Conformational changes induced by polyanions in haemoglobin from Camelus dromedarius. Circular-dichroism study on the oxy derivative.

The c.d. spectrum of oxyhaemoglobin from Camelus dromedarius is significantly affected by the presence of inositol hexakisphosphate. Correlation with O2-binding measurements shows that these dichroic changes parallel the functional properties of the protein. The optical modifications suggest that, in contrast with human haemoglobin, the conformational changes induced by inositol hexakisphosphate on dromedary oxyhaemoglobin are mainly attributable to a local change of the tertiary structure reminiscent of that of the deoxy derivative, the quaternary conformation seeming to be almost unaffected. The results provide direct evidence of the existence on the protein of two distinct sites for polyanions.     

Effect of polyanions on the spectroscopic properties of the nitric oxide derivative of ferrous dromedary (Camelus dromedarius) hemoglobin

The effect of inositol hexakisphosphate, 2,3-diphosphoglycerate, dextran sulphate, and heparin on the spectroscopic (absorbance, circular dichroism, EPR) properties of the nitric oxide derivative of ferrous dromedary (Camelus dromedarius) hemoglobin was investigated. The results obtained show that: (i) all polyanions bind to the protein at the same sites, but with different affinities; (ii) polyanions affect the protein conformation of the ferrous nitrosyl derivative in a different way with respect to aquo-ferric and ferrous oxy dromedary hemoglobin; and (iii) the data obtained provide further independent evidence for the existence in dromedary hemoglobin of two functionally distinct polyanion binding sites that affect the conformational equilibrium of the protein in opposite ways.     

Effect of inositol hexakisphosphate on the EPR properties of the nitric oxide derivative of ferrous dromedary (Camelus dromedarius) hemoglobin. Evidence for two polyanion binding sites

The effect of inositol hexakisphosphate on the EPR properties of the nitric oxide derivative of ferrous dromedary (Camelus dromedarius) hemoglobin has been investigated at 110 K. In the absence of inositol hexakisphosphate, the nitrosyl derivative of dromedary hemoglobin shows an EPR spectrum with a rhombic shape and a weak hyperfine splitting in the gz region, a feature that is generally taken as characteristic of the high-affinity state of tetrameric hemoproteins. On addition of 1 mole of inositol hexakisphosphate/tetramer, three new hyperfine lines (Az = 1.7 mT), centered at gz = 2.01, appear; this type of spectrum is indicative of the low-affinity state of hemoglobins. A further addition of inositol hexakisphosphate, corresponding to a 20-fold molar excess, completely reverses the polyphosphate-dependent transition, giving an EPR spectrum that is exactly superimposable to that observed in the absence of the allosteric effector, i.e., is typical of the high-affinity state of the macromolecule. Both in the absence and presence of inositol hexakisphosphate, the EPR spectra are virtually independent of pH in the range explored (from 5.5 to 7.5). These results, taken together with the behavior of the nitric oxide derivative of human hemoglobin, provide further evidence for the existance in dromedary hemoglobin of two polyanion binding sites that affect in an opposite way the conformational equilibrium of the macromolecule.     

Modulatory effects on proteinase kinetics caused by association of both enzyme and substrate to heparin

Heparin is shown to produce modulatory effects on the amidolytic activity of trypsin, thrombin and plasmin with various synthetic peptide substrates. Simple Michaelis-Menten kinetics are observed in the absence of heparin. In its presence an enhancement effect is observed at low substrate concentrations, and an inhibitory effect is observed at high substrate concentrations. Other polyanions like dextran sulphate, phosvitin and inositol hexakisphosphate produces a similar effect. The modulatory effect of heparin is abolished when it binds cations. Co-binding of both substrate and enzyme to heparin seems to be a necessary requirement for the effect to occur. A model is proposed which can account semiquantitatively for the kinetics observed. It is suggested that the mechanism, which involves co-binding of substrate and enzyme in an competitive manner to a macromolecular structure, may be of primary importance as a regulatory mechanism in blood coagulation and fibrinolysis.     

Binding of inositol phosphates to arrestin.

Arrestin binds to phosphorylated rhodopsin in its light-activated form (metarhodopsin II), blocking thereby its interaction with the G-protein, transducin. In this study, we show that highly phosphorylated forms of inositol compete against the arrestin-rhodopsin interaction. Competition curves and direct binding assays with free arrestin consistently yield affinities in the micromolar range; for example, inositol 1,3,4,5-tetrakisphosphate (InP4) and inositol hexakisphosphate (InP6 bind to arrestin with dissociation constants of 12 microM and 5 microM, respectively. Only a small control amount of inositol phosphates is bound, when arrestin interacts with phosphorylated rhodopsin. This argues for a release of bound inositol phosphates by interaction with rhodopsin. Transducin, rhodopsin kinase, or cyclic GMP phosphodiesterase are not affected by inositol phosphates. These observations open a new way to purify arrestin and to inhibit its interaction with rhodopsin. Their physiological significance deserves further investigation.     

Simple synthesis of <Superscript>32</Superscript>P-labelled inositol hexakisphosphates for study of phosphate transformations

Background and aims

In many soils inositol hexakisphosphate in its various forms is as abundant as inorganic phosphate. The organismal and geochemical processes that exchange phosphate between inositol hexakisphosphate and other pools of soil phosphate are poorly defined, as are the organisms and enzymes involved. We rationalized that simple enzymic synthesis of inositol hexakisphosphate labeled with ³²P would greatly enable study of transformation of soil inositol phosphates when combined with robust HPLC separations of different inositol phosphates.

Methods

We employed the enzyme inositol pentakisphosphate 2-kinase, IP5 2-K, to transfer phosphate from [γ-³²P]ATP to axial hydroxyl(s) of myo-, neo- and 1D-chiro-inositol phosphate substrates.

Results

³²P-labeled inositol phosphates were separated by anion exchange HPLC with phosphate eluents. Additional HPLC methods were developed to allow facile separation of myo-, neo-, 1D-chiro- and scyllo-inositol hexakisphosphate on acid gradients.

Conclusions

We developed enzymic approaches that allow the synthesis of labeled myo-inositol 1,[³²P]2,3,4,5,6-hexakisphosphate; neo-inositol 1,[³²P]2,3,4,[³²P]5,6–hexakisphosphate and 1D-chiro-inositol [³²P]1,2,3,4,5,[³²P]6-hexakisphosphate. Additionally, we describe HPLC separations of all inositol hexakisphosphates yet identified in soils, using a collection of soil inositol phosphates described in the seminal historic studies of Cosgrove, Tate and coworkers. Our study will enable others to perform radiotracer experiments to analyze fluxes of phosphate to/from inositol hexakisphosphates in different soils.

     

Effect of polyanions on the kinetics of the reaction of apohemoglobin with carbonmonoxy heme.

The reaction of apohemoglobin with carbonmonoxy heme and with carbonmonoxy heme dimethyl ester was investigated in the presence and absence of inositol hexaphosphate. The binding stoichiometry of both heme derivatives to apohemoglobin was not affected by the presence of the polyphosphate, while, in both cases, the overall rate of recombination was substantially decreased. The absence of the negatively charged carboxyl groups in the dimethyl ester derivative of the heme indicated that the effect of inositol hexaphosphate on the reaction of apohemoglobin with heme was not due to electrostatic repulsions and resulted from conformational changes occurring upon the interaction of apohemoglobin with inositol hexaphosphate. Qualitative treatment of the kinetic data suggests that these conformational changes destabilize the intermediates of the reaction by increasing their redissociation into the original components. Also, benzenehexacarboxylate produced conformational changes in apohemoglobin and decreased its rate of reaction with carbonmonoxy heme, proving the aspecificity of the interaction of apohemoglobin with polyanions.     

Polyanionic inhibitors of phosphoglycerate mutase: combined structural and biochemical analysis.

The effects that the inhibitors inositol hexakisphosphate and benzene tri-, tetra- and hexacarboxylates have on the phosphoglycerate mutases from Saccharomyces cerevisiae and Schizosaccharomyces pombe have been determined. Their Kivalues have been calculated, and the ability of the inhibitors to protect the enzymes against limited proteolysis investigated. These biochemical data have been placed in a structural context by the solution of the crystal structures of S. cerevisiae phosphoglycerate mutase soaked with inositol hexakisphosphate or benzene hexacarboxylate. These large polyanionic compounds bind to the enzyme so as to block the entrance to the active-site cleft. They form multiple interactions with the enzyme, consistent with their low Kivalues, and afford good protection against limited proteolysis of the C-terminal region by thermolysin. The inositol compound is more efficacious because of its greater number of negative charges. The S. pombe phosphoglycerate mutase that is inherently lacking a comparable C-terminal region has higher Kivalues for the compounds tested. Moreover, the S. pombe enzyme is less sensititive to proteolysis, and the presence or absence of the inhibitor molecules has little effect on susceptibility to proteolysis.     

Structural analysis of the asparagine-linked oligosaccharides of human complement component C3.

The role of chloride ions in modulating polyanion-induced conformational changes in haemoglobin from the dromedary (Camelus dromedarius) has been investigated. The results obtained have shown that: in the ferric derivative at pH 6.5 the effect of single polyanion (dextran sulphate and inositol hexakisphosphate) on the conformation is essentially local, thus involving only the tertiary structure of the protein; the presence of chloride ions at a concentration close to the physiological value (i.e. 150 mM) is essential to induce quaternary conformational changes in the polyanion-ferric protein system; comparison between structural and functional data correlates polyanion-induced tertiary conformational changes with changes in the value of midpoint potential, E'0, and quaternary changes with co-operativity.     

Levels of inositol metabolites within normal myeloid blast cells and changes during their differentiation towards monocytes.

A homogeneous population of undifferentiated myeloid blast cells was purified from human fetal liver by rosette sedimentation of erythroblasts and macrophages, after coating these cells with monoclonal antibodies, followed by a cell elutriation step. The undifferentiated blast cells were maintained in culture, in a serum-free medium containing 1 mg l-1 inositol, by the presence of a high concentration of interleukin-3 (100 U ml-1). This allowed equilibrium labelling of cells with [2-3H]myo-inositol and analysis of the concentrations of inositol metabolites. The myeloid blast cells contained high concentrations of an unidentified inositol metabolite, possibly sn-glycero-3-phospho-1-inositol (GroPIns, 22 microM), inositol monophosphate (InsP, 16 microM), an unidentified inositol bisphosphate (InsP2, 9.4 microM), inositol pentakisphosphate (InsP5, 37 microM) and inositol hexakisphosphate (InsP6, 31 microM). These high concentrations are similar to those reported in the promyeloid cell line, HL60. Treatment of the blast cells with 10 nM phorbol myristate acetate (PMA) resulted in rapid differentiation of 48% of the cells towards monocytes. Notable changes in the levels of inositol metabolites included an increase in the putative GroPIns peak (to 73 microM) and decreases in the concentrations of InsP4 (from 4 microM to 1 microM) and InsP5 (to 21 microM). These changes in response to PMA, with the exception of the rise in the putative GroPIns, are similar to those reported in HL60 cells undergoing monocyte differentiation. These observations suggest that the abundant inositol polyphosphates may have an as yet unknown role in myeloid differentiation.     

Activation of the calcium release channel (ryanodine receptor) by heparin and other polyanions is calcium dependent.

Heparin has been used as a potent competitive inhibitor of inositol 1,4,5-trisphosphate (IP3)-binding to IP3 receptors and to block IP3-gated calcium channels in bilayer experiments. In contrast to the effect on the IP3-gated channel, heparin (0.1-1 micrograms/ml) opened the Ca release channel (ryanodine receptor). Other polyanions such as pentosan polysulfate and polyvinyl sulfate also activated the Ca release channel. The effect of polyanions on the Ca release channel was Ca dependent. Polyanion addition activated the Ca release channel when free Ca was > 80 nM, but was ineffective when free Ca was < 20 nM. The level of channel activation could be altered by manipulating the free Ca concentration. These results suggest that the polyanions act by increasing the local concentration of Ca near regulatory sites on the channel complex. As most cells have both types of intracellular channels, the opposite effects of the polyanions on the two channel types suggests that addition of polyanions to intact cells may produce multiple effects.     

Mass changes in inositol tetrakis- and pentakisphosphate isomers induced by chemotactic peptide stimulation in HL-60 cells

Absolute concentrations of inositol phosphate isomers (InsP(s] were quantified in the myeloid cell line HL-60 using the metal-dye detection technique. Stimulation with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) led to distinct alterations in at least seven different inositol phosphate species. Whereas the intracellular concentrations of the tetrakisphosphate isomers (InsP4(s] were found below the micromolar range, inositol 1,3,4,5,6-pentakis- and hexakisphosphate levels were about two orders of magnitude higher (36 and 54 +/- 2 microM (mean +/- S.D.), respectively). The three InsP4(s) showed distinct kinetic pattern upon receptor activation, the transient elevation of inositol 1,3,4,5-tetrakisphosphate being faster both in onset and in redecrease than inositol 1,3,4,6-tetrakisphosphate. Whereas the two latter isomers reached maximally 2.75 and 2.9 +/- 0.2 microM, respectively, 1 min after stimulation, inositol 3,4,5,6-tetrakisphosphate remained elevated (3.5 +/- 0.4 microM) up to 5 min after fMLP. Unexpected changes in highly phosphorylated InsP(s) were observed, notably a rise in inositol 1,3,4,5,6-pentakisphosphate and in inositol hexakisphosphate to 52 +/- 3 and 60 +/- 1 microM, respectively. In terms of mass, the increases in highly phosphorylated inositols are by far highest among all InsP(s). Combining radiotracer method with mass determination it was observed that the specific radioactivity of various InsP(s) was different and changed markedly upon fMLP stimulation, in spite of a prolonged labeling period leading to apparent isotopic steady state. The data presented demonstrate agonist-induced elevations of highly phosphorylated InsP(s) and suggest that inositol 1,4,5-trisphosphate, product of receptor-activated phospholipase C, is metabolized rather via phosphorylation than only by dephosphorylation pathways.     

Stereo- and regiospecificity of yeast phytases-chemical synthesis and enzymatic conversion of the substrate analogues neo- and L-chiro-inositol hexakisphosphate

Phytases are enzymes that catalyze the hydrolysis of phosphate esters in myo-inositol hexakisphosphate (phytic acid). The precise routes of enzymatic dephosphorylation by phytases of the yeast strains Saccharomyces cerevisiae and Pichia rhodanensis have been investigated up to the myo-inositol trisphosphate level, including the absolute configuration of the intermediates. Stereoselective assignment of the myo-inositol pentakisphosphates (D-myo-inositol 1,2,4,5,6-pentakisphosphate and D-myo-inositol 1,2,3,4,5-pentakisphosphate) generated was accomplished by a new method based on enantiospecific enzymatic conversion and HPLC analysis. Via conduritol B or E derivatives the total syntheses of two epimers of myo-inositol hexakisphosphate, neo-inositol hexakisphosphate and L-chiro-inositol hexakisphosphate were performed to examine the specificity of the yeast phytases with these substrate analogues. A comparison of kinetic data and the degradation pathways determined gave the first hints about the molecular recognition of inositol hexakisphosphates by the enzymes. Exploitation of the high stereo- and regiospecificity observed in the dephosphorylation of neo- and L-chiro-inositol hexakisphosphate made it possible to establish enzyme-assisted steps for the synthesis of D-neo-inositol 1,2,5,6-tetrakisphosphate, L-chiro-inositol 1,2,3,5,6-pentakisphosphate and L-chiro-inositol 1,2,3,6-tetrakisphosphate.     

Effect of polyanions on the refolding of human acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) is unstable at physiological temperatures in the absence of polyanions such as heparin. Therefore, the effect of temperature on the kinetics of refolding of aFGF has been examined in the presence and absence of several polyanions. The protein folds into its native state at temperatures up to 30 degrees C without polyanions with an activation energy of approximately 14 kcal/mol, but does not acquire native structure above this temperature. When heparin, inositol hexasulfate, or sulfate ion are present, aFGF refolds below 30 degrees C with a slightly reduced activation energy (10-11 kcal/mol). In addition, the protein now also renatures between 30 and 50 degrees C with activation energies of 1-2 (heparin), 16 (inositol hexasulfate), and 7 (sulfate) kcal/mol. Trace heavy metals appear to inhibit the refolding process, but a molecular chaperone (bovine 70-kDa heat shock cognate protein) and a peptidylprolyl isomerase (the FK506-binding protein) have no effect. It is concluded that the rate of refolding of aFGF at physiological temperatures is probably controlled by the interaction of a native-like state of the protein with an unknown polyanionic species.     

Purification of an inositol (1,3,4,5)-tetrakisphosphate 3-phosphatase activity from rat liver and the evaluation of its substrate specificity.

Hepatic inositol (1,3,4,5)-tetrakisphosphate 3-phosphatase activity was detected in a 100,000 x g soluble fraction and a detergent-solubilized particulate fraction. Activity in both fractions increased up to 40-fold after anion-exchange chromatography due to removal of endogenous inhibitors (Hodgson, M.E., and Shears, S.B. (1990) Biochem. J. 267, 831-834); at this stage the detergent-solubilized particulate activity comprised over 90% of total activity. The particulate phosphatase was further purified by affinity chromatography using heparin-agarose and red-agarose. The latter column resolved two peaks of enzyme activity (designated 1 and 2 by their order of elution from the column). Their proportions varied between experiments, but peak 2 generally predominated and so this was further purified by hydroxylapatite chromatography. The final preparation was typically 38,000-fold purified with a 7% yield. The apparent molecular mass of this enzyme was 66 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The enzyme had little or no affinity for the following: inositol (1,3,4,6)-tetrakisphosphate, inositol (1,3,4)-trisphosphate, inositol (1,3)-bisphosphate, inositol (3,4)-bisphosphate, and para-nitrophenylphosphate. At pH 7.4 the Km for inositol (1,3,4,5)-tetrakisphosphate was 130 nM and the Vmax was 4250 nmol/mg protein/min. The purified enzyme also dephosphorylated inositol (1,3,4,5,6)-pentakisphosphate to inositol (1,4,5,6)-tetrakisphosphate (Km = 40 nM, Vmax = 211 nmol/mg protein/min), and inositol hexakisphosphate to at least five isomers of inositol pentakisphosphate (Km = 0.3 nM, Vmax = 12 nmol/mg protein/min). The latter affinity is the highest yet defined for an enzyme involved in inositol phosphate metabolism. Determinations of IC50 values, and Dixon plots, revealed that with the (1,3,4,5)-tetrakisphosphate as substrate, the pentakis- and hexakisphosphates were potent competitive inhibitors; the Ki values (25 and 0.5 nM, respectively) were similar to their substrate Km values. The kinetic properties of this enzyme, as well as estimates of the cellular levels of its potential substrates, indicate that inositol pentakisphosphate and inositol hexakisphosphate are likely to be the preferred substrates in vivo.     

Inositol polyphosphates regulate zebrafish left-right asymmetry

Vertebrate body plans have a conserved left-right (LR) asymmetry manifested in the position and anatomy of the heart, visceral organs, and brain. Recent studies have suggested that LR asymmetry is established by asymmetric Ca2+ signaling resulting from cilia-driven flow of extracellular fluid across the node. We report here that inositol 1,3,4,5,6-pentakisphosphate 2-kinase (Ipk1), which generates inositol hexakisphosphate, is critical for normal LR axis determination in zebrafish. Zebrafish embryos express ipk1 symmetrically during gastrulation and early segmentation. ipk1 knockdown by antisense morpholino oligonucleotide injection randomized LR-specific gene expression and organ placement, effects that were associated with reduced intracellular Ca2+ flux in cells surrounding the ciliated Kupffer's vesicle, a structure analogous to the mouse node. Our data suggest that the pathway for inositol hexakisphosphate production is a key regulator of asymmetric Ca(2+) flux during LR specification.     

The function of inositol high polyphosphate binding proteins

The inositol phosphate metabolism network has been found to be much more complex than previously thought, as more and more inositol phosphates and their metabolizing enzymes have been discovered. Some of the inositol phosphates have been shown to have biological activities, but little is known about their signal transduction mechanisms except for that of inositol 1,4,5-trisphosphate. The recent discovery, however, of a number of binding proteins for inositol high polyphosphate [inositol 1,3,4,5-tetrakisphosphate (IP₄), inositol 1,3,4,5,6-pentakisphosphate, or inositol hexakisphosphate] enables us to speculate on the physiological function of these compounds. In this article we focus on two major issues: (1) the roles of inositol high polyphosphates in vesicular trafficking, especially exocytosis, and (2) pleckstrin homology domaincontaining IP4 binding proteins involved in the Ras signaling pathway.     

Inositol hexakisphosphate increases L-type Ca2+ channel activity by stimulation of adenylyl cyclase.

Inositol hexakisphosphate (InsP6) is a most abundant inositol polyphosphate that changes simultaneously with inositol 1,4,5-trisphosphate in depolarized neurons. However, the role of InsP6 in neuronal signaling is unknown. Mass assay reveals that the basal levels of InsP6 in several brain regions tested are similar. InsP6 mass is significantly elevated in activated brain neurons and lowered by inhibition of neuronal activity. Furthermore, the hippocampus is most sensitive to electrical challenge with regard to percentage accumulation of InsP6. In hippocampal neurons, InsP6 stimulates adenylyl cyclase (AC) without influencing cAMP phosphodiesterases, resulting in activation of protein kinase A (PKA) and thereby selective enhancement of voltage-gated L-type Ca2+ channel activity. This enhancement was abolished by preincubation with PKA and AC inhibitors. These data suggest that InsP6 increases L-type Ca2+ channel activity by facilitating phosphorylation of PKA phosphorylation sites. Thus, in hippocampal neurons, InsP6 serves as an important signal in modulation of voltage-gated L-type Ca2+ channel activity.     

Effect of inositol hexakisphosphate on the spectroscopic properties of the nitric oxide derivative of ferrous naturally glycated human hemoglobin HbA1c

The effect of inositol hexakisphosphate (IHP) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous naturally glycated human hemoglobin HbA1c (HbA1cNO) has been investigated quantitatively. The results obtained show that 1) both in the absence and presence of IHP, the EPR and absorbance spectra of HbA1cNO show the same basic characteristics described for the nitrosyl derivative of ferrous HbA0, the nonglycated major component of human hemoglobin (HbA0NO); and 2) HbA1cNO binds IHP with an apparent dissociation equilibrium constant (upsilon = 1.8 x 10(-2) M), which is at least four orders of magnitude higher than that estimated for the polyphosphate interaction with HbA0NO (less than or equal to 3 x 10(-6) M). These data provide further independent evidence that interaction(s) of polyphosphates at the specific cleft between beta-chains along the dyad-axis is sterically hindered in HbA1c by the presence of the two glucose residues covalently bound to the N-termini of beta-chains, this finding being in agreement with the reduced effect of polyanions on HbA1c spectral and ligand-binding properties.     

A novel metal-dye detection system permits picomolar-range h.p.l.c. analysis of inositol polyphosphates from non-radioactively labelled cell or tissue specimens.

A novel complexometric dye- and transition-metal-based post-column detection system for polyanions, called 'metal-dye detection' has been developed. This technique, combined with a new h.p.l.c. separation protocol, permits a direct highly-isomer-selective determination of bis- to poly-phosphorylated non-radioactively labelled compounds in the picomolar range, a sensitivity hitherto unknown for these substances. The application of the technique in the quantitative microanalysis of inositol polyphosphates from milligram amounts of cells or tissue specimens is described. The technique promises to answer hitherto unresolved questions about the role of inositol phosphates, especially those in intact tissues, which are not readily amenable to analysis by radioisotopic techniques.