 3 capsular polysaccharide of Streptococcus pneumoniae requires UDP-glucose (UDP-Glc) and UDP-glucuronic acid (UDP-GlcUA) for production of the [3)-beta-D-GlcUA-(1-->4)-beta-D-Glc-(1-->](n) polymer. The generation of UDP-Glc proceeds by conversion of Glc-6-P to Glc-1-P to UDP-Glc and is mediated by a phosphoglucomutase (PGM) and a Glc-1-P uridylyltransferase, respectively. Genes encoding both a Glc-1-P uridylyltransferase (cps3U) and a PGM homologue (cps3M) are present in the type 3 capsule locus, but these genes are not essential for capsule production. In this study, we characterized a mutant that produces fourfold less capsule than the type 3 parent. The spontaneous mutation resulting in this phenotype was not contained in the type 3 capsule locus but was instead located in a distant gene (pgm) encoding a second PGM homologue. The function of this gene product as a PGM was demonstrated through enzymatic and complementation studies. Insertional inactivation of pgm reduced capsule production to less than 10% of the parental level. The loss of PGM activity in the insertion mutants also caused growth defects and a strong selection for isolates containing second-site suppressor mutations. These results demonstrate that most of the PGM activity required for type 3 capsule biosynthesis is derived from the cellular PGM.     

Observation of uridine triphosphate:glucose-1-phosphate uridylyltransferase activity in maize root tips by saturation transfer 31P-NMR. Estimation of cytoplasmic PP

Saturation transfer 31P nuclear magnetic resonance was used to estimate the unidirectional rate of phosphorus exchange between Glc-1-P and UDPGlc in maize root tips. The rate was determined to be approx. 4 mumol.min-1 per g fresh weight. This estimated rate is much higher than net rates of other reactions in glucose metabolism (e.g., more than 10-times faster than the maximal glycolytic flux in this tissue). Furthermore, exchange between Glc-1-P and UDPGlc was not significantly inhibited by the metabolic poison KCN. We conclude that the unidirectional rate of conversion of Glc-1P to UDPGlc is much faster than the net rate of UDPGlc synthesis--the UTP:Glc-1-P uridylyltransferase reaction is near-equilibrium in vivo. From the equilibrium constant for this transferase reaction and the concentrations of Glc-1-P, UTP and UDPGlc, the level of cytoplasmic PPi was estimated to be approx. 10 nmol.g-1.     

Brain hexokinase has no preexisting allosteric site for glucose 6-phosphate

Difference spectroscopic investigations on the interaction of brain hexokinase with glucose and glucose 6-phosphate (Glc-6-P) show that the binary complexes E-glucose and E-Glc-6-P give very similar UV difference spectra. However, the spectrum of the ternary E-glucose-Glc-6-P complex differs markedly from the spectra of the binary complexes, but resembles that produced by the E-glucose-Pi complex. Direct binding studies of the interaction of Glc-6-P with brain hexokinase detect only a single high-affinity binding site for Glc-6-P (KD = 2.8 microM). In the ternary E-glucose-Glc-6-P complex, Glc-6-P has a much higher affinity for the enzyme (KD = 0.9 microM) and a single binding site. Ribose 5-phosphate displaces Glc-6-P from E-glucose-Glc-6-P only, but not from E-Glc-6-P complex. It also fails to displace glucose from E-glucose and E-glucose-Glc-6-P complexes. Scatchard plots of the binding of glucose to brain hexokinase reveal only a single binding site but show distinct evidence of positive cooperativity, which is abolished by Glc-6-P and Pi. These ligands, as well as ribose 5-phosphate, substantially increase the binding affinity of glucose for the enzyme. The spectral evidence, as well as the interactive nature of the sites binding glucose and phosphate-bearing ligands, lead us to conclude that an allosteric site for Glc-6-P of physiological relevance occurs on the enzyme only in the presence of glucose, as a common locus where Glc-6-P, Pi, and ribose 5-phosphate bind. In the absence of glucose, Glc-6-P binds to the enzyme at its active site with high affinity. We also discuss the possibility that, in the absence of glucose, Glc-6-P may still bind to the allosteric site, but with very low affinity, as has been observed in studies on the reverse hexokinase reaction.