B. B. Biswas

Purification and characterization of myo-Inositol hexaphosphate-adenosine diphosphate phosphotransferase from Phaseolus aureus

Abstract myo-Inositol hexaphosphate adenosine diphosphate phosphotransferase transfers phosphate from myo-inositol hexaphosphate to adenosine diphosphate to synthesize adenosine triphosphate. This enzyme has been isolated and purified from ungerminated mungbean seeds and found to be different from guanosine diphosphate phosphotransferase. A purification of about 200-fold with 15% recovery has been obtained. The optimal pH of the reaction is 7.0 and is dependent on the presence of a divalent cation, i.e., Mg2+ and Mn2+. The Km value for myo-inositol hexaphosphate has been found to be 0.41 × 10−4 m and V is 90.0 nmol of Pi transferred per milligram of protein per 20 min. Km for ADP is 0.88 × 10-4 m and V is 83.3 nmol of phosphorus transferred to ADP per milligram of protein per 20 min. The ADP phosphotransferase reaction is reversible to the extent of about 50% of the forward reaction. dADP is partly effective as an acceptor but other ribonucleoside mono- and diphosphates cannot substitute for ADP. The products ATP and myo-inositol pentaphosphate have been confirmed by several criteria. It has also been shown that this enzyme transfers phosphate only from a specific phosphoryl group (C-2 position) of myo-inositol hexaphosphate for the synthesis of ATP and 1,3,4,5,6-myo-inositol pentaphosphate or pentakis (dihydrogen phosphate).

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.

myo-inositol phosphates, phosphoinositides, and signal transduction

History of Phosphoinositide Research L.E. Hokin Phosphoinositide and Synaptic Transmission J.N. Hawthorne Control of the Ca2+ Release Induced by Myoinositol Trisphosphate and the Implication in Signal Transduction L. Missiaen, et al. Regulation of the Actin Cytoskeleton by Inositol Phospholipid Pathways D.E. Kandzari, P.J. Goldschmidt-Clermont Protein Phosphorylation and Signal Transduction S. Barik Structural and Functional Roles of Glycosylphosphoinositides A.R. Saltiel InsP5 and InsP6 Metabolism Adds Versatility to the Actions of Inositol Polyphosphates: Novel Effects upon Ion Channels and Vesicle Traffic S.B. Shears Inositol Phosphates and Their Metabolism in Plants P.P.N. Murthy Genetics of Myoinositol Phosphate Synthesis and Accumulation V. Raboy Metabolism of Myoinositol Phosphates and Alternative Pathway in Generation of Myoinositol Trisphosphate Involved in Calcium Mobilization in Plants S. Biswas, B.B. Biswas Phosphoinositide Turnover and Its Role in Plant Signal G.G. Cote, et al. Light-induced Signal Transduction Pathway Involving Inositol Phosphates S.K. Sopory, M.R. Chandok Synthesis, Separation and Identification of Different Inositol Phosphates C. Schultz, et al. Index.

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 μM with Ins(1,4,5)P3 and 0.68 μM 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.

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.

Metabolism of myo-Inositol Phosphates and the Alternative Pathway in Generation of myo-Inositol Trisphosphate Involved in Calcium Mobilization in Plants

The myo-inositol polyphosphates and their role in cellular metabolism and signal transduction processes in plants have now attracted the attention of scientists in growing numbers. Phytic acid, myo-inositol-1,2,3,4,5,6-hexakisphosphate has long been known as the storage form of phosphorus in seeds (see Cosgrove, 1980). The calcium and magnesium salt of phytic acid is also known as phytin. Of the total phosphorus, 50–80% has been found to be associated with phytin in different seeds. Although the composition of phytic acid has been known for more than 100 years, many problems concerning its metabolism and functions are not fully solved (see Loewus and Loewus, 1983; B. Biswas et al., 1984; Raboy, 1990; Drobak, 1992). What is apparent is that plant cells representing different tissue types synthesize phytic acid both to sequester phosphorus and also to chelate different metallic cations such as Ca2+ and others. Several reviews have dealt with the occurrence, chemistry, and nutritional implications of phytates in legumes and cereals (see Reddy et al., 1982; B. Biswas et al., 1984). Phytate in general rapidly accumulates during the development of seeds and disappears during germination of seeds.

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.

Myoinositol Phosphates as Implicated in Metabolic Signaling and Calcium Homeostasis in Plants

Myoinositol trisphosphate, particularly Ins(l,4,5)P3 is the intracellular messenger that mediates the effects of many cell surface receptors on the intracellular Ca2+ stores (Berridge and Irvine 1989). Although specific assays have identified high affinity Ins(l,4,5)P3-binding sites in many animal tissues (Supattapone et al 1988, Mignery and Sudhof 1990), these have not been convincingly shown in all cases to be the receptors that mediate Ca2+ mobilization, nor it is clear whether the binding sites are different from one tissue to another. Myoinositol trisphosphate receptor from plants has also been reported and characterized (Biswas et al 1995).