Susweta 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).

A NOVEL METABOLIC CYCLE INVOLVING myo -INOSITOL PHOSPHATES DURING FORMATION AND GERMINATION OF SEEDS

SUMMARY: myo-Inositol-hexakis-phosphate (IP 6 ) has been found to be synthesized during formation of mung bean seeds from glucose-6-phosphate through myo-inositol-1-phosphate synthase, phosphoinositol kinase and IP 6 -phosphotransferase system. During germination of mung bean seeds a portion of IP 6 appeared to be converted by IP 6 -ADP-phosphotransferase and phytase to myo-inositol-1-phosphate. Through the action of myo-inositol-1-phosphate dehydrogenase and hitherto unknown steps myo-inositol-1-phosphate has been converted back to glucose-6-phosphate. Thus a novel metabolic cycle, involving glucose-6-phosphate and myo-inositol phosphates during formation and germination of seeds has been proposed.

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.

An ATPase in sensitive plant Mimosa pudica. I. Purification and characterization.

Abstract Leaflets from the primary, secondary, and tertiary pulvini of Mimosa pudica contain a highly active ATPase. The level of this enzyme is highest in actively responding healthy fresh leaflets; no activity can be detected in the insensitive plant extracts. This enzyme is dependent on cation for its activity, the optimal concentration of Mg2+ being 5 × 10−3 m . It has been purified approximately 60-fold. The optimal activity of the enzyme is pH 5.5. The enzyme hydrolyzes dATP and ITP at a much slower rate than ATP. Other nucleoside triphosphates and phosphocompounds are not hydrolyzed. In the final step of purification five distinct protein fractions are obtained after passage through a DEAE cellulose column, and fraction III exhibits the ATPase activity and shows a single peak of 4.3 S by sedimentation analysis. This activity is increased several-fold after the addition of fraction IV and V, the latter being most effective. This ATPase is not inhibited by pCMB, iodoacetamide, and dinitrophenol. The crude enzyme preparation exhibits a viscosity change upon the addition of ATP; however, this viscosity change is not observed with the purified enzyme (fraction III) until fraction IV and V are added. The presence of ATPase in the sensitive leaflets of Mimosa, its increased activity and the viscosity change in presence of ATP with other protein fractions suggest that the ATPase is involved in the rapid movement of Mimosa leaflets.

Effect of polyribonucleotides on amino acid incorporation by chloroplast ribosomes.

Partikel mit Ribonucleoprotein aus Spinatehloroplasten wurden isoliert und als wahrscheinliche Loci der Proteinsynthese bestimmt. Nach Chloroplastenaufschluss hemmte die Granafraktion mit hoher Ribonucleaseaktivität den Aminosäureneinbau. Der Einbau von Phenylalanin wird durch Polyuridylsäure gefördert und durch Polyadenylsäure nur nach Vorbehandlung der Ribosomen-Aggregate mit Ribonuclease gehemmt.

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.

Tubulin from Mimosa pudica and its involvement in leaf movement

Abstract The sensitive plant Mimosa pudica is made insensitive by a brief treatment with colchicine. A high concentration of colchicine binding protein is present in the fresh actively moving leaves of M. pudica . This protein was partially characterized and compared with the animal brain tubulin. This colchicine binding activity is very low in the insensitive variety of Mimosa , namely Mimosa rubricaulis .

MIMOSA PUDICA APYRASE REQUIRES POLYSACCHARIDE AND CA2+ FOR THE ACTIVITY

Mimosa pudica Linn leaves with pulvini contain unique isoforms (I and II) of apyrase enzyme (EC 3.6.1.5). The activity of isoform I depends on divalent cation Mn2+. This isoform is associated noncovalently with the polysaccharide, containing mainly of galactose and arabinose sugars. The apparent molecular mass of these 2 isoforms are 36 and 34 Kd respectively. The association of the polysaccharide with the isoform I has been found to be Ca2+ dependent which is endogenously present in this isoform. Removal of Ca2+ and polysaccharide from the enzyme (isoform I) leads to an inactivation. The enzyme activity can be restored when both Ca2+ and endogenous polysaccharide fraction were added at an optimal molar ratio of Ca2+:protein of 7:1. The endogenous polysaccharide can be replaced by the standard arabinogalactan. No other sugar or polysaccharide except the arabinogalactan can restore the apyrase activity. Calcium mediates a conformational change in the protein which helps in association of polysaccharide as evidenced from fluorometric and far UV-CD studies to restore the enzymic activity. Neither any interaction of the polysaccharide with the protein is detected in absence of Ca2+ nor the enzyme activity could be recovered under such condition.

Protein A-activated rat splenic lymphocyte proliferation involves tyrosine kinase-phospholipase C-protein kinase C pathway.

Abstract Protein A (PA) of Staphylococcus aureus was long been known for its affinity towards the Fc domain of immunoglobulin G. It is now well established that PA is a potent biological response modifier showing simultaneously antitumor, antitoxic, and anticarcinogenic properties. This bacterial protein was also observed to stimulate production of cytokines. But the molecular mechanism(s) of immunocyte activation by PA still remained essentially unknown. In this report, we demonstrate a hitherto undescribed role of PA as a signal inducer in rat splenic lymphocytes. Our studies describe that PA induces transition of G0/G1 to S and G2/M phases of cell cycle, thus ultimately stimulating splenic lymphocyte proliferation. It has also been revealed that PA binds to rat splenic lymphocytes in a dose dependent manner and stimulates proliferation via tyrosine kinase-phospholipase C (PLC)-Ca2+-dependent protein kinase C (PKC) pathway. These observations will be of valid help in correlating the immunostimulatory activities of PA with the molecular mechanism(s) of its action.

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.