Inositol Tetraphosphate Research Articles

Evolution of E. coli Phytase Toward Improved Hydrolysis of Inositol Tetraphosphate

Protein engineering campaigns are driven by the demand for superior enzyme performance under non-natural process conditions, such as elevated temperature or non-neutral pH, to achieve utmost efficiency and conserve limited resources. Phytases are industrial relevant feed enzymes that contribute to the overall phosphorus (P) management by catalyzing the stepwise phosphate hydrolysis from phytate, which is the main phosphorus storage in plants. Phosphorus is referred to as a critical disappearing nutrient, emphasizing the urgent need to implement strategies for a sustainable circular use and recovery of P from renewable resources. Engineered phytases already contribute today to an efficient phosphorus mobilization in the feeding industry and might pave the way to a circular P-bioeconomy. To date, a bottleneck in its application is the drastically reduced hydrolysis on lower phosphorylated reaction intermediates (lower inositol phosphates, ≤InsP4) and their subsequent accumulation. Here, we report the first KnowVolution campaign of the E. coli phytase toward improved hydrolysis on InsP4 and InsP3. As a prerequisite prior to evolution, a suitable screening setup was established and three isomers Ins(2,4,5)P3, Ins(2,3,4,5)P4 and Ins(1,2,5,6)P4 were generated through enzymatic hydrolysis of InsP6 and subsequent purification by HPLC. Screening of epPCR libraries identified clones with improved hydrolysis on Ins(1,2,5,6)P4 carrying substitutions involved in substrate binding and orientation. Saturation of seven positions and screening of, in total, 10,000 clones generated a dataset of 46 variants on their activity on all three isomers. This dataset was used for training, testing, and inferring models for machine learning guided recombination. The PyPEF method used allowed the prediction of recombinants from the identified substitutions, which were analyzed by reverse engineering to gain molecular understanding. Six variants with improved InsP4 hydrolysis of >2.5 were identified, of which variant T23L/K24S had a 3.7-fold improved relative activity on Ins(2,3,4,5)P4 and concomitantly shows a 2.7-fold improved hydrolysis of Ins(2,4,5)P3. Reported substitutions are the first published Ec phy variants with improved hydrolysis on InsP4 and InsP3.

A C terminus–dependent conformational change is required for HDAC3 activation by nuclear receptor corepressors

Histone deacetylase 3 (HDAC3) plays an important role in signal-dependent transcription and is dysregulated in diseases such as cancer. Previous studies have shown that the function of HDAC3 requires an activation step, which is mediated by the interactions of HDAC3 with the deacetylase-activation domain (DAD) of nuclear receptor corepressors and inositol tetraphosphate (IP4). However, the role of the unique HDAC3 C-terminal region in HDAC3 activation is elusive. Here multiple biochemical, structural, and functional studies show that HDAC3 activation requires a priming step mediated by the C terminus to remodel HDAC3 conformation. We show that multiple C-terminal mutations prevent HDAC3 activation by preventing this C terminus–dependent conformational change. Mechanistically, we demonstrate that the C terminus–mediated function in altering HDAC3 conformation is required for proper complex formation of HDAC3 with DAD and IP4 by allowing HDAC3 to undergo IP4-dependent interaction with DAD. Remarkably, we found that this C terminus function is conformation dependent, being necessary for HDAC3 activation prior to but not after the conformational change. Together, our study defines two functional states of free HDAC3, reveals the complete HDAC3 activation pathway, and links the C terminus function to the specific interaction between HDAC3 and DAD. These results also have implications in how signaling pathways may converge on the C terminus to regulate HDAC3 and suggest that the C terminus–mediated conformational change could represent a new target for inhibiting HDAC3 in diseases such as cancer.

NADPH levels affect cellular epigenetic state by inhibiting HDAC3-Ncor complex.

NADPH has long been recognized as a key cofactor for antioxidant defence and reductive biosynthesis. Here we report a metabolism-independent function of NADPH in modulating epigenetic status and transcription. We find that the reduction of cellular NADPH levels, achieved by silencing malic enzyme or glucose-6-phosphate dehydrogenase, impairs global histone acetylation and transcription in both adipocytes and tumour cells. These effects can be reversed by supplementation with exogenous NADPH or by inhibition of histone deacetylase 3 (HDAC3). Mechanistically, NADPH directly interacts with HDAC3 and interrupts the association between HDAC3 and its co-activator nuclear receptor corepressor 2 (Ncor2; SMRT) or Ncor1, thereby impairing HDAC3 activation. Interestingly, NADPH and the inositol tetraphosphate molecule Ins(1,4,5,6)P4 appear to bind to the same domains on HDAC3, with NADPH having a higher affinity towards HDAC3 than Ins(1,4,5,6)P4. Thus, while Ins(1,4,5,6)P4 promotes formation of the HDAC3-Ncor complex, NADPH inhibits it. Collectively, our findings uncover a previously unidentified and metabolism-independent role of NADPH in controlling epigenetic change and gene expression by acting as an endogenous inhibitor of HDAC3.

P0884MYO-INOSITOL HEXAPHOSPHATE PEGYLATION IMPROVES BIOAVAILABILITY BUT REDUCES ITS ANTI-VASCULAR CALCIFICATION EFFECT IN RATS

Abstract Background and Aims Vascular calcification (VC) is a major contributor to increased morbidity and mortality in End Stage Kidney Disease (ESKD) patients undergoing dialysis. SNF472, a salt of inositol hexaphosphate (InsP6), is a selective calcification inhibitor that interferes in the formation and growth of ectopic hydroxyapatite (HAP). SNF472 is currently in Phase 3 clinical trials for the treatment of calciphylaxis in ESKD patients on dialysis. Inositol-1,2,3,5-tetraphosphate-4,6-bisPEG100 (InsP4bisPEG or INS3001) results from the PEGylation of inositol tetraphosphate (InsP4) with polyethylene glycol (PEG) 100. Our aim was to compare the relative bioavailability of SNF472 and InsP4bisPEG and their efficacy in the inhibition of calcification in silico, in vitro and in vivo. Method Subcutaneous (10 mg/kg) pharmacokinetics of InsP4bisPEG and SNF472 were assessed in Sprague Dawley (SD) rats. To evaluate the adsorption binding affinity (Eads) of SNF472, InsP4bisPEG and other inositol phosphates to the HAP crystal surface, computational studies were performed using Density Functional Theory calculations with DMOL3 (MS2016). The in vitro efficacy of the compounds was evaluated using a pharmacodynamic assay to measure the calcification potential of human plasma. An in vivo efficacy study (calcification induced by 3 consecutives daily s.c. administrations of 150 kIU/kg vitamin D3) was performed with SD rats receiving s.c. vehicle, or equimolar doses (36 µmol/kg) of SNF472 or InsP4bisPEG once daily. Results The PEGylation of inositol tetraphosphate in positions 4 and 6 increased the exposure and t1/2 of the compound when given subcutaneously compared to SNF472. Molecular modelling revealed that SNF472 binds to the HAP surface with higher affinity than InsP4bisPEG and INSP4 (ΔEads=-352 kcal/mol for SNF472, ΔEads=-177 kcal/mol for InsP4bisPEG and ΔEads=-146 Kcal/mol for InsP4, taking inositol as reference). These results were correlated with the inhibition of calcium phosphate crystallization in plasma in vitro. SNF472 treated animals presented significantly lower calcium levels in aorta, which were 38% and 55% lower than placebo and InsP4bisPEG treated animals, respectively. Conclusion The differential pharmacokinetic profile of InsP4bisPEG (INS3001) does not translate into higher, but lower, efficacy than SNF472 against vascular calcification when comparing equimolar doses.

Variation in chemical composition and physical characteristics of cereal grains from different genotypes

ABSTRACTGenotypes of cereal grains, including winter barley (n = 21), maize (n = 27), oats (n = 14), winter rye (n = 22), winter triticale (n = 21) and winter wheat (n = 29), were assayed for their chemical composition and physical characteristics as part of the collaborative research project referred to as GrainUp. Genotypes of one grain species were grown on the same site, except maize. In general, concentrations of proximate nutrients were not largely different from feed tables. The coefficient of variation (CV) for the ether extract concentration of maize was high because the data pool comprised speciality maize bred for its high oil content. A subset of 8 barley, 20 rye, 20 triticale and 20 wheat samples was analysed to differ significantly in several carbohydrate fractions. Gross energy concentration of cereal grains could be predicted from proximate nutrient concentration with good accuracy. The mean lysine concentration of protein was the highest in oats (4.2 g/16 g N) and the lowest in wheat (2.7 g/16 g N). Significant differences were also detected in the concentrations of macro elements as well as iron, manganese, zinc and copper. Concentrations of arsenic, cadmium and lead were below the limit of detection. The concentration of lower inositol phosphates was low, but some inositol pentaphosphates were detected in all grains. In barley, relatively high inositol tetraphosphate concentration also was found. Intrinsic phytase activity was the highest in rye, followed by triticale, wheat, barley and maize, and it was not detectable in oats. Substantial differences were seen in the thousand seed weight, test weight, falling number and extract viscoelasticity characteristics. The study is a comprehensive overview of the composition of different cereal grain genotypes when grown on the same location. The relevance of the variation in composition for digestibility in different animal species will be subject of other communications.

Isolation and Characterization of Inositol Tetraphosphate 1-Kinase (AhITPK1) and Inositol 1,4,5-Tris-Phosphate Kinase (AhIPK2) Gene in Peanut

In the current study, partial cDNA clones of inositol tetraphosphate 1-kinase (ITPK1) and inositol 1,4,5-trisphosphate kinase/inositol polyphosphate multikinase (IPK2), were isolated from embryo using RT-PCR and designated as AhITPK1 and AhIPK2 isoforms of the gene. The partial cDNA sequence of AhITPK1 and AhIPK2 genes have an open reading frame (ORF) of 1146 and 891bp respectively and showed high similarity to other plant genes. AhITPK1 shared high homology with Aradu. Q95MC of Arachis duranensis, had a single exon with no introns and belonged to ATP-grasp family of proteins. AhIPK2 shared high similarity with Aradu.24V9G of A. duranensis and contained three exons with 5’ and 3’ UTR’s on either side. Unlike other IPK2 genes, AhIPK2 possessed conserved domains such as PxxxDxKxG and [L/M][I/V]D[F/L][A/G][H/K]. Phylogenetic analysis grouped AhITPK1 with A. duranensis, A. ipinensis and Oryza brachyantha into one cluster, whereas AhIPK2 was grouped along with Cucumis melo and C. sativus. Evolutionarily, AhITPK1 and AhIPK2 were genetically distinct from other plant genera. Furthermore, real-time PCR analysis revealed high expression of AhITPK1 and AhIPK2 genes in the peanut embryo and flower bud. For the first time AhITPK1 (KR778986) and AhIPK2 (KR778988) genes belonging to phytic acid pathway from Arachis hypogaea were identified and characterized the expression pattern of these two isoforms on different tissues. These genes were found to be abundant in flower bud and embryo. Results suggest that embryo development significantly influences the expression of the two AhIPK isoforms in peanut. Evolutionarily they were found to be distinct from their parental species. This study is an important step toward understanding the role of these two AhIPK isoforms in phytic acid synthesis. However, future research involving RNAi-based functional characterization is warranted to establish their link to embryo development in peanut.

Novel fiber-rich lentil flours as snack-type functional foods: an extrusion cooking effect on bioactive compounds.

Novel snack-type functional foods based on extruded lentil flours could convey the related health benefit of their bioactive compounds, provide a gluten-free alternative to consumers, and potentially increase the consumption of pulses. Extrusion treatment promoted an increase in galactopinitol, ciceritol, raffinose, stachyose and total α-galactoside content, in most lentil flours. As α-galactosides may act as prebiotics, they could convey beneficial effects to human and monogastric animals. Conversely, extrusion significantly (p < 0.05) reduced the inositol hexaphosphate content to less phosphorylated phytates (inositol pentaphosphate and inositol tetraphosphate), which provide health effects. The gluten-free formulation (control formulation #3) presented the highest significant (p < 0.05) drop in the inositol hexaphosphate of 14.7-fold decrease, but had a large increase in inositol pentaphosphate, due to extrusion processing. These two results are desirable in the finished product. Extrusion also caused a significant (p < 0.05) reduction in the trypsin content and completely inactivated lectin, in all processed samples.

Histone deacetylase (HDAC) 1 and 2 are essential for accurate cell division and the pluripotency of embryonic stem cells.

Histone deacetylases 1 and 2 (HDAC1/2) form the core catalytic components of corepressor complexes that modulate gene expression. In most cell types, deletion of both Hdac1 and Hdac2 is required to generate a discernible phenotype, suggesting their activity is largely redundant. We have therefore generated an ES cell line in which Hdac1 and Hdac2 can be inactivated simultaneously. Loss of HDAC1/2 resulted in a 60% reduction in total HDAC activity and a loss of cell viability. Cell death is dependent upon cell cycle progression, because differentiated, nonproliferating cells retain their viability. Furthermore, we observe increased mitotic defects, chromatin bridges, and micronuclei, suggesting HDAC1/2 are necessary for accurate chromosome segregation. Consistent with a critical role in the regulation of gene expression, microarray analysis of Hdac1/2-deleted cells reveals 1,708 differentially expressed genes. Significantly for the maintenance of stem cell self-renewal, we detected a reduction in the expression of the pluripotent transcription factors, Oct4, Nanog, Esrrb, and Rex1. HDAC1/2 activity is regulated through binding of an inositol tetraphosphate molecule (IP4) sandwiched between the HDAC and its cognate corepressor. This raises the important question of whether IP4 regulates the activity of the complex in cells. By rescuing the viability of double-knockout cells, we demonstrate for the first time (to our knowledge) that mutations that abolish IP4 binding reduce the activity of HDAC1/2 in vivo. Our data indicate that HDAC1/2 have essential and pleiotropic roles in cellular proliferation and regulate stem cell self-renewal by maintaining expression of key pluripotent transcription factors.

Differential expression of structural genes for the late phase of phytic acid biosynthesis in developing seeds of wheat (Triticum aestivum L.)

In cereals, phytic acid (PA) or inositol hexakisphosphate (IP6) is a well-known phosphate storage compound as well as major chelator of important micronutrients (iron, zinc, calcium, etc.). Genes involved in the late phases of PA biosynthesis pathway are known in crops like maize, soybeans and barley but none have been reported from wheat. Our in silico analysis identified six wheat genes that might be involved in the biosynthesis of inositol phosphates. Four of the genes were inositol tetraphosphate kinases (TaITPK1, TaITPK2, TaITPK3, and TaITPK4), and the other two genes encode for inositol triphosphate kinase (TaIPK2) and inositol pentakisphosphate kinase (TaIPK1). Additionally, we identified a homolog of Zmlpa-1, an ABCC subclass multidrug resistance-associated transporter protein (TaMRP3) that is putatively involved in PA transport. Analyses of the mRNA expression levels of these seven genes showed that they are differentially expressed during seed development, and that some are preferentially expressed in aleurone tissue. These results suggest selective roles during PA biosynthesis, and that both lipid-independent and -dependent pathways are active in developing wheat grains. TaIPK1 and TaMRP3 were able to complement the yeast ScΔipk1 and ScΔycf1 mutants, respectively, providing evidence that the wheat genes have the expected biochemical functions. This is the first comprehensive study of the wheat genes involved in the late phase of PA biosynthesis. Knowledge generated from these studies could be utilized to develop strategies for generating low phyate wheat.

Inactivating mutation in histone deacetylase 3 stabilizes its active conformation.

Histone deacetylases (HDACs), together with histone acetyltransferases (HATs), regulate gene expression by modulating the acetylation level of chromatin. HDAC3 is implicated in many important cellular processes, particularly in cancer cell proliferation and metastasis, making inhibition of HDAC3 a promising epigenetic treatment for certain cancers. HDAC3 is activated upon complex formation with both inositol tetraphosphate (IP4) and the deacetylase-activating domain (DAD) of multi-protein nuclear receptor corepressor complexes. In previous studies, we have shown that binding of DAD and IP4 to HDAC3 significantly restricts its conformational space towards its stable ternary complex conformation, and suggest this to be the active conformation. Here, we report a single mutation of HDAC3 that is capable of mimicking the stabilizing effects of DAD and IP4, without the presence of either. This mutation, however, results in a total loss of deacetylase activity, prompting a closer evaluation of our understanding of the activation of HDAC3.

Structural insight into the separate roles of inositol tetraphosphate and deacetylase‐activating domain in activation of histone deacetylase 3

Histone deacetylases (HDACs) repress transcription by deacetylating acetyllysines on specific histone tails. HDAC3 is implicated in neurodegenerative diseases, certain leukemias, and even in disrupting HIV-1 latency. A recent crystal structure of HDAC3 in complex with the deacetylase-activating domain (DAD) of its corepressor complex revealed an inositol tetraphosphate (IP4) molecule at the protein-protein interface. IP4 was shown to play an important, yet mechanistically ambiguous, role in the activity of HDAC3. The goal of this article is to explore the conformational ensemble of HDAC3 in its inactive apo state and in the presence of each or both of DAD and IP4. Using triplicate, 100 ns molecular dynamic simulations, we study the apo, ternary, and intermediate DAD-bound or IP4-bound HDAC3 states. We find that a population-shift effect is induced by the presence of each corepressor, and is most notable in the presence of both. Our results offer new insights into the change in dynamics necessary for the activation of HDAC3 and highlight the roles of IP4 and DAD in this process.

Effect of Several Germination Treatments on Phosphatases Activities and Degradation of Phytate in Faba Bean (Vicia faba L.) and Azuki Bean (Vigna angularis L.)

Two assays were conducted to investigate the changes of faba bean (Vicia faba L.) and azuki bean (Vigna angularis L.) phosphatases (phytase [Phy] and acid phosphatase [AcPh]) and the degradation of its substrates (inositol phosphate esters) during seed germination. The 1st assay was to establish the optimal germination conditions of faba bean and azuki bean to improve the endogenous phosphatases and increase the hydrolysis of phytate and, in the second assay, to determine the different lower phosphate esters of myo-inositol produced during the germination process. In the 1st assay, seeds were soaked for 12 and 24 h and germinated for 3 and 5 d with and without the addition of gibberellic acid (GA(3) ). In the second assay, seeds were soaked for 12 h and germinated for 1, 3, and 5 d with GA(3) . Phy (up to 3625 and 1340 U/kg) and AcPh (up to 9456 and 2740 U/g) activities, and inositol hexaphosphate (IP6) (8.23 and 7.46 mg/g), inositol pentaphosphate (IP5) (0.55 and 0.82 mg/g), and inositol tetraphosphate (IP4) (0.26 and 0.01 mg/g) were detected in ungerminated faba bean and azuki bean, respectively. The germination process caused a significant increase of Phy and AcPh activities in faba bean (up to 147% and 210%) and azuki bean (up to 211% and 596%) and a reduction in the phytate phosphorus content (up to 81% and 63%, respectively). Phytate phosphorus content was affected only by soaking time in the case of faba bean. Finally, during the course of germination, IP6 and IP5 were rapidly degraded in faba bean (88% and 39%) and azuki bean (55% and 56%), and IP4 was only a short-living intermediate, which was increased during hydrolysis and degraded to inositol triphosphate. In this manner we could obtain a low-phytate, endogenous phosphatase-rich ingredient for enhancing human nutrition.

IP4 is an epigenetic coregulator

Inositol tetraphosphate is required for both the incorporation of the histone deacetylase HDAC3 into a repressive complex and its enzymatic activity.

Structure of HDAC3 bound to co-repressor and inositol tetraphosphate

SummaryHistone deacetylase enzymes (HDACs) are emerging cancer drug targets. They regulate gene expression by removing acetyl groups from lysine residues in histone tails resulting in chromatin condensation. The enzymatic activity of most class I HDACs requires recruitment to corepressor complexes. We report the first structure of an HDAC:corepressor complex - HDAC3 with the deacetylase-activation-domain (DAD) from the SMRT corepressor. The structure reveals two remarkable features. First the SMRT-DAD undergoes a large structural rearrangement on forming the complex. Second there is an essential inositol tetraphosphate molecule, Ins(1,4,5,6)P4, acting as an ‘intermolecular glue’ between the two proteins. Assembly of the complex is clearly dependent on the Ins(1,4,5,6)P4, which may act as a regulator – potentially explaining why inositol phosphates and their kinases have been found to act as transcriptional regulators. This mechanism for the activation of HDAC3 appears to be conserved in class I HDACs from yeast to man and opens novel therapeutic opportunities.

The type of thermal feed treatment influences the inositol phosphate composition

The content and composition of inositol phosphate phosphorus (InsP-P) in maize, wheat, barley and heat treated soybean meal, rapeseed meal and sunflower meal was determined by high-performance ion chromatography (HPIC). Approximately 0.88–0.96 of the InsP-P in the feedstuffs was present in the inositol hexaphosphate (InsP 6) form, whereas the rest was in the inositol pentaphosphate (InsP 5) form and for oilseeds a very small amount was present as inositol tetraphosphate (InsP 4). Rapeseed differed from this pattern by having as much as 300 and 60 g InsP 4-P/kg of the total InsP-P pool. The effect of pelleting (90 °C) and extrusion cooking (130–140 °C, 6.5 MPa) on the composition of InsP-P was investigated. Neither treatment had any major effect on the total content of InsP-P in the feedstuffs. However, as indicated by the statistically significant effects on the proportion of the inositol phosphates, extrusion cooking shifted the inositol phosphates from InsP 6-P towards InsP 5-P both in cereals (P=0.002) and in oilseeds (P<0.001), which show a slight degradation of phytate during this treatment. The degradation of InsP 6 to InsP 5 appeared to be unspecific with regard to isomers in all feedstuffs, indicating that the degradation was non-enzymatic, i.e. a result of the high temperature and pressure during the extrusion cooking. The degradation of InsP 6 in the feedstuffs during extrusion is too limited to have any nutritional effect on the availability of phosphorous and minerals.

Inositol Phosphate Profiling of Fermented Cowpeas by 1H NMR Spectroscopy

The inositol phosphate content of naturally fermented cowpeas (Vigna sinensis var. Carilla) was studied using ion-pair HPLC and 1H NMR spectroscopy. The fermented flour was extracted with 0.5 M HCl, and the extract was purified and fractionated by ion-exchange chromatography. 1H NMR allowed for the identification of two monophosphates [Ins(1 or 3)P1 and Ins(4 or 6)P1], one inositol diphosphate [Ins(1,4)P2], three inositol triphosphates [Ins(1,2,6)P3, Ins(1,5,6)P3, and Ins(1,4,5)P3], one inositol tetraphosphate [Ins(1,3,4,5)P4], and one inositol pentaphosphate [Ins(1,2,3,5,6)P5]. Some of these isomers [Ins(1,4,5)P3 and Ins(1,3,4,5)P4] are considered to play important biological roles in intracellular signaling.

Fermentation of Vigna sinensis var. carilla Flours by Natural Microflora and Lactobacillus Species

Natural fermentation and an inoculum containing 10% (vol/vol) Lactobacillus fermentum or Lactobacillus plantarum were used to obtain fermented flours from Vigna sinensis L. var. carilla seeds that had been washed with distilled water and dried at 55°C for 24 h. To optimize the fermentation parameters (lactic acid bacterium level, bean flour concentration, and fermentation time), several small-scale fermentation processes were carried out. On the basis of the results obtained, fermentor-scale bean fermentation by microorganisms present on the seeds (natural fermentation [NF]) or by inoculation with L. plantarum (PF) was carried out at 37°C for 48 h with a concentration of 300 g of bean flour per liter. The fermented flours (NF and PF) were also autoclaved. The levels of α-galactosides, inositol phosphates, trypsin inhibitor activity (TIA), soluble carbohydrates, starch (total and available), total available carbohydrates, thiamin, and riboflavin were determined for the processed cowpea flours, and microbiological studies were also carried out. The beans' levels of α-galactosides, TIA, and inositol hexaphosphate decreased by 95, 50, and 85%, respectively, for the NF flour and by 87, 27, and 85%, respectively, for the PF flour, while inositol pentaphosphate and inositol tetraphosphate were present in both fermented flours. The sucrose content decreased, and glucose, fructose, and galactose appeared as a result of fermentation. The levels of total available sugars and thiamin decreased by 2 and 12% and by 69 and 43%, respectively, while the riboflavin content increased by 106 and 94% for NF and PF flours, respectively. When NF and PF cowpea flours were heated in an autoclave for 20 min, TIA decreased further (by 80 and 56%, respectively). According to the chemical and microbiological results obtained in this study, fermentation with L. plantarum and autoclaving is an excellent process by which to produce a new functional food from the seed of a cheap legume (Vigna sinensis L. var. carilla).

Controlling the Frequency

Oscillations in calcium released from intracellular stores regulate cellular responses to increases in intracellular calcium concentration [Ca 2+ ] I . The amplitude of the calcium spikes can be independent of agonist concentration, but the frequency of the spikes is often a function of agonist concentration. Zhu et al. electroporated HeLa cells to deliver specific inositol phosphate compounds or antibodies against phosphoinositol kinase, which altered the intracellular concentrations of specific polyphosphoinositides, and monitored calcium oscillations in response to histamine with the calcium-sensitive dye Fluo-3. Their data are consistent with inositol (1,3,4,5) tetraphosphate (Ins(1,3,4,5)P 4 ) being the frequency regulator in vivo and with Ins(1,3,4,5)P 4 being produced through the action of inositol (1,4,5) trisphosphate kinase (IP3K) on inositol (1,4,5) trisphosphate. Furthermore, IP3K is a calcium/calmodulin-activated enzyme providing a possible mechanism for the oscillatory nature of the activity of IP3K. Zhu, D.-M., Tekle, E., Huang, C.Y., and Chock, P.B. (2000) Inositol tetrakisphosphate as a frequency regulator in calcium oscillations in HeLa cells. J. Biol.Chem. 275 : 6063-6066. [Abstract] [Full Text]

Changes in quantities of inositol phosphates during maturation and germination of legume seeds

The changes in the quantities of inositol phosphates during the maturation and germination of pea, faba bean and lupin seeds were determined in two consecutive (1993 and 1994) years of differing weather conditions. Irrespective of the year, all seeds accumulated predominantly inositol hexaphosphate (IP6). The weather conditions influenced the accumulation of inositol phosphates in maturing seeds, but they did not influence the total content. Gradual degradation of inositol phosphates occurred during seed germination. After 8 days of germination, IP6 was degraded by some 80% in peas, 78% in faba beans and 42% in lupin seeds. The enzymic hydrolysis of higher forms of inositol phosphates (IP6 and IP5) in germinating seeds was assumed to yield inositol tetraphosphate (IP4) and inositol triphosphate (IP3), because the quantities of these compounds increased during seed germination.

The analysis for inositol phosphate forms in feed ingredients

Limited data are available on the content of inositol phosphates in feed ingredients. The doubtfulness over the identity and literature values of the inositol hexaphosphate (phytate) analysed in feed ingredients by the classical iron precipitation method prompted the analysis for inositol phosphates in some feed ingredients using a modified high-performance liquid chromatography method. The inositol phosphates analysed were inositol triphosphate (IP3), inositol tetraphosphate (IP4), inositol pentaphosphate (IP5) and inositol hexaphosphate (IP6). At least 97% of the inositol phosphates in grains occurred as IP6 and the remainder as IP5. In milled products the relative percentage of IP5 was higher (up to 14·5% in wheat bran) but the overall IP6 relative percentage was still high (more than 82%). IP3 and IP4 were not present in grains, and their amounts were very low when present in their milling or processed fractions. The absolute IP6 content was highest in rice bran and wheat bran at 68·31 and 47·63 g kg−1, respectively. The results indicate that processed plant products may contain hydrolysis products of IP5 and IP6. These products contain greater percentages of IP3, IP4 and IP5 than in grains or milling products. © 1998 SCI.