Purification of myo-Inositol 1-Phosphate Synthase from Rice Cell Culture by Affinity Chromatography

Abstract

myo-Inositol 1-phosphate synthase (EC 5.5.1.4) is the enzyme which catalyzes the synthesis of the precursor for the myo-inositol oxidation pathway. Rice callus grown in suspension culture provides a good source of plant enzyme. Use has been made of a noncompetitive inhibitor to prepare an affinity column for this enzyme. With this column, the enzyme from rice callus has been purified 1500-fold in a single step, about 9000-fold over-all, to a specific activity of 0.078 units per milligram of protein. This is an order of magnitude greater than previous purifications of the plant enzyme.MI4 is an intermediate in one of the two pathways by which the carbon skeletons of cell wall uronic acids and pentoses are derived from D-glucose (21). MI 1-phosphate synthase5 (EC 5.5.1.4) catalyzes conversion of glucose to MI via their phosphorylated intermediates. In plants, it provides the immediate precursor of the MI oxidation pathway (22)(23)(24). If this enzyme can be purified to homogeneity, then significant experiments involving enzymatic regulation and control of the endogenous pool of MI can be designed.Synthase activity has been observed in all plant material examined so far (16,17,19,25,29,35). It has also been found in fungi (32), yeast (5), rat testes (9), bovine testes (36), chicken erythrocytes (35), rat mammary gland (3, 4, 31) and rat liver and plasma (3). Enzyme from plant sources studied in this laboratory offers a distinct advantage over the others in that it is extremely stable. No loss of activity was noted after 1 week at 4 C or 8 months at -20 C (28).In previous attempts to purify the synthase from Acer pseudoplatanus, ammonium sulfate fractionation and chromatography on DEAE cellulose and Sephadex G-200 columns were used (25). This gave 500-to 1000-fold purification. At least seven electrophoretically distinct protein bands remained in the final preparation. At this point, insufficient material was available for

L-myo-lnositol 1-Phosphate Synthase from Plant Sources (Characteristics of the Chloroplastic and Cytosolic Enzymes)

“…Such results are comparable to those obtained for I-1-P synthases from pine pollen (Gumber et al, 1984), yeast (Chen and Charalampous, 1964), and the rat mammary gland (Naccarato et al, 1974). Inositol inhibited the activity of both cytosolic and chloroplastic I-1-P synthases, as reported earlier for yeast (Hirsch and Henry, 1986), Arabidopsis (Johnson and Sussex, 1995;Johnson and Wang, 1996), and O. safiva (Funkhouser and Loewus, 1975). This possible regulatory role of inositol in I-1-P synthase activities needs further investigation.…”

Myo-inositol content in pteridophytes and the isolation and characterization of L-myo-inositol-1-phosphate synthase from Diplopterygium glaucum

“…Omission of NAD + resulted in a loss of enzyme activity of about 67 %. In a similar study it was found that when NAD + was not added to the rice enzyme, its activity was reduced to 55 % of the maximum (Funkhouser and Loewus, 1975).…”

Comparative Proteomic and Physiological Analysis Reveals the Variation Mechanisms of Leaf Coloration and Carbon Fixation in a Xantha Mutant of Ginkgo biloba L.

“…Myo-inositol-1-phosphate synthase (MIPS, spot 2133) also declined in abundance. MIPS catalyses the conversion of d -glucose 6-phosphate to 1- l -myo-inositol-1-phosphate (MIP), providing the immediate precursor to myo-inositol, which is an intermediate carbon skeleton of cell wall uronic acids and pentoses derived from d -glucose [58]. MIPS has been found to play a key role in the biosynthesis of inositol and inositol phosphate and to be important in plant development and environmental stress tolerance [59].…”