B.B. Biswas

Isolation, purification and characterization of phytase from germinating mung beans☆

Abstract Phytase isolated from mung bean cotyledons was purified about 80-fold with a recovery of 28%. The enzyme is stable at 0°, has a pH optimum at 7·5 and optimal temperature of 57°. The energy of activation is approximately 8500 cal/mole between 37° and 57°. Inhibition by Pi has been found to be competitive, the Ki value being 0·40–0·43 × 10−3 M; the Km value with phytate is 0·65 × 10−3 M. Divalent cations are not required for activity. Lower members of inositol phosphates are better substrates, as shown by their Vmax and Km values. When subjected to polyacrylamide gel electrophoresis two bands have been resolved; one (major) corresponds to phytase and the other (minor) to phosphatase and pyrophosphatase activity. Filtration through Biogel P-200 partially resolves phytase from phosphatase and pyrophosphatase. The molecular weight of phytase is approximately 160,000.

Purification and mode of action of phytase from Phaseolus aureus

Phytase isolated from germinated mung bean cotyledons was further purified and migrated as a single protein band in polyacrylamide gel electrophoresis.

Biology of inositols and phosphoinositides

Chapter 1: Structure and Nomenclature of Inositol Phosphates, Phosphoinositides, and Glycosylphospatidylinositols, Pushpalatha P.N. Murthy Chapter 2: Inositol and Plant Cell Wall Polysaccharide Biogenesis Frank A. Loewus Chapter 3: Functional Genomics of Inositol Metabolism Javad Torabinejad and Glenda E. Gillaspy Chapter 4: Genetics of Inositol Polyphosphates Victor Raboy and David Bowen Chapter 5: Inositol in Bacteria and Archaea, Mary F. Roberts Chapter 6: Regulation of 1D-myo-inositol-3-phosphate synthase in yeast Lilia R. Nunez and Susan A. Henry Chapter 7: The structure and mechanism of myo-inositol-1-phosphate synthase, James H. Geiger and Xiangshu Jin Chapter 8: Phosphoinositide Metabolism: Towards an Understanding of Subcellular Signaling, Wendy F. Boss, Amanda J. Davis, Yang Ju Im, Rafaelo M. Galvn o and Imara Y. Perera Chapter 9: Cracking the green paradigm: Functional coding of phosphoinositide signals in plant stress responses, Laura Zonia and Teun Munnik Chapter 10: Inositols and their metabolites in abiotic and biotic stress responses, Teruaki Taji1,2, Seiji Takahashi3 and Kazuo Shinozaki1 Chapter 11: Inositol Phosphates and Phosphoinositides in Health and Disease, Yihui Shi, Abed N Azab, Morgan Thompson and Miriam L Greenberg Chapter 12: Mammalian Inositol 3-phosphate Synthase: Its Role in the Biosynthesis of Brain Inositol and its Clinical Use as a Psychoactive Agent * Latha K. Parthasarathy, Ratnam S. Seelan, Carmelita R. Tobias, Manuel F. Casanova, and Ranga N. Parthasarathy, Chapter 13: Evolutinary divergence of L-myo-inositol-1-phosphate synthase: Significance of a Core catalytic structure Krishnarup Ghosh Dastidar, Aparajita Chatterjee, Anirban Chatterjee and Arun Lahiri Majumder.

An inhibitor of phosphoinositol kinase from ungerminated mung bean seeds

Abstract A protein inhibitor of phosphoinositol kinase has been detected in the later stages of ripening of mung bean seeds. This has been isolated and purified from the ungerminated seeds. It migrated as a single protein band when subjected to polyacrylamide gel electrophoresis. The MW of the inhibitor is approx. 86 000. The phosphoinositol kinase inhibition has been found to be dependent on the protein concentration of the purified inhibitor. It seems that 1 molecule of the inhibitor is necessary to inhibit 1 molecule of enzyme. The nature of the inhibition has been found to be non-competitive, the K i of which is around 1·47 × 10 −6 M. The enzyme inhibitor complex dissociates on gel electrophoresis without any loss of enzyme activity.

Further characterization of phosphoinositol kinase isolated from germinating mung bean seeds

Abstract Phosphoinositol kinase isolated and purified from germinating mung bean seeds has been further characterized. The rate of phosphorylation varies with different inositol phosphates and this is consistent with the K m and V max for each of the substrates. The phosphate transfer from ATP has been found to be mediated by a phosphoprotein intermediate. In a particular step of the reaction the immediate product of the reaction has been found to be most inhibitory, other products being less or non-inhibitory. The inhibition has been found to be competitive in nature. The K i s have been found to range between 0.6 and 1 × 10 −4 M. ADP also inhibited non-competitively with respect to IP 5 . K i for this has been found to be 2.3 × 10 −4 M. The purified enzyme migrated as a single protein band on polyacrylamide gel electrophoresis. In the presence of sodium dodecyl sulphate it is dissociated into 3 subunits in the ratio 1 : 1 : 1. The MW of the three subunits are approx. 86 000, 56 000 and 35 000. The MW of the enzyme has been found to be approx. 177 000.

Two forms of phosphoinositol kinase from germinating mung bean seeds

Abstract Phosphoinositol kinase, the key enzyme responsible for the biosynthesis of higher inositol phosphates has been isolated from the cotyledons of mung beans germinated for 24 hr and has been resolved into two different forms, phosphoinositol kinase A and phosphoinositol kinase B. Both forms were purified to homogeneity and characterized. The Km values for ATP with phosphoinositol kinase A (1.78 × 10−4 M) and phosphoinositol kinase B (3.12 × 10 −5 M) showed that phosphoinositol kinase B had a greater affinity for ATP. ATP could be partially replaced as phosphate donor by UTP and phosphoenolpyruvate in the case of phosphoinositol kinase A but not in the case of phosphoinositol kinase B.

Further characterization of phytase from Phaseolus aureus

Abstract Phytase purified to homogeneity from germinated mungbean cotyledons was inhibited by EDTA although it did not show any absolute requirement for divalent cations. Sodium fluoride, sodium citrate, mercaptoethanol and p CMB also inhibit the phytase activity but l -phenylalanine has no effect on activity. The phytase has a low affinity for inositol monophosphate. The relative rate of dephosphorylation of myo-inositol-1 -phosphate and myo-inositol-5 phosphate by phytase is 6 and 18% respectively of that of myo-inositol-hexaphosphate. Mungbean phytase cannot cleave myo-inositol-2-phosphate, 1,2-cyclic inositol phosphate, Na-β-glycerophosphate or p -nitrophenylphosphate. The relative rates of hydrolysis of different isomers of inositol hexaphosphate are in the following order: myo-IP 6 , > neo-IP 6 > scyllo-IP 6 = d -chiro-IP 6 , > l -chiro-IP 6 . This enzyme seems to be most active with myo-inositol hexaphosphate.

Inositol hexaphosphate guanosine diphosphate phosphotransferase from Phaseolus aureus

Abstract Inositol hexaphosphate guanosine diphosphate phosphotransferase which transfers phosphate from inositol hexaphosphate to guanosine diphosphate, synthesizing guanosine triphosphate, has been isolated from germinating mung bean. A purification of 86-fold with 33% recovery has been obtained and the protein was made homogeneous after polyacrylamide gel electrophoresis. The MW of this enzyme was ca 92000. The optimal pH was 7·0 and Mn 2+ was stimulatory. Inositol hexaphosphate was the most active donor of the phosphoryl group (P) to GDP. Inositol penta- or tetra-phosphate (mixed) was partially active, but inositol pentaphosphate produced in this reaction did not act further as phosphate donor. The transfer of P from inositol hexaphosphate was mediated through a phosphoprotein. Polyphosphate (poly Pi), pyrophosphate (PPi) and orthophosphate (Pi) were inactive in this reaction. ADP, CDP and UDP could not substitute for GDP, neither could dGDP nor GMP accept P from inositolphosphate. GTP inhibited the reaction, but ATP did not interfere with the reaction. The products have been shown to be [GMP- 32 P] and inositol pentaphosphate by several criteria. The reaction is practically irreversible. K m values for GDP and inositol hexaphosphate were 1·1 × 10 −4 M and 1·6 × 10 −6 M respectively.

Induction of a high affinity binding site for auxin in Avena root membrane

Abstract A membrane preparation from Avena sativa root has been found to contain only one low-affinity IAA-binding site having a K d value of 8.4 ×

myo-Inositol Polyphosphates and Their Role in Cellular Metabolism

Life is characterized by complex organization, precise regulation, and colorful diversity. It is a biochemical system that is always far from thermodynamic equilibrium. The maintenance of such a state demands a constant expenditure of energy to maintain a unidirectional flow of metabolites. A network of anabolic and catabolic pathways operates in every living cell. It is well known that phosphate esters in living organisms are essential intermediates in metabolic transformations and these phosphorylated compounds are invariably associated with energy balance, which is fundamental to life processes. The sources of energy for living organisms are extremely diverse, but it is not clear why phosphate esters rather than the esters of other inorganic acids predominate in biological systems. It is probably significant that phosphate anhydrides combine high activation energies of nonenzymatic hydrolysis with large negative free energies of hydrolysis. This permits controlled enzymatic cleavage of the anhydride, rather than spontaneous hydrolysis. Lipmann (1951) has pointed out that acetic anhydride is hydrolyzed rapidly in neutral conditions, acetylphosphate is more stable, and pyrophosphate is resistant to hydrolysis at neutral pH. Phosphates are probably protected from hydroxyl attack by their negative charge. The discussion in this chapter is centered around a biologically important phosphocompound that was discovered as early as 1872 by Pfeffer (1872). This was subsequently identified as a salt of phytic acid (or more correctly myo-inositol hexakisphosphate), which is the major phosphorus constituent of cereal grain (Williams, 1970).