Effect of myo-inositol(1,4,5)trisphosphate on the hydrolysis of phytic acid by phytase.

Phytase is a monomeric enzyme of molecular mass 160 kDa which catalyzes the hydrolysis of phytic acid (D-myo inositol hexakisphosphate, InsP6) in a stepwise manner to myo-inositol. The enzyme-InsPn (n = 1-6) interaction at the catalytic site has a dissociation constant in the micro molar range. There also exists in the enzyme, a non-catalytic site specific for InsP3 with dissociation constant in the nano molar range. We have probed the effect of the high affinity InsP3 binding on the dissociation constant (Kd) of the phytase-InsP6 interaction and the kinetics of hydrolysis. These studies demonstrate the effect exerted by the high affinity InsP3 binding on the catalytic site of the enzyme.

High affinity association of myo-inositol trisphosphates with phytase and its effect upon the catalytic potential of the enzyme.

A neutral phytase from germinating mung bean (Vigna radiata) seeds dephosphorylates myo-inositol hexakisphosphate sequentially to myo-inositol. The enzyme also binds with higher affinity to myo-inositol trisphosphates (1,4,5), (2,4,5), and (1,3,4) isomers without catalysis. The high affinity complex elicits Ca(2+) mobilization in vitro from microsomes/vacuoles via the formation of a ternary complex with the receptor for Ins(1,4,5)P(3). As a sequel to our previous report, we have carried out a detailed characterization of the two sites and examined the mutual interactions between them. Presaturation of the high affinity site leads to an increase in the affinity of the enzyme for phytic acid and its rate of dephosphorylation as well. From the products of limited tryptic cleavage of phytase, two peptides, each with one activity, have been isolated. The larger peptide ( approximately 66 kDa) contains the catalytic site, and the smaller peptide ( approximately 5 kDa) has the high affinity myo-inositol trisphosphate-binding site. The interaction between the dual activities of phytase has been observed also at the level of the two peptides. A sequence homology search using N-terminal 12 amino acid residues of the 5-kDa fragment has revealed significant homology with the Homer class of proteins implicated in signaling pathways involving metabotropic glutamate receptor and myo-inositol 1,4,5-trisphosphate receptor. These results indicate a second role of phytase in Ca(2+) mobilization during germination of mung been seed via a salvage pathway that involves allosteric activation by myo-inositol trisphosphate.

Conformational changes in plant Ins(1,4,5)P3 receptor on interaction with different myo-inositol trisphosphates and its effect on Ca2+ release from microsomal fraction and liposomes.

The interaction of the only reported plant inositol trisphosphate receptor with different myo-inositol trisphosphates (InsP3 species), namely Ins(1,4,5)P3, Ins(1,3,4)P3, Ins(1,5,6)P3, and Ins(2,4,5)P3, were studied to assess the extent of Ca2+ mobilization from microsomes/vacuoles as well as liposomes in vitro. Ins(1,4,5)P3 and Ins(2,4,5)P3 bind with the receptor with comparable affinities, as evidenced from their dissociation constants (Kd approx. 100 nM at 5 degrees C), whereas the interaction between Ins(1,3,4)P3/Ins(1,5,6)P3 and the receptor was not detected even with these ligands at 5 microM. Ins(1,3,4)P3/Ins(1,5,6)P3 isomers also do not elicit Ca2+ release from liposomes or microsomes/ vacuoles. The ability of any InsP3 to bind the receptor for Ins(1,4,5)P3 is a prime requirement for Ca2+ release. However, the comparison of binding affinities at a single temperature does not help to correlate it directly with the extent of Ca2+ release from the intracellular stores because the concentration of Ca2+ released by Ins(1,4,5)P3 as estimated over a period of 20 s is 3500 +/- 200 nM/mg of protein and is about 4-fold higher than that by Ins(2,4,5)P3 under identical conditions. To understand the role of the receptor conformation in Ca2+ release by different isomers, we have probed the conformational change of the receptor when the different isomers bind to it. Accessibility of the tryptophan residues in the free and Ins(1,4,5)P3/Ins(2,4,5)P3-bound receptor was monitored by a neutral fluorescence quencher, acrylamide. The resulting Stern-Volmer-type quenching plots of the internal fluorescence indicate a change in the conformation of the receptor on binding to Ins(1,4,5)P3 and Ins(2,4,5)P3. It is also detected when far-UV CD spectra (205-250 nm) of the free and ligand [Ins(1,4,5)P3/Ins(2,4,5)P3]-bound receptor are compared. The results from CD spectroscopic studies further indicate that the conformational changes induced by the two isomers are different in nature. When thermodynamic parameters, such as enthalpy (delta H), entropy (delta S) and free energy (delta G), for the formation of the two InsP3-receptor complexes are compared, a major difference in the extent of changes in enthalpy and entropy is noted. All these findings taken together support the proposition that it is the overall interaction leading to the requisite conformational change in the receptor that determines the potency of the InsP3 isomers in their abilities of Ca2+ mobilization from the intracellular stores or reconstituted liposomes.

Relative importance of inositol (1,4,5)trisphosphate and inositol (1,3,4,5)tetrakisphosphate in Entamoeba histolytica.

[3H]Inositol tetrakisphosphate (Ins(1,3,4,5)P4) binding sites which were poorly displaced by unlabelled inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) were detected in membrane fractions of Entamoeba histolytica. Similarly, unlabelled Ins(1,3,4,5)P4 was 30-fold less efficient in displacing [3H]Ins(1,4,5)P3 binding. pH sensitivities of binding of the two isomers were markedly different. Scatchard analysis of the data revealed single binding sites and similar receptor densities for each of the two isomers. Formation of both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in E. histolytica was also demonstrated. Calcium release studies showed that after treatment with a saturating dose of either Ins(1,4,5)P3 or Ins(1,3,4,5)P4 the other inositol polyphosphate could partially revive the response to a subsequent addition of the first inducer. Our data clearly demonstrate that Ins(1,4,5)P3 and Ins(1,3,4,5)P4 are two equally important but independent second messengers in E. histolytica.

Interaction of myoinositoltrisphosphate-phytase complex with the receptor for intercellular Ca2+ mobilization in plants.

One of the myoinositol trisphosphates produced by the phytase-myoinositol hexakisphosphate (InsP6) reaction is Ins(2,4,5)P3. That Ins(2,4,5)P3 can elicit Ca2+ mobilization from intracellular stores in plants [Samanta, S., Dalal, B., Biswas, S., & Biswas, B.B.(1993) Biochem. Biophys. Res. Commun. 191,427] prompted us to elucidate the mechanism. The InsP3 [Ins(1,4,5)P3/Ins(2,4,5)P3]-phytase complex has been found to interact with the receptor for InsP3 in vitro forming a ternary complex, and a nanomolar concentration of InsP3 is required. For enzymatic cleavage of InsP3 by phytase, micromolar concentrations are needed, and the affinities of the phytase for different myoinositol phosphates have been found to depend upon the number of phosphate groups present in the substrate. Fraction accessibility of tryptophan residues to a neutral fluorescence quencher, acrylamide in free and myoinositol phosphates bound phytase, as determined by Stern-Volmer plot, records a progressive decrease starting from InsP6 to InsP with the notable exceptions of both Ins (1,4,5)P3 and Ins(2,4,5)P3. This deviation from the trend of change in the accessibility of tryptophan residues in myoinositol phosphate bound phytase is recorded from the fact that there is a high affinity (dissociation constant of the nanomolar order) and noncatalytic binding site in phytase for the two isomers of InsP3. In the nanomolar range of concentrations, both isomers of InsP3 bind to a second site of phytase having about 40-fold higher affinity than the normal substrate binding site. InsP3, when bound to noncatalytic site in phytase is not hydrolyzed but induces a significant change in the conformation of phytase as assayed from the relative accessibility of tryptophan residues. This conformational change in phytase is recognized by the receptor for InsP3, because in absence of InsP3 no interaction between the receptor and phytase is detected. However, InsP3-phytase complex is a better elicitor of Ca2+ efflux from microsomal/vacuolar fractions than free InsP3. This is further confirmed by the fact that when Ins(1,3,4)P3-phytase complex can elicit Ca2+ efflux from intracellular stores, Ins(1,3,4)P3 per se is minimally effective.

Inositol(1,3,4,5) tetrakisphosphate plays an important role in calcium mobilization from Entamoeba histolytica.

Calcium release from internal stores of Entamoeba histolytica, a parasitic protozoan, was observed by measuring fluorescence of Fura-2. Emptying of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3)-sensitive calcium pools in permeabilized E. histolytica did not significantly affect subsequent calcium release by inositol(1,3,4,5)tetrakis-phosphate (Ins(1,3,4,5)P4). Similarly, prior depletion of Ins(1,3,4,5)P4-sensitive stores did not have any influence on subsequent calcium release by Ins(1,4,5)P3. The EC50 for calcium release was 0.15 microM with Ins(1,4,5)P3 and 0.68 microM with Ins(1,3,4,5)P4. In conclusion, the Ins(1,3,4,5)P4-sensitive calcium store in E. histolytica is separate and independent from the Ins(1,4,5)P3-sensitive pool.

Receptor for myo-inositol trisphosphate from the microsomal fraction of Vigna radiata.

The microsomal fraction from mung-bean (Vigna radiata) hypocotyl was found to contain Ins (1,4,5)P3- and Ins(2,4,5)P3-binding activity. Preincubation of the microsomal fraction with thiol-containing reagents reduced specific InsP3 binding. A single class of binding site with a Kd value of 1.5 nM and Bmax. of 1.1 pmol/mg of protein was detected. Other myo-inositol phosphates exhibited little affinity for this protein. The binding protein was purified to homogeneity and the molecular mass of the native form recorded as 400 kDa. However, under denaturing conditions the molecular mass was 110 kDa, suggesting that the protein is a homotetramer. That this protein is associated with Ca2+ release was confirmed by including it in proteoliposomes and adding Ins(1,4,5)P3 or Ins(2,4,5)P3. The affinity of Ins(1,4,5)P3 is 3-fold higher than that of Ins(2,4,5)P3. The binding affinity of InsP3 is also reflected in the extent of Ca2+ released from the microsomal fraction. Heparin inhibits binding of InsP3 to the protein, the K1/2 being 0.26 microM. It is also shown that the protein acts as a receptor for InsP3 with characteristics of high affinity and low density.

Expression of the Arabidopsis thaliana 2S albumin gene 3 in Saccharomyces cerevisiae.

The Arabidopsis thaliana (At) 2S albumin gene 3 (At2S3) has been cloned in YEp13 as a 3.5-kb genomic fragment. To study its expression in Saccharomyces cerevisiae, the accumulation in saturated cultures reached about 0.032% of the yeast total protein, and the product was localized in vacuolar bodies within the cell. The 13-kDa protein was processed to 9- and 4-kDa proteins, as obtained in transgenic tobacco plants.

Myo-inositol trisphosphate-mediated calcium release from internal stores of Entamoeba histolytica.

Calcium mobilisation from internal stores of the parasitic protozoan Entamoeba histolytica was studied by fluorescence measurements of the calcium indicator quin 2 and 45Ca2+ incorporation studies in saponin-permeabilised amoebae. Prior energy-dependent calcium sequestration was found to be necessary for subsequent release of calcium by inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Both Ins(1,4,5)P3 and inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) could release calcium equally well from permeabilised E. histolytica with similar EC50 (concentration which produced half maximal release) values for calcium release. Ins(1,4,5)P3-mediated calcium release occurred from a vesicular store, was sensitive to prior treatment by heparin and was attenuated by prior addition of a lower concentration of Ins(1,4,5)P3. cAMP failed to influence inositol trisphosphate induced calcium release, indicating the absence of control mechanisms through cAMP-dependent phosphorylation. GTP neither induced calcium release nor could potentiate inositol trisphosphate mediated calcium mobilisation. A saturating concentration of Ins(1,4,5)P3 could release 50% of radiolabelled calcium sequestered by energy-dependent mechanisms in E. histolytica. The energy-dependent calcium sequestration was inhibited by vanadate and the calcium antagonist Diltiazem but not by dicyclohexylcarbodiimide (DCCD), suggesting the involvement of an endoplasmic reticulum-like structure in calcium storage. Binding studies showed specific association of [3H]Ins(1,4,5)P3 to crude membrane fractions of E. histolytica, which was significantly inhibited by heparin in a dose-dependent manner. IC50 (concentration which produced half-maximal inhibition) values for displacement of radiolabelled Ins(1,4,5)P3 binding by unlabelled Ins(1,4,5)P3 and Ins(2,4,5)P3 were estimated to be 0.99 microM for both isomers. Our results suggested that Ins(1,4,5)P3-mediated calcium release from internal stores of E. histolytica most probably occurred in an inositol trisphosphate receptor-dependent manner.

Myoinositol tris-phosphate-phytase complex as an elicitor in calcium mobilization in plants.

Mobilization of Ca2+ from microsomal/vacuolar fractions was detected when InsP6-phytase was added after a definite time of hydrolysis which coincides with the time (20-30 min) of optimal production of Ins(2,4,5)P3 bound to phytase. The in vitro constituted Ins(1,4,5)P3 or Ins(2,4,5)P3-phytase complex is also effective in releasing Ca2+. InsP3-phytase complex releases 45% more microsomal Ca2+ than that released by free InsP3 under identical conditions. Other inositol-phytase complexes are ineffective. Furthermore InsP3-phytase complex is recognised by putative receptor associated with microsomal fraction suggesting that the myoinositol tris-phosphate-phytase complex can act as an elicitor in Ca2+ mobilization in plant systems where phytate and phytase occur.

Transformation and regeneration of mung bean (Vigna radiata).

The procedure relied on a protocol in which shoot organogenesis was induced on cotyledons of mung bean genotypes selected for susceptibility to agrobacterium seems to work reproducibly if not efficiently. Approximately 4-5% of the shoots produced on the kanamycin selected cotyledons are transgenic based on assays on kanamycin resistance and GUS activity. This demonstrated that transformation and regeneration in mung bean are possible. However, raising the transformed plants in field condition is yet to be perfected.

Characterisation of a repetitive DNA family from Entamoeba histolytica containing Saccharomyces cerevisiae ARS consensus sequences.

Several repetitive DNA families were identified in Entamoeba histolytica DNA digested with Sau3AI. Characterisation of one of these repetitive DNA families showed the presence of multiple copies of Saccharomyces cerevisiae autonomously replicating sequence (ARS) core consensus sequences. The E. histolytica ARS consensus sequences allowed a yeast-integrating plasmid, YIP5, to replicate autonomously in S. cerevisiae. A 'bent DNA' fragment was located in one member of this E. histolytica repetitive DNA family.

Purification and characterization of high salt-soluble vicilin from mung bean (Vigna radiata).

We report a method for the purification of vicilin from mung bean (Vigna radiata) mainly on the basis of solubility of mung bean vicilin even in high salt. Mung bean vicilin remains in solution even after 90% relative saturation of ammonium sulphate. The resulting supernatant after dialysis was subjected to gel filtration (Sephadex G-150) to remove other contaminant polypeptides, and finally the protein was purified by DEAE cellulose chromatography. This purified fraction exhibited 3 bands on SDS-PAGE compared with vicilin from other legumes which exhibite more than 3 bands generally. The results raise the possibility that the presence of the two small polypeptides in vicilin preparations is the breakdown product of the major larger one of mol.wt. 52 K and that vicilin may be a tetramer of four subunits of Mr 52000. That the high salt-soluble protein containing 52 K subunit is vicilin has been determined by several criteria.

Isolation, characterization and sequencing of a novel repetitive DNA from the mung bean Vigna radiata.

A family of highly reiterated, small (approx. 300 bp) sequences has been identified in DNA of the mung bean Vigna radiata. The members are extensively interspersed throughout the chromosomes with some clustering. They also occur extrachromosomally. There is no tissue-specificity to the repeat family but it is highly species-specific. The repetitive DNA hybridizes to total RNA as well as to polyadenylated RNA isolated from germinated mung beans. It has analogy with the human AluI family in the mode of isolation, size, genomic distribution, copy number and transcribability though they do not share any sequence homology. A repetitive DNA clone was selected from a shotgun genomic library of mung bean DNA in pBR322. The average copy number of the cloned repeat is estimated to be 8 x 10(4) per haploid genome, and thus constitutes approx. 5% of the total mung bean genome. The genomic organization and transcription of the cloned repeat is reported. Sequencing of the cloned repetitive DNA reveals the presence of the number of direct and inverted repeats and some short palindromic sequences.

Ontogenically regulated expression of metallothionein and its messenger RNA in chick liver.

By electrophoretic and immunological assay the concentration of hepatic metallothionein in new born chick liver was found to be ontogenically modulated, reaching a peak accumulation per gram liver in fourth day of hatching and declining below the detection limit after second week postnatal. The protein was undetectable upto second week of incubation in egg-embryonic stage. The concentration of metallothionein mRNA shows drastic change during first few days after hatching. The greatest accumulation of metallothionein mRNA was detected in the one day new born chicks, which declined rapidly there after, and reduced to a barely detectable level. Metallothionein was also detected in the in vitro translated product of one day neonatal chick hepatic poly(A+) RNA by S-cysteine labelling and immunoprecipitation. The naturally occurring new-born chick liver metallothionein was found to be a zinc-metallothionein and the concentration of hepatic zinc in new-born chick was found to undergo drastic modulation during development, unlike some other chick tissues. Endogenous zinc ion mobilization can thus play a significant role in the developmental regulation of chick metallothionein expression.