The Isolation and Characterization of d-Glucose 6-Phosphate Cycloaldolase (NAD-Dependent) from Acer pseudoplatanus L. Cell Cultures

Abstract

York at Buffalo, Buffalo, New York 14214 labile than the phosphatase, thus permitting the latter to be studied independently of the former (13). Chen and Charalampous (7) have achieved separation of these activities on DEAE'-cellulose using the yeast system. Partial purification of the cyclizing enzyme and related studies on cell-free systems from rat testis, yeast, and Neurospora have generated a wealth of information regarding the mechanism of action (3,8,13,16,27,31), physical and chemical properties of the soluble system (2, 5, 6, 12), and identity of substrate, intermediates, and product (4,6,13,15).In plants, cyclization of -glucose 6-P to myoinositol takes on added significance in light of the observation that oxidative cleavage of myoinositol to n-glucuronate and conversion of the latter to uronate and pentose components of cell wall polysaccharides provides an alternative route from 1-glucose to products of D-glucuronate metabolism that bypasses the oxidation of UDP-D-glucose (22). Further evidence of the prevalence of the cyclizing enzyme in plants and a comparative study of its properties and mechanism with those occurring in yeast and rat testis is desirable. We have conducted such a survey in the search for a dependable plant source of this enzyme. On the basis of this search we have chosen to isolate and purify the cycloaldolase from suspension cell cultures of Acer pseudoplantanus L.In 1962 Loewus and Kelly (23) demonstrated the cyclization of 1-glucose to myoinositol in detached parsley leaves. Since then, the enzyme system catalyzing this conversion has been isolated from several sources. 1-Glucose 6-P is the actual substrate (4). Cell-free preparations from rat testis (14) and Candida utilis (4) revealed that D-glucose 6-P is converted to myoinositol in two enzymatic steps; first, cyclization of the sugar phosphate to 1-L-myoinositol 1-P (7, 15), then hydrolysis of 1-L-myoinositol 1-P to myoinositol and P1 (6, 13). The crude systems required both NAD+ and Mge for activity, but resolution of the cycloaldolase from the phosphatase activity revealed that only NAD+ is needed by the cyclizing enzyme, and the Mg'+ requirement is associated with the phosphatase. Similar systems have been found in Neurospora, Oryza sativa, Phaseolus vulgaris, and Sinapis alba (19,28,30).In rat testis preparations, the cycloaldolase is more heat MATERIAL AND METHODSHerbaceous plants were grown under green house conditions. Leaves were harvested just prior to use.A sample of pollen (Lilium longiflorum, cv. Croft) that had been held in storage at -20 C for several months was freed of "pollen kitt," a gummy residue adhering to pollen grains, by rinsing in medium prepared according to Dickinson (11). Washed grains were divided into two portions, one which was homogenized immediately and the other after incubation for 1 hr at 25 C in fresh medium. Pollen tubes were not microscopically visible at the end of 1 hr, but prolonged incubation (6-8 hr) of separate aliquots showed about 70% germination.Corn seeds (Zea mays, Agway h...

Differentially Expressed Forms of 1-L- -Inositol-1-Phosphate Synthase (EC) in

“…D- [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]Glucose 6-phosphate (specific activity, 60.3 mCi/mmol) and [1,[2][3] H]myo-inositol (specific activity, 370 -740 GBq/mmol) were obtained from DuPont NEN. Glucose 6-phosphate, bacterial alkaline phosphatase, and phosphate standard were purchased from Sigma.…”

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

“…The rate of enzyme reaction proceeded linearly up to 90 min with G-6-P as the substrate. An earlier study with Acer pseudoplatanus showed time linearity up to 150 min with a G-6-P concentration under 4 mM (Loewus and Loewus, 1971) Effect of temperature and pH: The enzyme was highly active in the temperature range of 20-40ºC with 30ºC as the temperature maximum. Temperature maxima of 35ºC were found for the cytosolic enzymes from both higher and primitive plants (RayChoudhury et al, 1997).…”

“…It is formed by the conversion of DGlucose-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) and is subsequently dephosphorylated by inositol monophosphatase (EC: 3.1.3.25) to myo-inositol (Lohia et al, 1999). The MIPS reaction has been reported for archea (Chen et al, 2000), bacteria (Bachhawat and Mande, 1999;, protozoa (Lohia et al, 1999), lower plants (Donahue and Henry, 1981a;1981b;Escamilla et al, 1982;Dasgupta et al, 1984;RayChoudhury et al, 1997;Benaroya et al, 2004;Chhetri et al, 2005), higher plants (Loewus and Loewus, 1971;Loewus et al, 1978;Ogunyemi et al, 1978;Adhikari and Majumder, 1983;Johnson and Sussex, 1995;Johnson and Wang, 1996;Chun et al, 2003) and animals (Pittner and Hoffmann-Ostenhof, 1976;1979;Maeda and Eisenberg Jr., 1980;Adhikari and Majumder, 1988;Chiu et al, 2003). The present study reports the occurrences of free myo-inositol in different pteridophytes.…”

Occurrence of myo-inositol-1-phosphate phosphatase in pteridophytes: characteristics of the enzyme from the reproductive pinnules of Dryopteris filix-mas (L.) Schott

“…Generation of free myo-inositol may be blocked if MIPP is not active. The MIPS reaction has been reported to occur in a large number of living systems namely archea (Chen et al, 2000), bacteria (Bachhawat and Mande, 2000), protozoa (Lohia et al, 1999), lower plants (Donahue and Henry, 1981a,b;Escamilla et al, 1982;Dasgupta et al, 1984;RayChoudhury et al, 1997;Benaroya et al, 2004, Chhetri et al, 2005, higher plants (Loewus and Loewus, 1971;Loewus et al, 1978;Ogunyemi et al, 1978;Adhikari et al, 1987;Johnson and Sussex, 1995;Johnson and Wang, 1996;Chun et al, 2003), and animals (Pittner and Hoffmann-Ostenhof, 1976;Maeda and Eisenberg Jr., 1980;Majumder, 1983, 1988;Chiu et al, 2003). In pteridophytes, Benaroya et al (2004) and Chettri et al (2005Chettri et al ( , 2006 have recently documented the occurrence and characterization of L-myo-inositol-1-phosphate synthase.…”