Mapping of glucose and glucose-6-phosphate binding sites on bovine brain hexokinase. A 1H- and 31P-NMR investigation

Inhibition of bovine brain hexokinase by its product, glucose 6-phosphate, is considered to be a major regulatory step in controlling the glycolytic flux in the brain. Investigations on the molecular basis of this regulation, i.e. allosteric or product inhibition, have led to various proposals. Here, we attempt to resolve this issue by ascertaining the location of the binding sites for glucose and glucose 6-phosphate on the enzyme with respect to a divalent-cation-binding site characterized previously [Jarori, G. K., Kasturi, S. R. & Kenkare, U. W. (1981) Arch. Biochem. Biophys. 211, 258-268]. The paramagnetic effect of enzyme-bound Mn(II) on the spin-lattice relaxation rates (T-1(1] of ligand nuclei (1H and 31P) in E.Mn(II).Glc and E.Mn(II).Glc6P complexes have been measured. The paramagnetic effect of Mn(II) on the proton relaxation rates of C1-H alpha, C1-H beta and C2-H beta of glucose in the E.Mn(II).Glc complex was measured at 270 MHz and 500 MHz. The temperature dependence of these rates was also studied in the range of 5-30 degrees C at 500 MHz. The ligand nuclear relaxation rates in E.Mn(II).Glc are field-dependent and the Arrhenius plot yields an activation energy (delta E) of 16.7-20.9 kJ/mol. Similar measurements have also been carried out on C1-H alpha, C1-H beta and C6-31P at 270 MHz (1H) and 202.5 MHz (31P) for the E.Mn(II).Glc6P complex. The temperature dependence of 31P relaxation rates in this complex was measured in the range 5-30 degrees C, which yielded delta E = 9.2 kJ/mol. The electron-nuclear dipolar correlation time (tau c), determined from the field-dependent measurements of proton relaxation rates in the E.Mn(II).Glc complex, is 0.22-1.27 ns. The distances determined between Mn(II) and C1-H of glucose and glucose 6-phosphate are approximately 1.1 nm and approximately 0.8 nm, respectively. These data, considered together with our recent results [Mehta, A., Jarori, G. K. & Kenkare, U. W. (1988) J. Biol. Chem. 263, 15492-15498], suggest that glucose and glucose 6-phosphate may bind to very nearly the same region of the enzyme. The structure of the binary Glc6P.Mn(II) complex has also been determined. The phosphoryl group of the sugar phosphate forms a first co-ordination complex with the cation. However, on the enzyme, the phosphoryl group is located at a distance of approximately 0.5-0.6 nm from the cation.     

Insoluble complex formation between low density lipoprotein and heparin. A 31P-NMR study

31P-NMR has been used to probe the motions of the phosphate moiety of phospholipid head-groups in samples of human low density lipoprotein (LDL) in which particle tumbling has been greatly reduced by increasing the viscosity of the medium, by forming an LDL gel by ultracentrifugation, or by precipitation with heparin. The 31P-NMR spectra of LDL gel give broad "powder-like" lineshapes, with the sign and magnitude of the anisotropy characteristic of the bilayer mesophase, which narrow as the temperature is raised from 5 to 45 degrees C. This narrowing occurs over the same temperature range as the core cholesteryl ester liquid-crystalline to liquid phase transition, suggesting interactions between the surface and core. The 31P lineshapes of LDL-heparin insoluble complexes are also "powder-like", but are broader than native LDL at all temperatures studied. The spectra were simulated assuming an axially-symmetric shielding tensor motionally narrowed by Brownian isotropic diffusion [Burnell et al. (1980) Biochim. Biophys. Acta 603, 63-69], allowing determination of the lateral diffusion coefficients, DT, and the chemical shift anisotropy, delta sigma, of the monolayer phospholipids. Relative to LDL gel, the temperature-dependence of DT was reduced in the LDL-heparin insoluble complexes, and delta sigma was increased from 50 to 60 ppm. The results suggest that insoluble complex formation slows phospholipid lateral diffusion in the LDL monolayer and alters the orientation and/or order of the head-group.     

Regulation of glucose metabolism in oral streptococci through independent pathways of glucose 6-phosphate and glucose 1-phosphate formation

In vivo rates of glucose uptake and acid production by oral streptococci grown in glucose- or nitrogen-limited continuous culture and batch culture were compared with the glucose phosphorylation activities of harvested, decryptified cells. The strains examined contained significant phosphoenolpyruvate-phosphotransferase system (PTS) activity, measured by a glucose 6-phosphate (G6P) dehydrogenase-linked assay procedure, but this activity was insufficient to account for the in vivo glucose uptake rates. However, ATP was a superior phosphoryl donor to phosphoenolpyruvate, and unlike the PTS, phosphoryl transfer with ATP was insensitive to bacteriostatic concentrations of chlorhexidine, suggesting glucokinase-mediated G6P formation. Again, G6P formation from the PTS and glucokinase reactions was not commensurate with some of the glucose uptake rates observed, implying that other phosphorylation reactions must be occurring. Two novel reactions involving carbamyl phosphate and acetyl phosphate were identified in some of the strains. No G6P formation was detected with these potential phosphoryl donors, but in the presence of phosphoglucomutase, glucose 1-phosphate (G1P) formation was evident, which was insensitive to chlorhexidine. G1P is a precursor of glycogen, and good correlation was obtained between G1P formation activity and endogenous metabolism of washed cells measured either as a rate of acid production at a constant pH 7 or as a decrease in pH with time in the absence of titrant. A "league table" of abilities to synthesize G1P and produce acid from endogenous metabolism was compiled for oral streptococci grown in batch culture. This indicated that Streptococcus mutans Ingbritt and Streptococcus sanguis Challis were unable to form G1P or produce much acid endogenously, whereas increasing activities were obtained with Streptococcus salivarius, Streptococcus sanguis, and Streptococcus mitis. In particular, S. mitis had the highest G1P formation activities and was able to decrease the pH to less than 5 in 15 min by endogenous metabolism alone. The data are consistent with the intracellular accumulation of free glucose driven by proton motive force when PTS activities are low and the subsequent phosphorylation to either G6P for metabolism via glycolysis or G1P for glycogen biosynthesis. The accumulation of acetyl phosphate during glucose-limited growth and the availability of arginine for catabolism to carbamyl phosphate provide an explanation as to why some glucose-limited oral streptococci continue to synthesize glycogen under these conditions, which might prevail in plaque.     

Effect of glycophorin on lipid polymorphism: A 31P-NMR study

(1) The effect of glycophorin, a major intrinsic glycoprotein of the human erythrocyte membrane, on lipid polymorphism has been investigated by ³¹P-NMR (at 36.4 MHz) and by freeze-fracture electron microscopy. (2) Incorporation of glycophorin into vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) results in the formation of unilamellar vesicles (1000–5000 Å diameter) which exhibit ³¹P-NMR bilayer spectra over a wide range of temperature. A reduction in the chemical shift anisotropy (Δσ_csa^eff) and an increase in spectral linewidth in comparison to dioleoylphosphatidylcholine liposomes may suggest a decrease in phospholipid headgroup order. (3) 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), in the presence of excess water, undergoes a bilayer to hexagonal (H_II) phospholipid arrangement as the temperature is increased above 0°C. Incorporation of glycophorin into this system stabilizes the bilayer configuration, prohibiting the formation of the H_II phase. (4) Cosonication of glycophorin with DOPE in aqueous solution (pH 7.4) produces small, stable unilamellar vesicles (300–1000 Å diameter), unlike DOPE alone which is unstable and precipitates from solution. (5) The current study demonstrates the bilayer stabilizing capacity of an intrinsic membrane protein, glycophorin, most likely by means of a strong hydrophobic interaction between the membrane spanning portion of glycophorin and the hydrophobic region of the phospholipid.     

Accumulation of 2-deoxy-D-glucose-6-phosphate as a measure of glucose uptake in the isolated perfused heart: a 31P NMR study

The accumulation of 2-deoxy-D-glucose-6-phosphate (2DG6P), detected using 31P NMR spectroscopy, has been used as a measure of the rate of glucose uptake, yet the accuracy of this measurement has not been verified. In this study, isolated rat hearts were perfused with different substrates or isoproterenol for 30 min before measurement of either 2DG6P accumulation or [2-3H]glucose uptake, without and with insulin. Basal contractile function and metabolite concentrations were the same for all hearts. The basal rates of 2DG6P accumulation differed significantly, depending on the preceding perfusion protocol, and were 38-60% of the [2-3H]glucose uptake rates, whereas insulin-stimulated 2DG6P accumulation was the same or 71% higher than the [2-3H]glucose uptake rates. Therefore the ratio of 2DG6P accumulation/[2-3H]glucose uptake rates varied from 0.38 to 1.71, depending on the prior perfusion conditions or the presence of insulin. The rates of 2DG6P hydrolysis were found to be proportional to the intracellular 2DG6P concentrations, with a K(m) of 17.5mM and V(max) of 1.4 micromol/g dry weight/min. We conclude that the rates of 2DG6P accumulation do not accurately reflect glucose uptake rates under all physiological conditions in the isolated heart and should be used with caution.     

31P-NMR study of pig intestinal brush-border membrane structure: effect of zinc and cadmium ions

³¹P-NMR experiments on intact pig small intestine brush-border membrane vesicles (BBMV) and detergent-solubilized membranes gave direct insights into the organization of the phospholipids (PL) and their interaction with zinc and cadmium ions. Various endogenous PL were identified from well resolved BBM micelle spectra. These experiments revealed a strong interaction of Zn²⁺ and Cd²⁺ with the negatively charged phosphatidylinositol and phosphatidylserine. In BBM micelles, a progressive time-dependent PL degradation occurred in the absence of ions and indicated the presence of active phospholipases. The presence of zinc inhibited the degradation process whereas cadmium had the opposite influence. ³¹P spectra of BBMV were carefully characterized. Neither zinc nor cadmium affected the PL bilayer structural organization. A degradation of PL, monitored by the increase of the inorganic phosphate (P _i) signal, also occurred in vesicles but to a lesser extent than in micelles. A 2/3 internal, 1/3 external PL asymmetry was observed in the absence and presence of ions. Offprint requests to: P. Ripoche     

A 31P-NMR study of bovine adrenocortical mitochondrial metabolic activities

High-field 31P-NMR spectroscopy has been used to study the metabolic activities of coupled bovine adrenocortical mitochondria in vitro. These differentiated organelles use oxygen as a substrate to support both oxidative phosphorylation and specific steroid hydroxylation reactions. The NMR technique allowed the resolution of two inorganic phosphate signals, attributed to the matrix and external medium phosphate pools, at low and high field, respectively. These signals were used to calculate the respective Pi concentrations and to obtain the pH of the two corresponding compartments. In addition, the NMR spectra displayed resonance signals corresponding to ADP added to the medium and to ATP synthesized during oxidative phosphorylation. NMR analysis of the mitochondrial perchloric acid extracts identified the major phosphate-containing metabolites, namely NADP+, NAD+, phosphocholine, phosphoethanolamine, sn-glycero-(3)phosphocholine, AMP, ADP, ATP and Pi. Upon addition of ADP and malate to the oxygenated suspension, the kinetics of mitochondrial external Pi consumption and of ATP synthesis, along with the intra- and extraorganelle pH variations could be monitored over time periods of approximately 30 min, in the absence and presence of different steroid hydroxylation substrates. A major observation was that oxidative phosphorylation, which takes place in the absence of steroid, was markedly inhibited as soon as steroid hydroxylation was operating. These observations show the potential of 31P-NMR spectroscopy in the study of metabolic activities of isolated intact mitochondrial organelles. Such an approach appears promising for further determination of the underlying mechanisms in the balance between vital oxidative phosphorylation and differentiated steroid hydroxylation which are under hormonal control in adrenocortical mitochondria as well as in other steroidogenic cell systems.     

The formation of cyclic inositol 1,2-monophosphate, inositol 1-phosphate, and glucose 6-phosphate by brain preparations stimulated with deoxycholate and calcium: a gas chromatographic study

A gas chromatographic method has been developed for the separation and isolation of water-soluble phosphates as trimethylsilyl ethers. With this method cyclic inositol 1,2-monophosphate and inositol 1-phosphate, derived from endogenous phosphatidylinositol, have been shown to increase when a particulate portion of brain homogenate is stimulated with deoxycholate and Ca⁺⁺, confirming earlier observations of Lapetina and Michell (1). Concomitant with the appearance of inositol phosphates is the stimulated formation of glucose 6-phosphate in the whole homogenate. Although ATP can replace deoxycholate and Ca⁺⁺ in a dialyzed homogenate, glucose 6-phosphate apparently does not arise by any known metabolic pathway but from another unidentified source.     

Dehydrogenation of the phosphonate analogue of glucose 6-phosphate by glucose 6-phosphate dehydrogenase.

6,7 -Dideoxy-alpha-D-gluco-heptose 7-phosphonic acid, the isosteric phosphonate analogue of glucose 6-phosphate, was synthesized in six steps from the readily available precursor benzyl 4,6-O-benzylidene-alpha-D-glucopyranoside. The analogue is a substrate for yeast glucose 6-phosphate dehydrogenase, showing Michaelis-Menten kinetics at pH7.5 and 8.0. At both pH values the Km values of the analogue are 4-5 fold higher and the values approx. 50% lower than those of the natural substrate. The product of enzymic dehydrogenation of the phosphonate analogue at pH8.5 is itself a substrate for gluconate 6-phosphate dehydrogenase.     

A 31P-NMR study on multilamellar liposomes formed from the lipids of a thermophilic bacterium

The membrane lipids of a thermophilic bacterium, Thermus SPS11, isolated from thermal springs in S?o Pedro do Sul, Portugal, were fractionated by chromatography on silica gel. The total lipid extract was found to contain one major phospholipid (PL), which accounts for about 90% of the total lipid phosphorous, and one major glycolipid (GL), which accounts for about 95% of the total carbohydrate in the non-phospholipid fraction. The membranes also contain about 11% by weight of a complex mixture of carotenoids (CA). Multilamellar liposomes, in excess water, formed from PL and mixtures of PL with GL and CA in proportions found in the natural membrane were investigated by proton-decoupled 31P-nuclear magnetic resonance (NMR) spectroscopy and X-ray diffraction. All mixtures examined were found to be in a lamellar phase with disordered hydrophobic chains with no evidence for "non-bilayer structures" between 23 degrees and 85 degrees C. Compared to bilayers formed from pure PL or mixtures of PL and CA, significantly larger values for the chemical shift anisotropy of the 31P-NMR powder patterns were obtained from bilayers formed from mixtures of PL and GL, at temperatures above 75 degrees C, and mixtures of PL, GL and CA at all temperatures examined. These differences are interpreted in terms of changes in the order of the bilayer and/or changes in the orientation of the phosphate moiety of PL. The significance of these results to the thermophily of the bacterium is discussed.     

31P-NMR studies of glucose and glutamine metabolism in cultured mammalian cells

31P-NMR measurements of the concentrations of phosphorus-containing metabolites in mammalian cells immobilised and perifused with glucose and glutamine as sole carbon source have shown that the intracellular Pi concentration is significantly higher in cells perifused with glutamine than with glucose. The data are consistent with the proposal that the rate of glutamine utilisation may be controlled by the activity of phosphate-activated glutaminase.     

Polyphosphoinositides undergo charge neutralization in the physiological pH range: a 31P-NMR study

The charge state of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was determined as a function of pH by way of 31P-NMR spectroscopy. The pK values for the first protonation of the phosphomonoester residues in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were found to be 6.2 and 6.6, respectively, for the 4-phosphate moiety, and 7.7 for the 5-phosphate moiety.     

Generation of phosphodiesters during fast-to-slow muscle transformation A 31P-NMR study

During rabbit fast-to-slow twitch muscle transformation, in response to electrical stimulation, the compound glycerophosphocholine can be detected in these muscles by ³¹P-NMR. This compound is not detectable in contralateral control muscles but is present in slow twitch soleus.     

Phosphocholine and phosphoethanolamine during chick embryo myogenesis: a (31)P-NMR study

Elevated contents of phosphoethanolamine (Etn-P) and/or phosphocholine (Cho-P), a common feature of most tumours with respect to normal counterparts, may also occur in non-cancerous proliferating tissues. The significance of these alterations in relation to cell proliferation, differentiation and maturation is scarcely understood. In this work, the Cho-P and Etn-P pools were measured by (31)P-NMR in extracts of chick embryo pectoral muscle at different days of development. The average concentration of these metabolites exhibited the highest values (respectively, 1.5 and 3.0 micromol/mg DNA) on days 9-11 and decreased at later stages of myogenesis. While, however, Cho-P maintained substantial levels (above 1.0 micromol/mg DNA) also during myotube formation (days 11-18) and stepwise decreased (to about 0.5 micromol/mg DNA) upon fibres' maturation, Etn-P gradually decreased between day 11 and hatching time (down to about 0.2 micromol/mg DNA). These results demonstrate that significant changes may occur in the steady-state pools of these metabolites during normal in vivo cellular development and differentiation, and are consistent with: (a) high rates of phospholipid biosynthesis reported in the literature for proliferating myoblasts; (b) sustained phosphatidylcholine synthesis maintained also during myoblast fusion; and (c) decreased requirement of phospholipid synthesis in the last phase of in ovo myofibre maturation.     

Muscle fatigue in McArdle's disease studied by 31P-NMR: effect of glucose infusion

In muscle phosphorylase deficiency (McArdle's disease) there is an abnormally rapid fatigue during strenuous exercise. Increasing substrate availability to working muscle can improve exercise tolerance but the effect on muscle energy metabolism has not been studied. Using phosphorus-31 nuclear magnetic resonance (31P-NMR) we examined forearm muscle ATP, phosphocreatine (PCr), inorganic phosphate (Pi) and pH in a McArdle patient (MP) and two healthy subjects (HS) at rest and during intermittent maximal effort handgrip contractions under control conditions (CC) and during intravenous glucose infusion (GI). Under CC, MP gripped to impending forearm muscle contracture in 130 s with a marked decline in muscle PCr and a dramatic elevation in Pi. During GI, MP exercised easily for greater than 420 s at higher tensions and with attenuated PCr depletion and Pi accumulation. In HS, muscle PCr and Pi changed more modestly and were not affected by GI. In MP and HS, ATP changed little or not at all with exercise. The results suggest that alterations in the levels of muscle PCr and Pi but not ATP are involved in the muscle fatigue in McArdle's disease and the improved exercise performance during glucose infusion.     

Subunit structure of human glucose 6-phosphate dehydrogenase and its genetic implication

Electrophoretic patterns of the subunits from the normal (B) and from the common A type variant of human glucose 6-phosphate dehydrogenase, following dissociation in the presence of 8m urea, were examined. A single protein band was found in both enzymes, indicating that the B and the A enzyme each consists of different subunits and do not have common subunits in their molecules. Since the A enzyme is a structural variant resulting from a single amino acid substitution, it can be concluded that the human G6PD consists of several identical subunits and that only one structural gene is involved in producing the enzyme. The findings of only a tyrosine residue as amino-terminal as well as a glycine residue as carboxy-terminal are in agreement with this conclusion.This study is supported by Public Health Service Grant GM 15253.