Plant Physiol. (1971) 48, 255-260

The Isolation and Characterization of D- 6- Cycloaldolase (NAD-Dependent) from Acer pseudoplatanus L. Cultures

ITS OCCURRENCE IN '

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 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 . 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 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 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 , 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 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 (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 -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. Comparison of the molecular size of purified cyclo- testing. Addition of Polyclar AT (a polyvinylpyrrolidone prod- aldolase with those of catalase (Worthington Biochem. Corp.), uct of General Aniline and Film Corp.),1 g/10 g tissue, fresh yeast dehydrogenase (Worthington Biochem. Corp.), weight, did not improve the yield or activity of enzyme from and bovine serum albumin (Nutritional Biochem. Corp.) was leaves or marine algae. The 2-min heating step at 60 C sug- made in this step. Blue Dextran 2000 (Pharmacia Fine Chem.) gested by Eisenberg(13), preliminary to addition of (NH)2SO04, wasused to determine the void volume. Proteins were applied appeared to be unnecessary with plant extracts and was in 2-ml volumes and fractions of 2.5 ml were collected. The omitted. elution was followed spectrophotometrically at 280 nm. The Enzyme Assay. The assay used by Chen and Charalampous procedure follows that described by Determann and Michel (5) was adopted for this study but modified by an additional (10). All manipulations were performed at 4 C. step involving two-dimensional thin layer cellulose chroma- Protein was determined by the method of Lowry as de- tography of the product, myoinositol. The assay consisted of scribed by Layne (20). Measurements of radioactivity were 0.2 to 0.4 mg protein (enzyme), 75,tmoles tris-HCl, pH 8, made on samples that had been diluted with water (0.5 ml) and 1.5,umoles NAD+, ,umoles2 MgCl2, 18,tmoles NH,CI, 9 then combined with liquid scintillation fluid (15 ml of a mix- 1-moles D-glucose-1-14C 6-P (2 x 100 cpm) in a total volume of ture of naphthalene (100 g) PPO (7 g) and dimethyl POPOP 1.5 ml. MgC12 was omitted in analyzes for cycloaldolase ac- (0.3 g) in p-dioxane to make I ). Samples were counted in a tivity alone. Samples were incubated for 2.5 hr at 30 C. The Packard TriCarb Spectrometer, Model 3324. reaction was stopped by plunging the reaction tubes into boiling water for 2 min. To the cooled reaction mixture was RESULTS added, unless otherwise stated, 0.1 mg of alkaline phosphatase (Worthington Biochem. Corp.), followed by incubation for Several species of plants were examined for the myoinositol 2 hr at 37 C. This reaction was stopped by placing the tubes synthesizing system, and the results of this survey are listed in in boiling water. An equal volume of 0.15M Ba(OH)2 was TableI. D-Glucose 6-P cycloaldolase is found in many higher added, and the mixture was heated in a boiling water bath for plant tissues as well as two algae, confirming its relatively gen- 20 min. The supernatant and two 0.5-ml water washes of the eral occurrence, suspected from previous work. The specific precipitate were combined and passed through an ex- activity of Fucus enzyme marked the lower limit of the assay. change column containing Dowex 1, formate form(3 ml, The enzyme system was undectable in three other marine algae upper half) and Dowex 50, hydrogen form (3 ml, lower half). that were examined: Cliordafiliurin, Codiumn fragile, and Planc- About 40 ml of effluent were collected, flash evaporated to taria latifola. dryness, redissolved in ,ul50 of water and applied to the Of the four herbaceous plants tested, parsley, the one first intersection of two lines drawn parallel to adjoining edges and used to examine myoinositol biosynthesis in plants (23), had Plant Physiol. Vol. 48, 1971 GLUCOSE 6-PHOSPHATE CYCLOALDOLASE FROM ACER 257

Table I. A Survey ofPlantt Tissues for the Myoinositol-synthesizing Enzyme System

Plant Tissue (NH4)2S04 Fraction Protein in Extract Specific Activity

% saturation mg/g wet wt. tissue tinits/mg protein X 106 Petroselinzum crispum (parsley) Leaf 30-50 1.54 4.6 Salvia officinalis (sage) Leaf 30-50 1.32 36.6 Rosemarinus officinalis (rosemary) Leaf 30-50 2.07 8.0 Thymus vulgaris (thyme) Leaf 30-50 1.62 15.6 Lemnia perpusilla (duckweed) Whole plant 30-90 0.07 2.2 Zea mays (corn) Seedling 30-90 0.45 0.8 Lilium longiflorum (Easter lily) Pollen, ungerminated 30-90 5.50 8.5 Pollen, germinated 30-90 4.10 22.9 Acer pseudoplatanus (sycamore maple) Cultured cells 30-50 0.60 36.2 Fucus vesiculosis (rockweed) Vegetative fronds 30-90 0.23 0.02 Clhontdrus crispus (Irish moss) Vegetative fronds 30-90 0.23 0.35 Enteromorpha linza Vegetative 30-90 0.10 2.5

Table II. Purificatioz of D-Glucose 6-Phosphate Cycloaldolase from Cell Cultures of A. Pseudoplatantus L.

Step Purification Protein Specific Activity Enzyme Recovery

mg units/mg protein X 106 units X 106 % I Crude extract 3,504 4.1 14,366 100 II (NH4) 2SO4 fraction (35-50%' saturation) 399 11.6 4,638 32 III DEAE-cellulosel Tube 46 8.4 36.5 306 2 Tube 47 12.0 275.0 3,300 23 Tube 48 12.2 97.3 1,187 8 Tube 49 12.7 30.5 387 3 Sum 45.3 5,180 36 Data in this table are from an experiment described in the text and Figure 1. the lowest specific activity. Sage was eight times more active.

Corn seedling had a very low level of enzyme activity. This C/) may reflect a process of enzyme synthesis or activation during 0 germination. The increase in specific activity of lily pollen C-) after 1 hr of incubation in germination medium is very interest- E o~~~~~~~~~~o - (-) ing and suggests that a profound change in the amount or 0 1.2 06 3tyI + ItaN' Ia9 --4 activity of this enzyme occurred very early in the germination CI-i II% process. A. pseudoplatanus cells were also rich in enzyme (I) 0.4 --4 and became the source of material for purification of the en- zyme. cM~~~~~~~~~~~~~~c0 Table II summarizes data taken from a purification of I- cycloaldolase from A. pseudoplatanus cells. As noted in "Mate- a- rials and Methods," about 65 to 70% of the activity was lost z~~~~I0o%O~0Vr in the (NH4)2S04 precipitation and subsequent dialysis. Chro- 0 matography on DEAE-cellulose brought a 25-fold increase in specific activity with no loss of enzyme. The enzyme eluted as 0 20 40 60 a narrow peak (Fig. 1) in the region of 0.125 M NaCl in 20 mm TUBE NUMBER tris-HCl buffer, pH 8, with the major portion being recovered FIG. 1. Chromatography of DEAE-cellulose of a 35 to 50% in a single fraction, No. 47. This fraction was used for kinetic saturated ammonium sulfate fraction of A. pseudoplatanus cell studies. For studies of cofactor requirements, all tubes con- extract. Protein, measured by absorbance at 280 rin, is indicated taining active protein were pooled and used. At this stage of by the dashed line and cycloaldolase activity by the solid line. The purification, the enzyme remained stable when stored as a separation described here refers to purification step III in Table frozen solution for at least 8 weeks. II. Fraction-~~~~~size was 8.5 ml. Additional purification of the enzyme was obtained by gel filtration on Sephadex G-200. Using a preparation from the Pifia et al. (27) have reported a value of 150,000 for yeast DEAE-cellulose step in which all fractions containing activity cycloaldolaseI as determined by meas- gradient were combined, a 10-fold increase in specific activity was urement. noted. Bovine serum albumin, yeast alcohol dehydrogenase Identification of Dephosphorylated Product as Myoinositol. and catalase, proteins with molecular weights of 67,000, A portion of the radioactive product (5490 cpm) recovered 151,000 and 225,000, respectively, were chromatographed on from thin layer chromatograms of assays involving a purified the same column. As seen in Figure 2, the cycloaldolase eluted enzyme fraction from DEAE-cellulose was diluted with un- in the same volume occupied by yeast alcohol dehydrogenase. labeled myoinositol (100 mg) and recrystallized. The specific 258 LOEWUS AND LOEWUS Plant Physiol. Vol. 48, 1971 the amount of free myoinositol produced by the crude extract to 34% of that found if it were added. If, in addition, Mge was BSA deleted, the recovery was only 23% of the complete assay. Apparently, traces of Mg'+ or a phosphatase lacking a Mg`

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FIG. 2. Further purification of cycloaldolase from A. pseudo- platanus cell extract on a Sephadex G-200 column after partial purification on DEAE-cellulose. Protein, measured by absorbance at 280 nm, is indicated by the dashed line and cycloaldolase activity FIG. 3. Effect of different concentrations of NAD+ on the specific by the solid line. The approximate molecular size of A. pseudo- activity of A. pseudoplatanus cycloaldolase. platanus cycloaldolase is obtained by comparing its elution profile to those of catalase, yeast alcohol dehydrogenase (YAD) and bovine serum albumin (BSA) on the same column, dotted line. 100 Table III. Requiremenits for Conversionz of D-Glucose-I -lC 6-Phosphate to Myoinositol by the Plurified A. pseudoplatanus Enzzyme 0.5 Assay Specific Activity Activity > 50 iunits/mg protein X 106 g Complete 44.6 100 - NAD+ 0.0 0 - NH4Cl 19.2 43 - mg2+1 0.0 0

0 3 6 9 12 15 ' No alkaline phosphatase was added to the assay mixture. NH4 Cl CONCENTRATION, mM FIG. 4. Stimulatory effect of increasing concentrations of am- radioactivity of samples taken from each of three successive monium ion on A. pseudoplatanus cycloaldolase activity. The en- recrystallizations was 5543 + 283 cpm/100 mg. When con- zyme used in this experiment had been purified on a Sephadex G-100 verted to the hexa-acetate and recrystallized, first from toluene, gel column to remove traces of ammonium salts that might have then from methanol-water, there was no change in specific accompanied it through earlier purification steps. radioactivity. Requirements and Properties of A. pseudoplatanus Cyclo- Table IV. Requirements for Conversion of Phosphorylated Product aldolase. As seen in Table III, NAD+ is required for activity. to Free Myoinositol at Separate Steps of Purification of Maximum activity occurred at 1.0 mm NAD+ (Fig. 3). Deleting A. psuedoplatanus cycloaldolase NH4+ reduced the activity to 43% of that obtained in its presence. The effect of increasing concentrations of NH4Cl on Alkaline activity is seen in Figure 4. Only 2 mm NHOC1 was required to Purification phatasePhos- AddedMg2e Specificpc Activity Activitytvt achieve maximum activity. With the yeast enzyme, maximum Added activity was not reached until 10 mm (5). In the absence of units/mg protein Mg'+ and without addition of alkaline phosphatase to the as- X 106 say, conversion to free myoinositol did not occur. The enzyme Crude Extract preparation used to obtain results given in Table III had been + + 22.6 100 partially purified on DEAE-cellulose and was devoid of the _ + 7.7 34 Mg2+-dependent phosphatase encountered in the crude ex- - - ~~~5.2 23 tract. The effect of purification on the ability of the enzyme (NH4)2SO4 fraction, + + 47.3 100 preparation to convert the phosphorylated product of cycliza- 35-50% - tion to free myoinositol is seen in Table IV. Separate samples + 4.8 10 of enzyme taken from the crude extract, the (NH4)2SO frac- - - ~~~0.8 2 tion and the most active fraction recovered from DEAE- DEAE-cellulose + + 431.0 100 cellulose were assayed with Mg`+ and externally added phos- phatase, with Mg`+ only and with both Mg` and externally _ + 26.3 94 added phosphatase absent. Deleting the phosphatase reduced _ _ 17.5 Plant Physiol. Vol. 48, 1971 GLUCOSE 6-PHOSPHATE CYCLOALDOLASE FROM ACER 259 requirement was responsible for the residual of 15 phosphorylated product to free myoinositol. Further purifica- tion progressively removed components responsible for hy- drolysis in the absence of added phosphatase. The final values 0 12 of 4 to 6% obtained with a purified fraction from DEAE- I . cellulose are within experimental error. Maximum activity with the assay used in this study was at 1.33 mm, similar to 9 that reported by Chen and Charalampous (5). The pH optimum of the A. pseudoplatanus enzyme was 6 approximately 8.0 (Fig. 5). Similar optima have been reported IL- for C. utilis, Neurospora, and rat testis . The rate of the reaction was linear for at least 2.5 hr with D-glucose 6-P concentrations under 4 mm. With higher con- 3- centrations, there was a slight decrease in rate after 1 hr. When LO) the dependence of initial velocity on substrate concentration I is plotted according to Michaelis-Menton kinetics (Fig. 6), a 7.0 8.0 9.0 Km of 1.77 mm is obtained, comparable to 1.9 mm reported pH for N. crassa enzyme (27) and 1.5 mm for C. utilis (7). FIG. 5. Effect of pH on the activity of A. pseudoplatanus cyclo- aldolase. The enzyme used in this experiment had been purified on DISCUSSION DEAE-cellulose. All points were determined with tris-HCl buffer. 1)-Glucose 6-P cycloaldolase, previously reported to be pres- ent in rice, , and mustard plants (19, 30) has now been shown to be present in other higher plants as well as algae. 200~~~~~~~~~~ Partial purification of this enzyme from A. pseudoplatanus had yielded preparations, the best of which has about one-third the specific activity reported for yeast (5) and about one-tenth -005 that of rat testis (13). Purified cycloaldolase as isolated from A. pseudoplatanus is free of phosphatase. The crude extract, however, appears to contain two phosphatase activities, one of which requires a Mg2+ concentration similar to that reported _°_ 100 -/ for the inositol-specific phosphatase of yeast and rat testis. The cycloaldolase described here requires NAD+ in the same con- T,IKm= 1.77 mM centration as that reported for the enzyme from other sources for maximum activity. It has approximately the same pH optimum. It is stimulated 2.3-fold by NH,+, but at a lower concentration than that which stimulates the C. utilis enzyme 0 II 5-fold. 0 0.3 0.6 0.9 Efforts to isolate and examine D-glucose 6-P cycloaldolase S m moles-' source, higher plants, are engendered by the from yet another FIG. 6. A double reciprocal plot of initial velocity versus D- knowledge that this enzyme may have a significant function in glucose 6-P concentration with A. pseudoplatanus cycloaldolase the metabolism of plants. Formation of UDP-n- after purification of the enzyme on DEAE-cellulose. Points on the glucuronate and related products of UDP-i)-glucuronate me- plot indicated by filled and open circles represent results from two tabolism in plants proceeds from hexose phosphate over two different enzyme preparations, corrected for enzyme concentration. possible pathways. One, through UDP-D-glucose, involves an enzyme that catalyzes oxidation of carbon 6 of the glucose examples galactinol (33), (9), and the phospho- moiety in two steps to the corresponding acid (32). The other, inositides (18). The functional role of cycloaldolase in these first proposed on the basis of studies involving distribution of interconversions must await a more complete examination of radioisotopes in products of intact plant tissues fed specifically the structure and properties of this interesting, bifunctional labeled , uronic acids and myoinositol (21), involves enzyme. cyclization of the carbon chain of D-glucose to myoinositol (23) followed by oxidative cleavage of myoinositol to form Acknowledgments-Able technical assistance was provided by Miss Anne Golebiewski. Greenhouse-grown plant material was supplied by Mr. Joseph D-glucuronate (24). Subsequent phosphorylation of D-glu- Nolan. A sample of Lemna perpusilla was obtained from Dr. W. S. Hillman. curonate to its 1-phosphate and conversion of the latter to UDP-D-glucuronate were first described in studies with mung LITERATURE CITED bean extracts (17, 26). Recently, Roberts and Rao (29) suc- ceeded in purifying the pyrophosphorylase from barley - 1. ATKINSON, D. E. 1970. Enzymes as control elements in metabolic regulation. reported a 12-fold increase in pyrophosphorylase In: P. D. Boyer, ed., The Enzymes, Vol. 1. Academic Press, New York. pp. lings. They 461-489. activity in the first 6 days following germination. At the same 2. BARNETT, J. E. G., R. E. BRICE, AND D. L. CORINA. 1970. A colorimetric time, only low levels of UDP-D-glucose dehydrogenase were determination of inositol monophosphates as an assay for D-glucose 6- detected in this tissue. phosphate-l-L-myo-isositol-l-phosphate cyclase. Biochem. J. 119: 183-186. D-Glucose 6-P is an intermediate common to both pathways. 3. BARNETr, J. E. G. AND D. L. CORINA. 1968. The mechanism of glucose-6- phosphate-D-mYo-inositol 1-phosphate cyclase of rat testis. Biochem. J. Beyond D-glucose 6-P, the cycloaldolase is the first enzyme of 108: 125-129. the myoinositol pathway and, as such, may possess an im- 4. CHEN, I. W. AND F. C. CHARALAMPOUS. 1964. Biochemical studies on inositol. portant regulatory function over the products of myoinositol VII. Biosynthesis of inositol by a soluble enzyme system. J. Biol. Chem. metabolism (22). These products include several, in addition 239: 1905-1910. 5. CHEN, I. W. -ND F. C. CHARALAMPOUS. 1965. Biochemical studies on inositol. to D-glucuronic acid, with established roles as key compounds VIII. Purification and properties of the enzyme system which converts in biochemical interconversions vital to plants. One may cite as glucose-6-phosphate to inositol. J. Biol. Chem. 240: 3507-3512. 260 LOEWUS Al.NMD LOEWUS Plant Physiol. Vol. 48, 1971

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