L-myo-Inositol-1-phosphate synthase from bovine testis: purification to homogeneity and partial characterization

Abstract

L-myo-Inositol-1-phosphate synthase has been purified to homogeneity from bovine testis by (NH4)2SO4 precipitation on Celite followed by reverse (NH4)2SO4 gradient elution, DEAE chromatography, gel filtration, and hydroxylapatite chromatography. The enzyme is then pure by the criteria of elution profile from the hydroxylapatite, electrophoresis, and sedimentation properties. We find no overall (gluconeogenic) reversibility of the enzyme using 6 mM DL-myo-inositol-1-P. The first three steps of the reaction are reversible as determined by uptake of isotope from a D2O incubation medium into the 6 position of D-glucose-6-P. Thus, substrate binding, dehydrogenation, and proton removal prior to the aldol cyclization are all reversible steps. The enzyme is less than 5% NAD+ independent and is not inhibited by substrate or product (5 mM D-glucose-6-P or 0.8 mM DL-myo-inositol-1-P). The enzyme is twofold stimulated by either 50 mM NH4+ or 50 mM K+; the activation by these ions is not additive. Sodium ions inhibit the enzyme by 78% at 153 mM. The effect of sodium and potassium is not on the Km of D-glucose-6-P but on Vmax. We propose that K+ activates the enzyme by stabilizing a carbanion intermediate. Ethanol stimulates the enzyme 2-fold and 2.5-fold with added K+. The effect of ethanol appears to be via lowering of the D-glucose-6-P Km. In the presence of ethanol the effect of salt on Vmax disappears.

TheArabidopsis thaliana Myo-Inositol 1-Phosphate Synthase1 Gene Is Required forMyo-inositol Synthesis and Suppression of Cell Death

“…The catalytic activity of the recombinant MIPS proteins was measured under steady state conditions (see Supplemental Figure 8B online; Table 1). It was determined that MIPS activity was enhanced by 10% glycerol and 20 mM NH 4 Cl, as previously observed for characterized MIPS enzymes (Mauck et al, 1980;RayChaudhuri et al, 1997;Ju et al, 2004). Therefore, NH 4 Cl and glycerol were included in our standard assay buffer.…”

Sodium- and energy-dependent uptake of myo-inositol by rabbit peripheral nerve. Competitive inhibition by glucose and lack of an insulin effect.

“…Sodium-dependent endoneurial myoinositol transport resembles saturable uptake systems in other tissues thought to concentrate myo-inositol, e.g., brain (40), lens (25,41), choroid plexus (36,42), ciliary body (24), small intestine (22), and kidney tubules (21, 22,43). Taken together, these observations suggest that carrier-mediated transport contributes to the maintenance of high myo-inositol concentrations in some endoneurial components(s).…”

“…Neither myo-inositol pool size nor synthetic or metabolic flux is sufficiently well characterized in peripheral nerve to permit meaningful quantitative comparison with sodium-dependent myoinositol uptake (17,18,26,42,44,45). Moreover, endoneurial fluid myo-inositol concentration, which may possibly differ from that of plasma (46,47), is unknown.…”

L-myo-InositoL-1- phosphate synthase from bryophytes: purification and characterization of the enzyme from Lunularia cruciata (L.) Dum

“…The de-novo synthesis of myo-inositol takes place by the conversion of D-glucose-6-phosphate (G-6-P) to L-myo-inositol-1-phosphate (I-1-P) by the enzyme L-myo-inositol-1-phosphate synthase (MIPS; EC 5.5.1.4) which is subsequently dephosphorylated by a specific Mg +2 dependent inositol monophosphatase to myo-inositol. The MIPS reaction has been reported from archea (Chen et al 2000); bacteria Mande, 1999, 2000); protozoa (Lohia et al 1999); animals (Maeda and Eisenberg, 1980;Mauck et al, 1980;Biswas et al, 1981); humans (Adhikari and Majumder, 1988) and plants. Among plants the occurrence of MIPS has been described and characterized from algae (Dasgupta et al, 1984;RayChaudhuri et al, 1997); fungi (Donahue and Henry, 1981a,b;Escamilla et al 1982;Dasgupta et al 1984); pteridophytes (Chhetri et al 2005(Chhetri et al , 2006a; gymnosperm (Gumber et al, 1984;Chhetri and Chiu, 2004) and angiosperm (Loewus and Loewus, 1971;Johnson and Sussex, 1995;Johnson and Wang, 1996;RayChaudhuri et al, 1997).…”