Cycloaldolase (NAD-Dependent) from Acer Pseudoplatanus L. Cell Cultures
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Plant Physiol. (1971) 48, 255-260 The Isolation and Characterization of D-Glucose 6-Phosphate Cycloaldolase (NAD-Dependent) from Acer pseudoplatanus L. Cell Cultures ITS OCCURRENCE IN PLANTS' Received for publication March 4, 1971 M ARY W. LOEWUS AND F. LOEWUS Department of Biology, State University of New York at Buffalo, Buffalo, New York 14214 ABSTRACT labile than the phosphatase, thus permitting the latter to be studied independently of the former (13). Chen and Charalam- A soluble enzyme system from suspension cultures of Acer pous (7) have achieved separation of these activities on pseudoplatanus L. converts D-glucose 6-phosphate to myoinosi- DEAE'-cellulose using the yeast system. Partial purification of tol. A Mg'-dependent phosphatase, present in the crude ex- the cyclizing enzyme and related studies on cell-free systems tract, hydrolyzes the product of the cyclization, myoinositol from rat testis, yeast, and Neurospora have generated a wealth monophosphate, to free myoinositol. Further purification of the of information regarding the mechanism of action (3, 8, 13, enzyme system by precipitation with (NEL)2SO& followed by 16, 27, 31), physical and chemical properties of the soluble diethylaminoethyl cellulose chromatography eliminates the system (2, 5, 6, 12), and identity of substrate, intermediates, phosphatase and makes it necessary to add alkaline phospha- and product (4, 6, 13, 15). tase to the reaction mixture in order to assay for free myo- In plants, cyclization of -glucose 6-P to myoinositol takes inositol. Gel filtration on Sephadex G-200 increases the specific on added significance in light of the observation that oxidative activity of the cycloaldolase to 8.8 X 10-' units per milligram cleavage of myoinositol to n-glucuronate and conversion of protein (1 unit = 1 micromole of myoinositol formed per min. the latter to uronate and pentose components of cell wall poly- ute). The cycloaldolase has an absolute requirement for nico- saccharides provides an alternative route from 1-glucose to tinamide adenine dinucleotide and a maximum activity at pH 8 products of D-glucuronate metabolism that bypasses the oxi- with 0.1 mM nicotinamide adenine dinucleotide. The reaction dation of UDP-D-glucose (22). Further evidence of the preva- rate is linear for 2.5 hours when D-glucose 6-phosphate is below lence of the cyclizing enzyme in plants and a comparative 4 mMand has a Km of 1.77 mm. The diethylaminoethyl cellu- study of its properties and mechanism with those occurring lose-purified enzyme is stable for 6 to 8 weeks in the frozen in yeast and rat testis is desirable. We have conducted such a state. survey in the search for a dependable plant source of this enzyme. On the basis of this search we have chosen to iso- late and purify the cycloaldolase from suspension cell cultures of Acer pseudoplantanus L. MATERIAL AND METHODS In 1962 Loewus and Kelly (23) demonstrated the cycliza- Herbaceous plants were grown under green house con- tion of 1-glucose to myoinositol in detached parsley leaves. ditions. Leaves were harvested just prior to use. Since then, the enzyme system catalyzing this conversion has A sample of pollen (Lilium longiflorum, cv. Croft) that had been isolated from several sources. 1-Glucose 6-P is the actual been held in storage at -20 C for several months was freed of substrate (4). Cell-free preparations from rat testis (14) and "pollen kitt," a gummy residue adhering to pollen grains, by Candida utilis (4) revealed that D-glucose 6-P is converted to rinsing in medium prepared according to Dickinson (11). myoinositol in two enzymatic steps; first, cyclization of the Washed grains were divided into two portions, one which was sugar phosphate to 1-L-myoinositol 1-P (7, 15), then hydroly- homogenized immediately and the other after incubation for sis of 1-L-myoinositol 1-P to myoinositol and P1 (6, 13). The 1 hr at 25 C in fresh medium. Pollen tubes were not micro- crude systems required both NAD+ and Mge for activity, but scopically visible at the end of 1 hr, but prolonged incubation resolution of the cycloaldolase from the phosphatase activity (6-8 hr) of separate aliquots showed about 70% germination. revealed that only NAD+ is needed by the cyclizing enzyme, Corn seeds (Zea mays, Agway hybrid M-4) were soaked for and the Mg'+ requirement is associated with the phosphatase. 15 min in 50% Clorox (2.5% hypochlorite), rinsed, and ger- Similar systems have been found in Neurospora, Oryza sativa, minated at 28 C in the dark in sterile Petri dishes containing Phaseolus vulgaris, and Sinapis alba (19, 28, 30). a 3 mm layer of 0.5% lonagar. Three-day-old seedlings were In rat testis preparations, the cycloaldolase is more heat separated from cotyledons and homogenized. Marine algae from the vicinity of Woods Hole, Massachu- setts, were harvested just before assay. 1This investigation was supported by Grant GM-12422 from the Division of Research Grants and Fellowships, National Institutes of Health, United States Public Health Service. 2Abbreviation: DEAE: diethylaminoethyl. 255 256 LOEWUS AND LOEWUS Plant Physiol. Vol. 48, 1971 Acer pseudoplatanus L. cell cultures were obtained from a 1 cm from the edge of a 20- X 20-cm thin layer cellulose plate line of cells that had been maintained continuously in the au- (Brinkman MN 300). Two furrows through the cellulose layer thors' laboratory over the past 7 years. The specific clone used were drawn parallel to the opposite two edges, about 2.5 cm in this study had been grown on Murashige and Skoog's me- from the edges. Standards were applied to the origins of each dium with 0.1% myoinositol (22) throughout that period. Sus- one-dimensional strip formed by furrowing. Each plate was pension cultures were prepared by transferring a 2 to 3 g developed in the first direction with ethyl acetate-pyridine- mass of cells from agar medium to 100 ml of fresh liquid water (10:6:5, v/v) and in the second with methanol-formic medium in a 300-ml DeLong flask (Belco Glass, Inc. No. 599). acid-water (80:15:5, v/v). The myoinositol region of the two- After 3 weeks of continuous gyrorotatory shaking (200 rpm), dimensional separation, located by means of standards run at the contents (30-40 g fresh weight of cells) were transferred the margins, was scraped off the plate and eluted from the to 1 liter of fresh medium in a low form culture flask (Corning cellulose powder by stirring with a Teflon-coated magnetic bar No. 4422) and gently agitated on a reciprocating shaker for 3 in 1 ml of water for 1 hr. Samples of the supernatant were as- weeks at 28 C. Yields averaged about 125 to 150 g of cells sayed for radioactivity. Controls involving authentic radio- fresh weight per flask. active myoinositol gave 98 to 100% recovery by this method. Preparation of Soluble Enzyme for Assay. Tissues were Purification of Enzyme from A. pseudoplatanus Cells. The homogenized for 2 min with 20 mm tris-HCl buffer, pH 8, 35 to 50% saturated (NH4)2SO4 fraction obtained from cen- containing 0.5 mM GSH (1:1, w/v) in a Sorvall Omnimixer. trifuged disrupted cells retained about 70% of the activity Acer cells, suspended in the same buffer, were disrupted by present in the extract. Widening this fraction did not improve release from a Parr cell disruption vessel (Parr Inst. Co. model yields. Overnight dialysis resulted in an unexplainable loss of No. 4635) at 1000 psi of N2 after holding the cells at that 50% of the activity present in the (NH4)2SO4 fraction. Subse- pressure forS min. Cell breakage was better than 90%. quent dialyses did not influence the activity beyond the effect Exploratory experiments revealed that the relatively high noted here. rate of centrifugation used by others (3, 4, 13, 19) was not After dialysis to remove (NH4)2S04, the enzyme was put on necessary in the preparation of plant enzyme. Homogenates a DEAE-cellulose column (I X 30 cm, Cellex D, BioRad Labs) spun at 15,000 rpm for 90 min (4 C) in a Sorvall RC-2 cen- that had been previously equilibrated with buffer containing trifuge gave solutions with protein concentration and enzyme 0.5 mM GSH. The column was washed with additional buffer assay similar to ones obtained at the higher speeds. After re- (100 ml) and eluted with a gradient that had been prepared moving material that precipitated when solid (NH,)2SO4 was by introducing into a solution of 20 mm tris-HCl, pH 8, con- added to bring the solution to 30% saturation, further taining 0.5 mM GSH (200 ml) a second solution (100 ml) of the (NH4)2SO4 was added until the solution was 50% saturated same buffer containing 0.625M NaCl. In more recent prepara- (90% in some survey studies). The protein that salted out was tions the enzyme was separated as adequately using a linear collected by centrifugation, redissolved in tris-HCl buffer dia- gradient in which 0.2M NaCl in 20 mm tris-HCl buffer. pH 8 lyzed overnight against 20 mm tris-HCl, pH 8, omitting GSH, (200 ml), was added to the same buffer (200 ml). and assayed for myoinositol synthesizing ability. Although Further purification was obtained by an additional step of GSH was required in the initial extraction for recovery of gel filtration on a column of Sephadex G-200 (0.9 x 90 cm) maximum activity, it has been shown to inhibit under assay that had been equilibrated, prior to loading with 50 mM tris- conditions (5); therefore, it was removed by dialysis prior to HCI, pH 8.