Structures of diphospho-myo-inositol pentakisphosphate and bisdiphospho-myo-inositol tetrakisphosphate from Dictyostelium resolved by NMR analysis.
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ABSTRACT:
SUBMITTER: Laussmann T
PROVIDER: S-EPMC1217265 | biostudies-other | 1996 May
REPOSITORIES: biostudies-other
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Project description:Two diphospho-myo-inositol phosphates from Dictyostelium were recently investigated by two-dimensional 1H/31P NMR analysis and assigned to be either D-4-diphospho-myo-inositol pentakisphosphate (D-4-PP-InsP5) and D-4,5-bisdiphospho-myo-inositol tetrakisphosphate (D-4,5-bis-PP-InsP4) or their corresponding enantiomers D-6-PP-InsP5 and D-5,6-bis-PP-InsP4. In the present study the naturally occurring enantiomers were identified by using defined synthetic PP-InsP5 isomers as substrates for a partially purified PP-InsP5 5-kinase from Dictyostelium. This enzyme specifically phosphorylates the naturally occurring PP-InsP5 and the synthetic D-6-PP-InsP5, leading to D-5,6-bis-PP-InsP4. In contrast, neither D-4-PP-InsP5 nor D-1-PP-InsP5 or D-3-PP-InsP5 are converted by the enzyme.
| S-EPMC1218154 | biostudies-other
Project description:Avian erythrocytes were incubated with myo-[3H]inositol for 6-7 h and with [32P]Pi for the final 50-90 min of this period. An acid extract was prepared from the prelabelled erythrocytes, and the specific radioactivities of the gamma-phosphate of ATP and of both the myo-inositol moieties (3H, d.p.m./nmol) and the individual phosphate groups (32P, d.p.m./nmol) of [3H]Ins[32P](1,3,4,6)P4,[3H]Ins[32P](1,3,4,5)P4, [3H]Ins[32P](3,4,5,6)P4 and [3H]Ins[32P](1,3,4,5,6)P5 were determined. The results provide direct confirmation that one of the cellular InsP4 isomers is Ins(1,3,4,5)P4 which is synthesized by sequential phosphorylation of the 1,4,5 and 3 substitution sites of the myo-Ins moiety, precisely as previously deduced [Batty, Nahorski & Irvine (1985) Biochem. J. 232, 211-215; Irvine, Letcher, Heslop & Berridge (1986) Nature (London) 320, 631-634]. This is compatible with the proposed synthetic route from PtdIns via PtdIns4P, PtdIns(4,5)P2 and Ins(1,4,5)P3. The data also suggest that, in avian erythrocytes, the principle precursor of Ins(1,3,4,5,6)P5 is Ins(3,4,5,6)P4. Furthermore, if the gamma- (and/or beta-) phosphate of ATP is the precursor of the phosphate moieties of Ins(3,4,5,6)P4, then this isomer must be derived from the phosphorylation of Ins(3,4,6)P3. If the gamma- (and/or beta-) phosphate of ATP similarly acts as the ultimate precursor to all of the phosphates of Ins(1,3,4,6)P4, then, in intact avian erythrocytes, the main precursor of Ins(1,3,4,6)P4 is Ins(1,4,6)P3. This contrasts with the expectation, based on results with cell-free systems, that Ins(1,3,4,6)P4 is synthesized by the direct phosphorylation of Ins(1,3,4)P3.
| S-EPMC1136905 | biostudies-other
Project description:myo-Inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P(5)), an inositol polyphosphate of emerging significance in cellular signalling, and its C-2 epimer scyllo-inositol pentakisphosphate (scyllo-InsP(5)) were synthesised from the same myo-inositol-based precursor. Potentiometric and NMR titrations show that both pentakisphosphates undergo a conformational ring-flip at higher pH, beginning at pH 8 for scyllo-InsP(5) and pH 9 for Ins(1,3,4,5,6)P(5). Over the physiological pH range, however, the conformation of the inositol rings and the microprotonation patterns of the phosphate groups in Ins(1,3,4,5,6)P(5) and scyllo-InsP(5) are similar. Thus, scyllo-InsP(5) should be a useful tool for identifying biologically relevant actions of Ins(1,3,4,5,6)P(5), mediated by specific binding sites, and distinguishing them from nonspecific electrostatic effects. We also demonstrate that, although scyllo-InsP(5) and Ins(1,3,4,5,6)P(5) are both hydrolysed by multiple inositol polyphosphate phosphatase (MINPP), scyllo-InsP(5) is not dephosphorylated by PTEN or phosphorylated by Ins(1,3,4,5,6)P(5) 2-kinases. This finding both reinforces the value of scyllo-InsP(5) as a biological control and shows that the axial 2-OH group of Ins(1,3,4,5,6)P(5) plays a part in substrate recognition by PTEN and the Ins(1,3,4,5,6)P(5) 2-kinases.
| S-EPMC1892220 | biostudies-literature
Project description:BACKGROUND:Pleckstrin homology (PH) domains are one of the most prevalent domains in the human proteome and represent the major phosphoinositide-binding module. These domains are often found in signaling proteins and function predominately by targeting their host proteins to the cell membrane. Inositol phosphates, which are structurally similar to phosphoinositides, are not only known to play a role as signaling molecules but are also capable of being bound by PH domains. RESULTS:In the work presented here it is shown that the addition of commercial myo-inositol hexakisphosphate (IP6) inhibited the binding of the carboxy terminal PH domain of pleckstrin (C-PH) to phosphatidylinositol 3,4-bisphosphate with an IC50 of 7.5 muM. In an attempt to characterize this binding structurally, C-PH was crystallized in the presence of IP6 and the structure was determined to 1.35 A. Examination of the resulting electron density unexpectedly revealed the bound ligand to be D-myo-inositol 1,2,3,5,6-pentakisphosphate. CONCLUSION:The discovery of D-myo-inositol 1,2,3,5,6-pentakisphosphate in the crystal structure suggests that the inhibitory effects observed in the binding studies may be due to this ligand rather than IP6. Analysis of the protein-ligand interaction demonstrated that this myo-inositol pentakisphosphate isomer interacts specifically with protein residues known to be involved in phosphoinositide binding. In addition to this, a structural alignment of other PH domains bound to inositol phosphates containing either four or five phosphate groups revealed that the majority of phosphate groups occupy conserved locations in the binding pockets of PH domains. These findings, taken together with other recently reported studies suggest that myo-inositol pentakisphosphates could act to regulate PH domain-phosphoinositide interactions by directly competing for binding, thus playing an important role as signaling molecules.
| S-EPMC2200656 | biostudies-literature
Project description:Inositol polyphosphates are ubiquitous molecular signals in metazoans, as are their pyrophosphorylated derivatives that bear a so-called 'high-energy' phosphoanhydride bond. A structural rationale is provided for the ability of Arabidopsis inositol tris/tetrakisphosphate kinase 1 to discriminate between symmetric and enantiomeric substrates in the production of diverse symmetric and asymmetric myo-inositol phosphate and diphospho-myo-inositol phosphate (inositol pyrophosphate) products. Simple tools are applied to chromatographic resolution and detection of known and novel diphosphoinositol phosphates without resort to radiolabeling approaches. It is shown that inositol tris/tetrakisphosphate kinase 1 and inositol pentakisphosphate 2-kinase comprise a reversible metabolic cassette converting Ins(3,4,5,6)P4 into 5-InsP7 and back in a nucleotide-dependent manner. Thus, inositol tris/tetrakisphosphate kinase 1 is a nexus of bioenergetics status and inositol polyphosphate/diphosphoinositol phosphate metabolism. As such, it commands a role in plants that evolution has assigned to a different class of enzyme in mammalian cells. The findings and the methods described will enable a full appraisal of the role of diphosphoinositol phosphates in plants and particularly the relative contribution of reversible inositol phosphate hydroxykinase and inositol phosphate phosphokinase activities to plant physiology.
| S-EPMC7115839 | biostudies-literature
Project description:The ubiquitous mammalian signaling molecule bis-diphosphoinositol tetrakisphosphate (1,5-(PP)2 -myo-InsP4 , or InsP8 ) displays the most congested three-dimensional array of phosphate groups found in nature. The high charge density, the accumulation of unstable P-anhydrides and P-esters, the lack of UV absorbance, and low levels of optical rotation constitute severe obstacles to its synthesis, characterization, and purification. Herein, we describe the first procedure for the synthesis of enantiopure 1,5-(PP)2 -myo-InsP4 and 3,5-(PP)2 -myo-InsP4 utilizing a C2 -symmetric P-amidite for desymmetrization and concomitant phosphitylation followed by a one-pot bidirectional P-anhydride-forming reaction that combines sixteen chemical transformations with high efficiency. The configuration of these materials is unambiguously shown by subsequent X-ray analyses of both enantiomers after being individually soaked into crystals of the kinase domain of human diphosphoinositol pentakisphosphate kinase 2.
| S-EPMC4153399 | biostudies-literature
Project description:myo-[3H]Inositol 1,3,4,5,6-pentakisphosphate can be made from myo-[3H]inositol 1,4,5-trisphosphate in a rat brain homogenate or soluble fraction. Although D-myo-inositol 3,4,5,6-tetrakisphosphate can be phosphorylated by a soluble rat brain enzyme to give myo-inositol 1,3,4,5,6-pentakisphosphate, it is not an intermediate in the pathway from myo-inositol 1,4,5-trisphosphate. The intermediates in the above pathway are myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4-trisphosphate and myo-inositol 1,3,4,6-tetrakisphosphate [Shears, Parry, Tang, Irvine, Michell & Kirk (1987) Biochem. J. 246, 139-147; Balla, Guillemette, Baukal & Catt (1987) J. Biol. Chem. 262, 9952-9955], and it is catalysed by soluble kinase activities of similar anion-exchange mobility and Mr value. Compounds with chromatographic and chemical properties consistent with the structures myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4,6-tetrakisphosphate and myo-inositol 3,4,5,6-tetrakisphosphate are present in avian erythrocytes, human 1321 N1 astrocytoma cells and primary-cultured murine bone-marrow-derived macrophages. The amounts of these inositol tetrakisphosphates rise upon muscarinic cholinergic stimulation of the astrocytoma cells or stimulation of macrophages with platelet-activating factor.
| S-EPMC1149364 | biostudies-other
Project description:Homogenates of primary-cultured murine bone macrophages contain an enzyme capable of synthesizing myo-[3H]inositol pentakisphosphate from myo-[3H]inositol tetrakisphosphate fractions derived from myo-[3H]inositol-labelled mouse macrophages and chick erythrocytes. D-myo-inositol 1,3,4,5-tetrakis[32P]-phosphate present in the same incubations was not phosphorylated. Since the myo-[3H]inositol-labelled tetrakisphosphate fractions used as substrates consist of a mixture of L-myo-inositol 1,4,5,6-tetrakisphosphate (60-85%) and a periodate-resistant tetrakisphosphate(s) whose characteristics are consistent with those of D-myo-inositol 1,3,4,5-tetrakisphosphate (the preceding paper [Stephens, Hawkins, Carter, Chahwala, Morris, Whetton & Downes (1988) Biochem. J. 249, 271-282] ), these data suggest the existence of a kinase that phosphorylates L-myo-inositol 1,4,5,6-tetrakisphosphate to give a myo-inositol pentakisphosphate. A similar activity was identified in homogenates of rat cerebrum, liver, heart and parotid gland. D-myo-Inositol 1,3,4,5-tetrakis[32P]phosphate in the same incubations was not a substrate. The activity was almost entirely soluble in all the tissues investigated and was found at its greatest specific activity in brain cytosol. The activity was purified 120-fold from a rat brain homogenate by (NH4)2SO4 fractionation and anion-exchange chromatography. The activity was clearly distinct from D-myo-inositol 1,4,5-trisphosphate (3-hydroxy)kinase. Incubation of this partially purified preparation with L-myo-[3H]inositol 1,4,5,6-tetrakisphosphate from chick erythrocytes and [gamma-32P]ATP resulted in the formation of L-myo-[3H]-inositol [1-32P]1,3,4,5,6-pentakisphosphate. The enzyme is therefore identified as an L-myo-inositol 1,4,5,6-tetrakisphosphate (3-hydroxy)kinase.
| S-EPMC1148695 | biostudies-other
Project description:1. We have studied the metabolism of Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) by rat liver homogenates incubated in a medium resembling intracellular ionic strength and pH. 2. Ins(1,3,4,5)P4 was dephosphorylated to a single inositol trisphosphate product, Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate), the identity of which was confirmed by periodate degradation, followed by reduction and dephosphorylation to yield altritol. 3. The major InsP2 (inositol bisphosphate) product was inositol 3,4-bisphosphate [Shears, Storey, Morris, Cubitt, Parry, Michell & Kirk (1987) Biochem. J. 242, 393-402]. Small quantities of a second InsP2 product was also detected in some experiments, but its isomeric configuration was not identified. 4. The Ins(1,3,4,5)P4 5-phosphatase activity was primarily associated with plasma membranes. 5. ATP (5 mM) decreased the membrane-associated Ins(1,4,5)P3 5-phosphatase and Ins(1,3,4,5)P4 5-phosphatase activities by 40-50%. This inhibition was imitated by AMP, adenosine 5'-[beta gamma-imido]triphosphate, adenosine 5'-[gamma-thio]triphosphate or PPi, but not by adenosine or Pi. A decrease in [ATP] from 7 to 3 mM halved the inhibition of Ins(1,3,4,5)P4 5-phosphatase activity, but the extent of inhibition was not further decreased unless [ATP] less than 0.1 mM. 6. Ins(1,3,4,5)P4 5-phosphatase was insensitive to 50 mM-Li+, but was inhibited by 5 mM-2,3-bisphosphoglycerate. 7. The Ins(1,3,4,5)P4 5-phosphatase activity was unchanged by cyclic AMP, GTP, guanosine 5'-[beta gamma-imido]triphosphate or guanosine 5'-[gamma-thio]triphosphate, or by increasing [Ca2+] from 0.1 to 1 microM. 8. Ins(1,3,4)P3 was phosphorylated in an ATP-dependent manner to an isomer of InsP4 that was partially separable on h.p.l.c. from Ins(1,3,4,5)P4. The novel InsP4 appears to be Ins(1,3,4,6)P4. Its metabolic fate and function are not known.
| S-EPMC1148250 | biostudies-other
Project description:Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are an important group of metabolites and mediate a wide range of processes in eukaryotic cells. To elucidate the functions of these molecules, robust techniques for the characterization of inositol phosphate metabolism are required, both at the biochemical and the cellular level. Here, a new tool-set is reported, which employs uniformly 13C-labeled compounds ([13C6]myo-inositol, [13C6]InsP5, [13C6]InsP6, and [13C6]5PP-InsP5), in combination with commonly accessible NMR technology. This approach permitted the detection and quantification of InsPs and PP-InsPs within complex mixtures and at physiological concentrations. Specifically, the enzymatic activity of IP6K1 could be monitored in vitro in real time. Metabolic labeling of mammalian cells with [13C6]myo-inositol enabled the analysis of cellular pools of InsPs and PP-InsPs, and uncovered high concentrations of 5PP-InsP5 in HCT116 cells, especially in response to genetic and pharmacological perturbation. The reported method greatly facilitates the analysis of this otherwise spectroscopically silent group of molecules, and holds great promise to comprehensively analyze inositol-based signaling molecules under normal and pathological conditions.
| S-EPMC6540952 | biostudies-literature