Osmotic regulation ofmyo-inositol uptake in primary astrocyte cultures

Uptake ofmyo-inositol by astrocytes in hypertonic medium (440 mosm/kg H₂O) was increased near 3-fold after incubation for 24 hours, which continued for 72 hours, as compared with the uptake by cells cultured in isotonic medium (38 nmoles/mg protein).myo-Inositol uptake by astrocytes cultured in hypotonic medium (180 mosm/kg H₂O) for periods up to 72 hours was reduced by 74% to 8 to 10 nmoles/mg protein. Astrocytes incubated in either hypotonic or hypertonic medium for 24 hours and then placed in isotonic medium reversed the initial down- or up-regulation of uptake. Activation of chronic RVD and RVI correlates with regulation ofmyo-inositol uptake. A 30 to 40 mosm/kg H₂O deviation from physiological osmolality can influencemyo-inositol homeostasis. The intracellular content ofmyo-inositol in astrocytes in isotonic medium was 25.6 ± 1.3 g/mg protein (28 mM). This level ofmyo-inositol is sufficient for this compound to function as an osmoregulator in primary astrocytes and it is likely to contribute to the maintenance of brain volume.     

Effect of Osmolality and myo-Inositol Deprivation on the Transport Properties of myo-Inositol in Primary Astrocyte Cultures

myo-Inositol uptake measured in primary astrocyte cultures was saturable in the presence of Na⁺ with a Km of 13–18 M and a Vmax of 9.4 nmoles/mg protein/hour in myo-inositol-fed cells, indicating a high affinity transport system. In myo-inositol-deprived cells, Km was about 53 M with a Vmax of 13.2 nmoles/mg protein/hour. Decreasing osmolality decreased the Vmax to about 1.9 nmoles/mg protein/hour whereas increasing osmolality increased Vmax about 5-fold, while Kms were essentially unchanged in myo-inositol fed cells. In cells deprived of myo-inositol, Vmax decreased in hypotonic medium and increased in hypertonic medium almost 10-fold, but with more than a doubling of the Km regardless of the osmolality. Glucose (25 mM) inhibited myo-inositol uptake 51% whereas the other hexoses used inhibited uptake much less. Our findings indicate that myo-inositol uptake in astrocytes occurs through an efficient carrier-mediated Na⁺-dependent co-transport system that is different from that of glucose and its kinetic properties are affected by myo-inositol availability and osmotic stress.     

Reduced Na+/K+ ATPase transport activity,resting membrane potential,and bradykinin-stimulated phosphatidylinositol synthesis by polyol accumulation in cultured neuroblastoma cells

In these studies we examined the effect of polyol accumulation on neural cellmyo-inositol metabolism and properties. Neuroblastoma cells were cultured for two weeks in media containing 30 mM glucose, fructose, galactose or mannose with or without 0.4 mM sorbinil or 250 Mmyo-inositol. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a decrease inmyo- inositol content and myo-[2-³H]inositol accumulation and incorporation into phosphoinositides compared to cells cultured in unsupplemented medium or medium containing 30 mM fructose as an osmotic control. These monosaccharides each caused an increase in intracellular polyol levels with galactitol > sorbitol = mannitol accumulation. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a significant decrease in Na⁺/K⁺ ATPase transport activity, resting membrane potential, and bradykinin-stimulated³²P incorporation into phosphatidylinositol compared to cells cultured in medium containing 30 mM fructose. In contrast, basal incorporation of³²P into phosphatidylinositol or basal and bradykinin-stimulated³²P incorporation into phosphatidylinositol 4,5-bisphosphate were not effected. Each of these cellular functions as well asmyo-inositol metabolism and content and polyol levels remained near control values when 0.4 mM sorbinil, an aldose reductase inhibitor, was added to the glucose, galactose, or mannose supplemented media.myo-Inositol metabolism and content and bradykinin-stimulated phosphatidylinositol synthesis were also maintained when media containing 30 mM glucose, galactose, or mannose was supplemented with 250 Mmyo-inositol. The results suggest that polyol accumulation induces defects in neural cellmyo-inositol metabolism and certain cell functions which could, if they occurred in vivo, contribute to the pathological defects observed in diabetic neuropathy.     

Purification and characterization of myo-inositol 6-O-methyltransferase from Vigna umbellata Ohwi et Ohashi

The cyclitol 1d-4-O-methyl-myo-inositol (d-ononitol) is accumulated in certain legumes in response to abiotic stresses. S-Adenosyl-l-methionine:myo-inositol 6-O-methyltransferase (m6OMT), the enzyme which catalyses the synthesis of d-ononitol, was extracted from stems of Vigna umbellata Ohwi et Ohashi and purified to apparent homogeneity by a combination of conventional chromatographic techniques and by affinity chromatography on immobilized S-adenosyl-l-homocysteine (SAH). The purified m6OMT was photoaffinity labelled with S-adenosyl-l-[¹⁴C-methyl]methionine. The native molecular weight was determined to be 106 kDa, with a subunit molecular weight of 40 kDa. Substrate-saturation kinetics of m6OMT for myo-inositol and S-adenosyl-l-methionine (SAM) were Michaelis-Menten type with K _m values of 2.92 mM and 63 M, respectively. The SAH competitively inhibited the enzyme with respect to SAM (K _i of 1.63 M). The enzyme did not require divalent cations for activity, but was strongly inhibited by Mn²⁺, Zn²⁺ and Cu²⁺ and sulfhydryl group inhibitors. The purified m6OMT was found to be highly specific for the 6-hydroxyl group of myo-inositol and showed no activity on other naturally occurring isomeric inositols and inositol O-methyl-ethers. Neither d-ononitol, nor d-3-O-methyl-chiro-inositol, d-1-O-methyl-muco-inositol or d-chiro-inositol (end products of the biosynthetic pathway in which m6OMT catalyses the first step), inhibited the activity of the enzyme.Abbreviations DTT dithiothreitol - m6OMT myo-inositol 6-O-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine We are greatful to Professor M. Popp (University of Vienna) for helpful discussion and comment. This work was supported by Grant P09595-BIO from the Austrian Science Foundation (FWF).     

Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus Oocytes

The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na⁺/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (V _m ), the external myo-inositol concentration and the external Na⁺ concentration, yielding the kinetic parameters: K _0.5 ^MI , K _0.5 ^Na , and the Hill coefficient n. At 100 mM NaCl, K _0.5 ^MI was about 50 m and was independent of V _m . At 0.5 mm myo-inositol, K _0.5 ^Na ranged from 76 mm at V _m =–50 mV to 40 mm at V _m =–150 mV. n was voltage independent with a value of 1.9±0.2, suggesting that two Na⁺ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent K _I of 64 m at V _m =–50 mV and 130 m at V _m = –150 mV. To examine sugar specificity, sugar-induced steady-state currents (at V _m =–150 mV) were recorded for a series of sugars, each at an external concentration of 50 mm. The substrate selectivity series was myo-inositol, scyllo-inositol > l-fucose > l-xylose > l-glucose, d-glucose, -methyl-d-glucopyranoside > d-galactose, d-fucose, 3-O-methyl-d-glucose, 2-deoxy-d-glucose > d-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na⁺/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at V _m =–150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: d-glucose, -methyl-d-glucopyranoside, d-galactose, d-fucose, 3-O-methyl-d-glucose > d-xylose, l-xylose, 2-deoxy-d-glucose > myo-inositol, l-glucose, l-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na⁺-dependent uptake of ³H-d-glucose and ³H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mm Na⁺ that was abolished by phlorizin (0.5 mm). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in V _m . The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model. Present address: W.M. Keck Biotechnology Resource Laboratory, Boyer Center for Molecular Medicine, Rm, 305A, Yale University, 295 Congress Ave., New Haven, Connecticut 06536-0812 Present address: National Institute for Physiological Sciences, Department of Cell Physiology, Okazaka, 444, JapanContributed equally to this workWe thank John Welborn for the HPLC analysis of the sugar substrates. This work was supported by grants from the National Institutes of Health DK19567, DK42479 and NS25554.     

Potentiometric and P NMR studies on inositol phosphates and their interaction with iron(III) ions

Potentiometric, conductometric and ³¹P NMR titrations have been applied to study interactions between myo-inositol hexakisphosphate (phytic acid), (±)-myo-inositol 1,2,3,5-tetrakisphosphate and (±)-myo-inositol 1,2,3-trisphosphate with iron(III) ions. Potentiometric and conductometric titrations of myo-inositol phosphates show that addition of iron increases acidity and consumption of hydroxide titrant. By increasing the Fe(III)/InsP₆ ratio (from 0.5 to 4) 3 mol of protons are released per 2 mol of iron(III). At first, phytates coordinate iron octahedrally between P2 and P1,3. The second coordination site represents P5 and neighbouring P4,6 phosphate groups. Complexation is accompanied with the deprotonation of P1,3 and P4,6 phosphate oxygens. At higher concentration of iron(III) intermolecular P–O–Fe–O–P bonds trigger formation of a polymeric network and precipitation of the amorphous Fe(III)–InsP₆ aggregates. ³¹P NMR titration data complement the above results and display the largest chemical shift changes at pD values between 5 and 10 in agreement with strong interactions between iron and myo-inositol phosphates. The differences in T₁ relaxation times of phosphorous atoms have shown that phosphate groups at positions 1, 2 and 3 are complexated with iron(III). The interactions between iron(III) ions and inositol phosphates depend significantly on the metal to ligand ratio and an attempt to coordinate more than two irons per InsP₆ molecule results in an unstable heterogeneous system.     

Uptake of auxotrophic cells of a heterocyst-forming cyanobacterium by tobacco protoplasts,and the fate of their associations

During imbibition, exogenous myo-inositol (MI) was readily introduced into the free MI pool of germinating wheat (Triticum aestivum L.). Maximum uptake, 70 g per caryopsis or 1.5 mg g^–1 of caryopsis, was reached at 0.05 M MI. Movement of free MI within the germinating caryopsis was traced with [2-³H]MI by two procedures, uptake by imbibition and injection into softened endosperm. The former procedure was useful during initial stages of germination; the latter provided a means of tracing the metabolic fate of MI generated by hydrolysis of phytate during mobilization of reserves within the caryopsis. In both procedures, the bulk of the added label was transferred to the seedling where it appeared in uronosyl and pentosyl units of 80% ethanol-insoluble polysaccharides, 2-O, C-Methylene-MI, an inhibitor of the MI oxidation pathway, blocked the utilization of [2-³H]MI as well as d-[1¹⁴C]glucose for biogenesis of pentose-and uronic-acid-containing polysaccharides.Abbreviations MI myo-inositol - OCM-MI 2-O, C-methylene-myo-inositol     

Attempts to produce solasodine in callus and suspension cultures of Solanum mauritianum Scop.

Callus cultures of Solanum mauritianum Scop. were initiated from green berry explants on a hormone-free Murashige and Skoog (1962) medium excluding glycine, and containing 0.1 g L^–1 myo-inositol and 3% sucrose. Such cultures contained 10.08±0.59 g g^–1 DW of solasodine, which is equivalent to that in the leaves of mature S. mauritianum plants, but far less than that extracted from the green berries (185 g g^–1 DW). In vitro solasodine productivity could be increased by reducing the strength of the medium by half, substituting 3% glucose for 3% sucrose as carbon source, or by the addition of certain combinations of BA and NAA. Phosphate limitation and alterations in the carbon: nitrogen ratio were not able to increase solasodine productivity. Suspension cultures of S. mauritianum were initiated and maintained in a Murashige and Skoog (1962) medium with the RT vitamins of Khanna and Staba (1968), 0.1 g L^–1 myo-inositol, 3% sucrose and 1 mg L^–1 2,4-D. No solasodine was detectable in these cultures, or slight modifications thereof.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium     

Degradation of <Emphasis Type="Italic">myo</Emphasis>-inositol Hexakisphosphate by a Phytate-degrading Enzyme from <Emphasis Type="Italic">Pantoea agglomerans</Emphasis>

High-pressure liquid chromatography (HPLC) analysis established myo-inositol pentakisphosphate as the final product of phytate dephosphorylation by the phytate-degrading enzyme from Pantoea agglomerans. Neither product inhibition by phosphate nor inactivation of the Pantoea enzyme during the incubation period were responsible for the limited phytate hydrolysis as shown by addition of phytate-degrading enzyme and phytate, respectively, after the observed stop of enzymatic phytate degradation. In additon, the Pantoea enzyme did not possess activity toward the purified myo-inositol pentakisphosphate. Using a combination of High-Performance Ion Chromatography (HPIC) analysis and kinetic studies, the nature of the generated myo-inositol pentakisphosphate was established. The data demonstrate that the phytate-degrading enzyme from Pantoea agglomerans dephosphorylates myo-inositol hexakisphosphate in a stereospecific way to finally D-myo-inositol(1,2,4,5,6)pentakisphosphate.     

Functional identification of sll1383 from<Emphasis Type="Italic"> Synechocystis</Emphasis> sp PCC 6803 as L-<Emphasis Type="Italic">myo</Emphasis>-inositol 1-phosphate phosphatase (EC 3.1.3.25): molecular cloning,expression and characterization

The genome sequence of the cyanobacterium Synechocystis sp. PCC6803 revealed four Open reading frame (ORF) encoding putative inositol monophosphatase or inositol monophosphatase-like proteins. One of the ORFs, sll1383, is ∼870 base pair long and has been assigned as a probable myo-inositol 1 (or 4) monophosphatase (IMPase; EC 3.1.3.25). IMPase is the second enzyme in the inositol biosynthesis pathway and catalyses the conversion of L-myo-inositol 1-phosphate to free myo-inositol. The present work describes the functional assignment of ORF sll1383 as myo-inositol 1-phosphate phosphatase (IMPase) through molecular cloning, bacterial overexpression, purification and biochemical characterization of the gene product. Affinity (K _m) of the recombinant protein for the substrate DL-myo-inositol 1-phosphate was found to be much higher (0.0034 ± 0.0003 mM) compared to IMPase(s) from other sources but in comparison V _max (∼0.033 μmol Pi/min/mg protein) was low. Li⁺ was found to be an inhibitor (IC₅₀ 6.0 mM) of this enzyme, other monovalent metal ions (e.g. Na⁺, K⁺ NH₄⁺) having no significant effect on the enzyme activity. Like other IMPase(s), the activity of this enzyme was found to be totally Mg²⁺ dependent, which can be substituted partially by Mn²⁺. However, unlike other IMPase(s), the enzyme is optimally active at ∼42°C. To the best of our knowledge, sll1383 encoded IMPase has the highest substrate affinity and specificity amongst the known examples from other prokaryotic sources. A possible application of this recombinant protein in the enzymatic coupled assay of L-myo-inositol 1-phosphate synthase (MIPS) is discussed.     

myo-Inositol homeostasis in foetal rabbit lung

In several species, lung maturation is accompanied by a decline in the phosphatidylinositol content of lung surfactant and a concomitant increase in its phosphatidylglycerol content. To examine the possibility that this developmental change is influenced by the availability of myo-inositol, potential sources of myo-inositol for the developing rabbit lung were investigated. On day 28 of gestation the myo-inositol content of foetal rabbit lung tissue (2.3±0.5μmol/g of tissue) was not significantly different from that of adult lung tissue but the activity of d-glucose 6-phosphate:1l-myo-inositol 1-phosphate cyclase (cyclase) in foetal lung tissue (81.0±9.0nmol·h⁻¹·g of tissue⁻¹) was higher than that found in adult lung tissue (23.2±1.0nmol·h⁻¹·g of tissue⁻¹). Day 28 foetal rabbit lung tissue was found also to take up myo-inositol by a specific, energy-dependent, Na⁺-requiring mechanism. Half-maximal uptake of myo-inositol by foetal rabbit lung slices was observed when the concentration of myo-inositol in the incubation medium was 85μm. When the myo-inositol concentration was 1mm (but not 100μm) the addition of glucose (5.5mm) stimulated myo-inositol uptake. myo-Inositol uptake was observed also in adult rabbit lung and was found to be sub-maximal at the concentration of myo-inositol found in adult rabbit serum. The concentration of myo-inositol in the serum of pregnant adult rabbits (47.5±5.5μm) was significantly lower than that of non-pregnant adult female rabbits (77.9±9.2μm). On day 28 of gestation the concentration of myo-inositol in foetal serum (175.1±12.0μm) was much less than on day 25, but more than that found on day 30. A transient post-partum increase in the concentration of myo-inositol in serum was followed by a rapid decline. Much of the myo-inositol in foetal rabbit serum probably originates from the placenta, where on day 28 of gestation a high cyclase activity (527±64nmol·h⁻¹·g of tissue⁻¹) was measured. The gestational decline in serum myo-inositol concentration, together with the decreasing cyclase activity of the lungs, is consistent with the view that maturation of the lungs is accompanied by decreased availability of myo-inositol to this tissue.     

Modulation of HERG channel inactivation by external cations

We investigated the effect of external cations on the permeability characteristics and gating kinetics of the human ether-à-go-go-related gene (HERG) current using the whole-cell patch-clamp technique. Inward HERG currents were recorded on hyperpolarization in 140 mM external Cs⁺ and Rb⁺, as well as K⁺. The permeability ratios of Rb⁺ and Cs⁺ relative to K⁺ were 1.25 and 0.56, respectively. Biphasic outward currents were recorded on depolarization in 140 mM Cs⁺ and in Rb⁺ with much smaller amplitude. The voltage dependence of inactivation was affected by external cations, such that the half-inactivation voltage shifted from –69.4±3.7 mV in K⁺ to –30.7±1.6 mV in Cs⁺ and to –35.8±1.9 mV in Rb⁺ (n=5). The time constants of inactivation were also changed significantly by external cations; of inactivation at +40 mV was 16.4±2.2 ms in 140 mM K⁺, 181±20.3 ms in Cs⁺, and 94.1±7.6 ms in Rb⁺ (n=5). Voltage dependence of activation was not altered significantly. The inhibition of the rapid inactivation mechanism by large cations may suggest that the foot-in-the-door model of gating is involved in HERG channel inactivation.     

The latency of the response of Limulus photoreceptors to inositol trisphosphate lacks the calcium-sensitivity of that to light

Summary The latent period before depolarization of Limulus ventral photoreceptors by light flashes was compared with that following brief, intracellular, pressure-injection of d-myo-inositol 1,4,5 trisphosphate. At temperatures between 18 °C and 22 °C and with an extracellular calcium concentration of 10 mM, the responses of 4 cells to light and to injections of 100 M inositol trisphosphate displayed average latencies of 71 and 56 ms, respectively. The latencies of responses to InsP₃ included an estimated 20 ms dead-time inherent in the injection method. Reducing the temperature lengthened the latency of the response to light (Q₁₀ approximately 3.2 between 7 and 22 °C) more than that to inositol trisphosphate (Q₁₀ approximately 2.3). Bathing the photoreceptors in seawater containing no added calcium and 1 mM of the calcium chelator EGTA greatly increased the latency of the light response at all temperatures, but did not increase the latency of the response to inositol trisphosphate. We conclude that the response to inositol trisphosphate lacks the calcium- and temperature-sensitive latent period which characterizes the response to light. If inositol trisphosphate acts, via the release of stored calcium, to stimulate an intermediate in the visual cascade, then that intermediate would appear to be downstream from the latency-generating mechanism.Abbreviations InsP ₃ D-myo-inositol 1,4,5 trisphosphate - ASW Artificial seawater - Ca _i Cytosolic free calcium ion concentration - Ca ₀ Extracellular calcium ion concentration     

Antioxidative Effect of an Isolate from a Culture Filtrate of Aspergillus niger

D-Ins(1,3,4,5)P₄ and unnatural L-Ins(1,3,4,5)P₄ were prepared in gram-quantities from D- and L-2,6-di-O-benzyl-myo-inositol by a chemical phosphorylation and deprotection step in high yield and purity without extensive purification. The optically pure benzyl derivatives were obtained by enzyme-catalyzed resolution of racemic 2,6-di-O-benzyl-myo-inositol under acyl-transfer conditions in vinyl acetate as the acyl donor. The lipase of Candida antarctica only acetylated regio- and enantio-selectively the L-enantiomer, providing exclusively L-5-acetyl-2,6-di-O-benzyl-myo-inositol, whereas the D-enantiomer remained unchanged.     

Down-regulation of the <Emphasis Type="Italic">myo</Emphasis>-inositol oxygenase gene family has no effect on cell wall composition in Arabidopsis

The enzyme myo-inositol oxygenase (MIOX; E.C. 1.13.99.1) catalyzes the ring-opening four-electron oxidation of myo-inositol into glucuronic acid, which is subsequently activated to UDP-glucuronic acid (UDP-GlcA) and serves as a precursor for plant cell wall polysaccharides. Starting from single T-DNA insertion lines in different MIOX-genes a quadruple knockdown (miox1/2/4/5-mutant) was obtained by crossing, which exhibits greater than 90% down-regulation of all four functional MIOX genes. Miox1/2/4/5-mutant shows no visible phenotype and produces viable pollen. The alternative pathway to UDP-glucuronic acid via UDP-glucose is upregulated in the miox1/2/4/5-mutant as a compensatory mechanism. Miox1/2/4/5-mutant is impaired in the utilization of myo-inositol for seedling growth. The incorporation of myo-inositol derived sugars into cell walls is strongly (>90%) inhibited. Instead, myo-inositol and metabolites produced from myo-inositol such as galactinol accumulate in the miox1/2/4/5-mutant. The increase in galactinol and raffinose family oligosaccharides does not enhance stress tolerance. The ascorbic acid levels are the same in mutant and wild type plants.     

The Role of Protein Kinase C and Cyclic AMP in the Ammonia-Induced Shift of the Taurine Uptake/Efflux Balance Towards Efflux in C6 Cells

A previous study showed that treatment of C6 glioma cells with 10 mM ammonium chloride (ammonia) for 24 h decreases taurine uptake and evokes sodium-dependent taurine efflux, indicating reversal of the taurine transporter (TauT)-mediated transport as an underlying mechanism. Consistent with the involvement of TauT we now show that the ammonia-induced changes in Tau uptake and efflux are inhibited by the protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu). Ammonia treatment of C6 cells resulted in increased intracellular accumulation of cAMP. Incubation of the cells with dibutyryl cAMP (dbcAMP) mimicked the effects of ammonia on both taurine uptake and efflux. The effects of dbcAMP on taurine uptake and efflux were additive to the effects of ammonia. Collectively, the results suggest that the effects of ammonia on taurine uptake and efflux may be partly mediated by cAMP. Consistent with this mechanism, the adenyl cyclase inhibitor, miconazole reduced the stimulation of efflux by ammonia.     

Partial purification and characterization of phytases from pollen of lily (Lilium longiflorum Thunb.)

Summary Two phytases from lily pollen (Lilium longiflorum Thunb.) were partially purified and characterized. The first (pH optimum 5.0) was purified 40-fold from ungerminated pollen. The second (pH optimum 6.5) appeared during germination and was purified 68-fold from pollen germinated 2 h. Molecular weight of the first was 72 kD, and the second was 36 kD as determined by gel filtration. Both were active against phosphate esters other than phytate, although purification of the first reduced its activity against AMP and myo-inositol 2-P to 10% of activity against phytate. Phytase from germinated pollen (but not ungerminated) was inhibited by the sulfhydryl agent parahydroxy mercuribenzoate; P _i inhibited phytase from ungerminated but not germinated pollen. Such different catalytic and physical properties may reflect different biochemical functions.Abbreviations HPLC High performance liquid chromatography - DEAE diethyl aminoethyl - P _i orthophosphate - PP _i pyrophosphate - p-NPP para-nitrophenyl phosphate - pNP para-nitrophenol - MI myo-inositol - MI 2-P myo-inositol 2-P - MI penta P myo-inositol pentakisphosphate - PHMB para-hydroxy mercuribenzoate - PMSF phenyl methyl sulfonyl fluoride - AMP adenosine monophosphate - GMP guanosine monophosphate - EGTA ethylene glycol-bis (-aminoethyl ether) N, N, N, N-tetraacetic acid     

An examination of some possible sources of soil inositol phosphates

Summary Examination of extracts prepared from soil fungi and yeast cells (S. cavlsbergensis) showed that phytate phosphorus was not present. Phytin was successfully extracted from the acorns ofQuercus robur andQ. hodgkinsonii but was apparently absent from acorns ofQ. ilex. Acorn phytin was shown to consist only of phosphate esters ofmyo-inositol although freescyllo-inositol is present in this plant tissue. Small amounts of phytate phosphorus were isolated from sand/clay cultures, after a period of incubation, but onlymyo-inositol derivatives appeared to be present.     

Kinetic characterization of unspecific alkaline phosphatase at different villus sites of rat jejunum

Summary A quantitative histochemical method to determine the apparent Km and V _max values of rat intestinal unspecific alkaline phosphatase at different sites of the villi is described. Naphthol-As-Bi-phosphate (0.025–1.5 mM) is employed as substrate and Fast Blue B as coupling reagent, and the resulting azo-dye in the brush border membrane has an absorbance maximum at 550 nm. The ratio between the absorbance at 550 and 500 nm is constant as calculated from automatically recorded spectra at different intense dye deposits. Its absorbance is a linear function of incubation time up to 3 min and thickness of the slices up to 10 m both with medium (0.5 mM) and high (1.5 mM) substrate concentrations. Using the histochemical assay under comparable conditions in test tube experiments with homogenates of intestinal mucosa an app. Km of 0.26±0.081 mM (weighted regression analysis) and 0.28–0.084 mM (direct linear plotting) is determined, demonstrating an affinity to the histochemical substrate, which is about 10 times higher than for p-Nitro-phenyl-phosphate with the purified enzyme.The results obtained by scanning the total dye deposits along jejunal villi show considerable differences in enzymatic activity between single villi and an increase from the villus base up to the transition between medium and apical villus third. As well in the apical region as at the villus base saturation curves are obtained by determining the relationship between the absorbance and the substrate concentration under standard conditions (slice thickness 10 m, incubation time 3 min, 37°C, pH 8.3). Calculated by weighted regression analysis and direct linear plotting from the absorbance data of six female rats the medium app. kinetic data ±SD from the jejunal villi read as follows. Apical: Km=0.81±0.438 mM, V _max=3.99±1.217 absorbance units (A) and Km=0.87±0.428 mM, V _max=4.02±1.191 A, respectively. Basal: Km=0.82±0.261 mM, V _max=3.26±0.719 A and Km=0.77±0.184 mM, V _max=3.04±0.518 AU, respectively. As demonstrated by factorial analysis of variance only V _max is influenced by the villus position.Supported by the Deutsche Forschungsgemeinschaft GU 184/1