Biochem. J. (1976) 153, 613-619 613 Printed in Great Britain

Inhibition of Spinach by DL-Glyceraldehyde By ANTONI R. SLABAS and DAVID A. WALKER Department ofBotany, University ofSheffield, Sheffield S1O 2TN, U.K. (Received 10 September 1975)

Spinach phosphoribulokinase is inhibited by DL-glyceraldehyde. The in- hibition is non-competitive with respect to ribulose 5- (Ki 19mM) and ATP (Ki 20mM). The inhibition is discussed in relation to a previously reported inhibition of CO2 assimilation in intact and envelope-free by DL-glyceraldehyde. It is concluded that the inhibition of phosphoribulokinase is insufficient to account for the inhibition, by DL-glyceraldehyde, of 02 evolution with ribose 5-phosphate as and that a further site of inhibition is also present in this system.

DL-Glyceraldehyde inhibits photosynthetic carbon glycylglycine buffer, pH7.4, in a total volume of assiniilation by intact chloroplasts and by the re- 13 ml. The reaction was terminated by the addition constituted chloroplast system (Stokes & Walker, of 2ml of 50 % (w/v) trichloroacetic acid and the pH 1972). The inhibition is most pronounced with intact was adjusted to pH8.0 with KOH. After the addition chloroplasts, a concentration of 1OmM being sufficient of Sml of 1.OM-barium acetate the solution was to suppress 02 evolution completely. It is important centrifuged and the precipitate discarded after because DL-glyceraldehyde is the only known inhibi- washing with Sml of water. Cold (4vol.) was tor of that is entirely without detect- added to the combined supernatants (total volume able effect on photophosphorylation or photo- 25.4ml); the new precipitate was recovered by synthetic electron transport. The earlier work (Stokes centrifugation and dried in a desiccator in vacuo & Walker, 1972) suggested that one possible site of overnight. The (1.45g) contained 0.795mmol action was the reaction catalysed by phosphoribulo- of ribulose 5-phosphate when assayed by the carb- (ATP-D-ribulose 5-phosphate 1-phospho- azole method (see below). TheBa2+ salt was converted , EC, but no direct evidence was into the Na+ form by exchange on a Dowex-50 obtained and the nature of tio inhibition was not (Na+ form) column. determined. The present investigation was undertaken Sephadex was obtained from Pharmacia (G.B.) in order to obtain such evidence. Ltd., London W5 SSS, U.K., and DEAE-cellulose from Whatman Ltd., Maidstone, Kent, U.K. (ATP-pyruvate , Experimental EC from rabbit skeletal muscle (type II) Plant material and (L-lactate-NAD oxido- reductase EC from rabbit skeletal muscle The spinach (Spinacia oleracea var. True Hybrid were obtained from Sigma. DL-Glyceraidehyde 102; Arthur Yates and Co., Sydney, N.S.W., Aust- was purchased either from Sigma or from BDH ralia) used in the preparations was grown Chemicals Ltd., Poole, Dorset BH12 4NN, U.K. in soil at the Tapton Experimental Gardens, Sheffield, All remaining reagents and biochemicals were pur- U.K. That used in the reconstituted chloroplast chased from Fisons Ltd., Loughborough, Leics., system was grown in water culture as previously U.K., or from Boehringer Corp., Lewes, East described (Lilley & Walker, 1974). Sussex BN7 1LG, U.K., and were of the highest purity available. Reagents Assays D-Ribulose 5-phosphate was prepared enzymically from ribose 5-phosphate (sodium salt) by using All enzyme assays were performed at 20°C, except phosphoriboisomerase (D-ribose 5-phosphate ketol- that for catalase. An enzyme unit is defined as the , EC from Sigma Chemical Co. Ltd., amount of enzyme that converts substrate into pro- Kingston-upon-Thames, Surrey KT2 7BH, U.K. duct at a rate of lumol/min under the conditions Ribose 5-phosphate (3.23 mmol) was incubated for of the assay. All of these conditions are as described 10min at 37°C with 1000 units (see under 'Assays') here, except for catalase, where the manufacturers' of spinach phosphoriboisomerase in 20mM-N- assay was conducted at 250C and pH7.0 and the Vol. 153 614 A. R. SLABAS AND D. A. WALKER

enzyme was used in the reconstituted system at 20°C zation buffer. Acetone (42.86ml/100ml) at -20°C and pH7.9. was added to make the solution 30% (v/v) and the Phosphoribulokinase. Unless otherwise specified precipitate removed by centrifugation at -10°C for this was assayed by following changes in E340 in a 15min at 10000g. The supernatant was made 50% reaction mixture containing 100lpmol of Tris/HCI, (v/v) with respect to acetone by adding acetone pH 8.0, 6,pmol of GSH, 0.14,umol of NADH, 6,umol (40ml/100ml) at -20°C. After centrifugation (IOOOOg of ATP, lOpmol of MgCl2, lOO,umol of KCI, 3,umol for 15 min at -20°C) the dark-brown precipitate was of phosphoenolpyruvate, 4.68 units of pyruvate resuspended in 20ml of 0.1 M-Tris/HCl, pH 8.0, kinase and 3.75 units of lactate dehydrogenase in a containing 10mM-EDTA and 1 mM-2-mercapto- final volume of 1 ml. The reaction mixure was ethanol. The solution was adjusted to 55 % saturation preincubated with the enzyme for 5 min at 20°C and with respect to (NH4)2SO4 by the addition of a the reaction was started by the addition of 0.8pumol saturated solution of (NH4)2SO4, pH8.0. The of ribulose 5-phosphate. precipitate was resuspended in the Tris/EDTA/ Phosphoriboisomerase. This was assayed directly mercaptoethanol buffer and applied, to a Sephadex by following the increase in E290 (Wood, 1970) in a G-200 columni (100cm x 3.5 cm) previously equi- reaction mixture containing lOO1mol of Tris/HCI, librated with 0.2M-Tris/HCI, pH8.0, containing pH8.0, and 10,umol of ribose 5-phosphate in a final 10mm-EDTA and 1 mM-2-mercaptoethanol. The volume of 1 ml. The reaction was started by the was eluted with the Tris/EDTA/mercapto- addition of enzyme. A millimolar extinction coeffici- ethanol buffer at a flow rate of 7.7mi/h and fractions ent of 0.072 was assumed for ribulose 5-phosphate (1.9ml) were collected. The phosphoribulokinase (Wood, 1970). emerged slightly before the phosphoriboisomerase, Ribulose 5-phosphate. This was measured by the but no clear separation could be effected. Fractions carbazole method. To 0.6ml of solution was added 95-112 were pooled and the solution was adjusted 6ml of H2SO4 (225ml of conc. H2SO4 plus 95ml of to 75% saturation by the addition of a saturated water followed by 0.2ml of a 0.12% solution of solution of (NH4)2SO4, pH8.0. The precipitate was carbazole in ethanol and 0.2ml of 1.5% (w/v) collected by centrifugation at IOOOOg for 30mn and hydrochloride. The latter two components was then resuspended in 10ml of 50mM-Tris/HCI, were added and mixed within 30s. After 30min at pH7.4, containing 0.65mM-. This was 37OC the Es40 was recorded. Fructose was used as applied to a Sephadex G-25 column (45cmx 2.5cm) standard. equilibrated with the same buffer, and the S042--free Protein. This was measured by the Lowry method protein fractions were pooled. The pooled protein as modified by Bailey (1962), after precipitation in was applied to a DEAE-cellulose column (30cmx 5% (w/v) trichloroacetic acid and resuspension in 2.5cm) previously equilibrated with 50mM-Tris/HCl, 5 % (w/v) NaOH. Bovine serum albumin, fraction V, pH7.4, and eluted with a linear gradient (4000ml) of previously dried in a desiccator, was used as standard. 0.02-0.2 M-KCI in the same buffer at a flow rate of 33ml/h. This procedure resulted in substantial Purification ofphosphoribulokinase separation of phosphoribulokinase activity from that of phosphoriboisomerase (Fig. 1). Fractions 74-85 Lavergne & Bismuth (1973) have described the were pooled and the resultant enzyme preparation preparation of spinach phosphoribulokinase free of was found to have a small (2%) content of phospho- contaminating phosphoriboisomerase, although they riboisomerase. Data for purification are given in did not indicate the stage at which the separation was Table 1. effected. Thepresent procedureyields phosphoribulo- kinaseonlyslightly(2%.)contaminatedwithphospho- Reconstituted chloroplast system riboisomerase. All operations were performed at 0-50C unless otherwise specified. Spinach (1.5kg) The chloroplasts and stromal were was macerated in a Waring Blendor for I min with prepared as before (Stokes & Walker, 1972; Lilley 2800ml of 2mM-triethanolamine buffer, pH8.5, et al., 1974), except that the chloroplast extract was containing lOmM-2-mercaptoethanol. The homo- concentrated by dialysis under reduced pressure genate was filtered through two layers of muslin and (Sartorius membrane filter) against a solution centrifuged at 100OOg for 30min. The supernatant containing: sorbitol, 33mM; EDTA, 0.2mM; MgC12, was adjusted to 30% saturation with solid (NH4)2SO4 5.1mM; MnCl2, 0.1mM; NaHCO3, 5mM; dithio- (16.4g/100ml) and the pH re-adjusted to pH8.0 with threitol, 1 mM; Hepes [2-(N-2-hydroxyethylpiper- KOH. After centrifugation (150OOg for 20min) the azin- N'-yl)ethanesulphonic acid], 5mM, pH7.6. precipitate was discarded and solid (NH4)2SO4 Photosynthetic O° evolution was measured in twin- (16.1 g/100ml) added to adjust the supernatantto 60% channel Clark-type oxygen electrodes (see Delieu & saturation. After centrifugation (150OOg for 30min) Walker, 1972) manufactured by Hansatech Ltd., the precipitate was resuspended in 60ml ofhomogeni- King's Lynn, Norfolk, U.K. 1976 INHIBMON OF PHOSPHORIBULOKINASE BY DL-GLYCERALDEHYDE 615

Reaction mixtures contained the following in a glyceraldehyde (Fig. 4) a K, of 21 mm is obtained for total volume of I ml; sorbitol, 330mm; EDTA, DL-glyceraldehyde. Freshly prepared solutions of 2mM; MgC12, 20mM; MnCJ2, 1 mM; Hepes/KOH, crystalline DL-glyceraldehyde were used throughout, 50mM; NaHCO3, 20mM; KH2PO4, 2mM; NADP+, but with a high K, the possibility of inhibition by a 0.1nmi; ferredoxin,; ADP, 0.2mM; dithio- potent toxic impurity cannot be entirely ruled out. threitol, 1 mM; catalase, 110 units; sodium iso- The degree of inhibition was not altered by pre- ascorbate, 4mM; osmotically shocked chloroplasts incubation with DL-glyceraldehyde. (Lffley et al., 1974) equivalent to 100g of chloro- phyll; chloroplast extract equivalent to 100jug of Experiments with the reconstituted chloroplast system chlorophyll. The final pH was 7.9. The reconstituted chloroplast system used by Stokes &Walker (1972), whichisessentiallya mixture Chlorophyll of envelope-free chloroplasts, stromal protein and This was measured as described by Arnon (1949). soluble coenzymes and co-factors, has been modified so that it will also support 02 evolution with fructose 6-phosphate and fructose 1,6-bisphosphate Results Inhibition studies with DL-glyceraldehyde Solutions of DL-glyceraldehyde were prepared immediately before use. (The pH was not adjusted, because it was found that, even at 40mm, glyceralde- hyde did not alter the pH of the reaction mixture.) The link used in the phosphoribulokinase assay were shown to be present in sufficient concen- tration to be non-limiting and also to be unaffected by DL-glyceraldehyde up to the highest concentration used. DL-Glyceraldehyde inhibits phosphoribulokinase in a non-competitive manner with respect to ribulose 5-phosphate, altering the V,.. but not the Km for ribulose 5-phosphate (Fig. 2). The Km observed for ribulose 5-phosphate (2.22 x 104M) is the same as that observed by Hurwitz etal. (1955). Replotting the slope of the Lineweaver-Burk plots from Fig. 2 against the concentration of DL-glyceraldehyde (Fig. 4) yields a K, value' of approx. 20mM-DL- D 60 80 too glyceraldehyde. Fraction no. The inhibition observed by I)L-glyceraldehyde is Pig. 1. Elution profile for phosphoribmlokinase (A) and also non-competitive with respect to ATP, altering phosphoriboisomerase (0) activity from DEAE-cellulose the Vmax. but not the Km for ATP (Fig. 3). The appar- ent Conditions are as described under 'Purification of Km for ATP observed in these experiments was phosphoribulokinase'. Fractions (3.35ml) were collected 0.625mM, which is close to the value reported by and assayed for activity by the procedure desribed under Lavergne et al. (1974). When the data from Fig. 3 are 'Assays'. The arrow represnts the fractions that were replotted as slope versus concentration of DL- pooled.

Table 1. Purtfication procedurefor spinach chloroplastphosphoribulokinase Total enzyme activity (units) Total Total volume protein Stage Phosphoribulokinase Phosphoriboisomerase (ml) (g) lst'supernatant 2800 8000 -3048 7.7 30Y%-satd.4NH4)2SO4 supernatant 14600 16700 3154 5.9 60%0-satd.-(NH4)2SO4 precipitate 14000 23400 62 4.9 5OY. (v/v) acetone precipitate 6500 5550 20 0.37 Sephadex G-200 eluate 1600 1020 34 0.10 DEAE-ellulose eluate 1000 23 40 0.018 Vol. 153 616 A. R. SLABAS AND D. A. WALKER

20 f

151 0~~~~ 0


-4 _,


-10 0 10 20 30 40 50 1/[ATP] (mm-') Fig. 3. Lineweaver-Burk plot for spinach phosphoribulo- kinase with ATP as the variable substrate The assay conditions were as described in the Experi- mental section, except thatthe assays were performed in the presence of (A) 0, (o) 5, (*) 10, (A) 20 and (U) 40mM-DL- glyceraldehyde. The intercept is at -16mM-1.

0 10 20 30 40 s50 1/[Ribulose 5-phosphate] (mm-') Fig. 2. Lineweaver-Burk plot for spinach phosphoribulo- 1.5I kinase with ribulose 5-phosphate as the variable substrate The assay conditions were as described in the Experimental section, except that the assays were performed in the presence of (A) 0, (o) 5, (*) 10, (A) 20 and (U) 40mM-DL- 1.0. glyceraldehyde. The intercept is at -4.5mM-'. 9d

04 as substrates. The modified system is also entirely ° 0. self-sufficient with regard to ATP and NADPH, which are generated by endogenous photophos- G- phorylation in the presence of ferredoxin plus cata- lytic ADP and NADP+. The previous work (Stokes & Walker, 1972) had -20 -10 0 10 20 30 40 (mM) pointed to a site (or sites) of inhibition by DL-glycer- [DL-Glyceraldehyde] aldehyde in the reductive pentose phosphate pathway Fig. 4. Secondaryplots ofslope versus [DL-glyceraldehydeJ lying between triose phosphate and ribulose 1,5- The data are from Fig. 2 (U; intercept -19mM) and Fig. 3 bisphosphate, but the reconstituted system then (@; intercept -20mM) for variable ribulose 5-phosphate available would only evolve 02 in the presence of and ATP concentrations respectively. pentose or 3-phosphoglycerate. Because the new system (Walker & Slabas, 1976) allows the use of fructose 1,6-bisphosphate (and even triose phosphate) as substrate it permitted the effect of DL-glyceraldehyde to be examined step by step on the whencatalyticNADP+,reducedby electron transport, sequence of events leading to the generation of the was reoxidized in the presence of 3-phosphoglycerate natural oxidant (glycerate 1,3-bisphosphate). In or ribulose 1,5-bisphosphate+CO2. accord with previous experience (Stokes & Walker, DL-Glyceraldehyde was also without immediate 1972) DL-glyceraldehyde had no discernible effect on effect when relatively high concentrations (approx. 02 evolution with NADP+ as the electron acceptor or 1mM) of ribose 5-phosphate were substituted for 1976 INHIBITION OF PHOSPHORIBULOKINASE BY DL-GLYCERALDEHYDE 617

Table 2. Inhibition by 30mM-DL-glyceraldehyde of C02-dependent 02 evolution in the reconstituted chloroplast system with ribose 5-phosphate as substrate In addition to the reagents listed in the Experimental section the reaction mixtures contained 0.2,umol ofribose 5-phosphate. Velocities are expressed in pmol of 0° evolved/h per mg of chlorophyll. Extent of 02 evolution Initial velocity in Initial velocity in before attainment of absence of presence of Initial rate as % of 100%/ inhibition (% of Expt. no. DL-glyceraldehyde DL-glyceraldehyde control control)* 1 74 29 39.2 52.6 2 78 24 30.8 41.2 3 75 24 32.0 44.7 * These values show the total extent of 02 evolution when the inhibited rate had fallen to zero and the control had run to completion (see the text).

Table 3. Inhibition by lOmM-DL-glyceraldehyde Of CO2- completely suppressed when the reoxidation of dependent 02 evolution in the presence of successive NADPH depended on the presence of fructose 6- additives phosphate. Similarly, when mixtures containing Reaction mixtures were essentially the same as for Table 2. 0.1 pmol of NADP+, 0.1,umol of 3-phosphoglycerate The 02 evolution associated with the reduction of added and 0.5 umol offructose 6-phosphate were illuminated NADP+ was allowed to proceed to completion before the in the presence and absence of IOmM-DL-glyceralde- of3-phosphoglycerate, and similarly the addition addition hyde the rates of 02 evolution were identical until of fructose 6-phosphate was delayed until 02 evolution approx. 0.1 pmol of 02 had been evolved (i.e. until had again ceased. Ribose 5-phosphate was added after a re- further 3min. The results indicate a major block between the NADP+ and 3-phosphoglycerate had been fructose 6-phosphate and triose phosphate, a minor block duced). This took approx. 1.5min; thereafter 02 between ribose 5-phosphate and triose phosphate and no evolution slowed in the presence of the inhibitor and effect of DL-glyceraldehyde on the reduction of NADP+ ceased entirely after a further 3.5min. In all experi- or 3-phosphoglycerate. Under these conditions the ments ofthis nature, evolution could be fully restored (maximal) rates -used in the computation of percentages by the further addition of 3-phosphoglycerate or were established after a few seconds and thereafter ribulose bisphosphate. remained more or less constant for several minutes. The percentages quoted are based on results from chloroplasts Discussion isolated on 3 successive days but are characteristic of a larger number of experiments. Photosynthesis by intact isolated chloroplasts is Inhibition completely inhibited by 10mM-DL-glyceraldehyde. Additive -(Y. of control) At this concentration the inhibitor is without effect NADP+ (0.1 mM) 0 on photosynthetic electron transport, photosynthetic 3-Phosphoglycerate (0.2mM) 0 , the carboxylation of ribulose Fructose 6-phosphate (1.0mM) 100 1,5-bisphosphate and the reduction of 3-phospho- Ribose 5-phosphate (0.5mM) 22 glycerate (Stokes &Walker, 1972). It is clear therefore that the site or sites of inhibition must be between glyceraldehyde 3-phosphate and ribulose bisphos- ribulose bisphosphate. However, at 30mM-glyceral- phate. The participation of DL-glyceraldehyde in the dehyde and low concentrations ofribose 5-phosphate reactions catalysed by aldolase (Tung et aL., 1954) and (0.2umol) there was a marked inhibition from the transketolase (Horecker et al., 1953; de la Haba et al., outset (Table 2). This inhibition also 'progressed' 1955) seemed to be the most likely possibilities (cf. Stokes & Walker, 1972), i.e. 02 evolution started (Stokes & Walker, 1972), but the inhibition of re- at 30-40% of the rate observed in the control but actions in which pentose monophosphates were slowed progressively until it ceased entirely after utilized as substrates also suggested an effect on about 3min illumination (Table 2). phosphoribulokinase (Stokes & Walker, 1972). With fructose 6-phosphate, fructose bisphosphate, The present results confirm this suggestion, but, or mixtures containing these hexose phosphates and although they show that DL-glyceraldehyde inhibits glyceraldehyde 3-phosphate, inhibition was virtually purified phosphoribulokinase in a non-competitive complete in the presence of only lOmM-DL-glycer- manner (with respect to both ribulose 5-phosphate aldehyde. For example, Table 3 shows the degree of and ATP), the K1 values of approx. 20mM could not inhibition observed in the presence of successive easily account for complete inhibition at 10mM in the oxidants and substrates, and 02 evolution was only intact plastid. The conditions used in the spectro. Vol. 153 618 A. R. SLABAS AND D. A. WALKER

X 3 Ribulose 5-phosphate

3 Pentose monophosphate 3


,ADP Product (triose phosphate) 6 NADP+ 6 NADPH

6 H20 3 02 Scheme 1. Sites ofaction of DL-glyceraldehyde Simplified Benson-Calvincycleshowing points at which DL-glyceraldehyde inhibits. At 10mM the inhibition of the phospho- ribulose kinase reaction, described in the present paper, is le"s important than inhibition of the conVersion of triose phosphate into pentose phosphate (probably involving transketolase). photometric assays were necessarily somewhat with fructose 6-phosphate plus glyceraldehyde 3- different from those in the reconstituted system, and phosphate as substrates is much more susceptible to there are clearly limits to the extent to which results inhibition by DL-glyceraldehyde than the correspond- from one can be applied to the other. Nevertheless ing process in which ribose 5-phosphate is the pre- the extent of inhibition by DL-glyceraldehyde ob- cursorof3-phosphoglycerate. The reactions catalysed served in the reconstituted system with ribose 5- bytransketolase therefore emerge as the sites at which phosphate as substrate (Tables 2 and 3) is consistent it is most likely that this inhibitor exerts its major with a high K;, and Table 3 leaves little-doubt that effects. The possibility that DL-glyceraldehyde also formation of ribulose 5-phosphate is much more inhibits by condensing with phos- susceptible to inhibition by glyceraldehyde than is its phate is made less likely by the observations of utilization. Leuthardt & Wolf (1955), which indicate that this In an earlier report (Stokes & Walker, 1972) reaction is only catalysed by fructose 1-phosphate attention was drawn to the progressive nature of the aldolase. Scheme 1 summarizes the sites of action inhibition with pentose phosphate as substrate and of L-glyceraldehyde on the Benson- to the discrepancy between the inhibition of C02 (Ba4sham & Calvin, 1957) in the reconstituted fixation and the inhibition of C02-dependent 02 chloroplast system. evolution in the reconstituted system. We now believe that inhibition of C02-dependent 02 evolution will Note Added in Proof (Received 25 November 1975) only become apparent when the supply of phospho- Bamberger & Avron (1975) have independently glycerate from pentose phosphate is limiting and that concluded that DL-glyceraldehyde acts at (a) a more this will only occur in the presence of low concentra- sensitive site in the conversion of fructose bisphos- tions of pentose phosphate. The inhibition would phate into ribulose:5-phosphate and (b) a less sensi- become progressive if, for example, glycetaldehyde tive site in the conversion of ribulose 5-phosphate were to serve as a sink for glyceraldehyde units in the into ribulose bisphosphate. transketolase reactions (thus giving rise to free xylulose). Certainly there is little doubt that 02 This work was supported by the Science Research evolution in the reconstituted chloroplast system Council (U.K.). 1976 INHIBITION OF PHOSPHORIBULOKINASE BY DL-GLYCERALDEHYDE 619

References Lavergne, D. & Bismuth, E. (1973) Plant Sci. Lett. 1, Arnon, D. I. (1949) Plant Physiol. 24, 1-15 229-236 Bailey, J. L. (1962) in Techniques in Protein Chemistry Lavergne, D., Bismuth, E. & Champigny, M. L. (1974) (Bailey, J. L., ed.), p. 293, Elsevier Publishing Co., Plant Sci. Lett. 3, 391-397 Amsterdam Leuthardt, F. & Wolf, H. P. (1955) Methods Enzymol. 1, Bamberger, E. S. & Avron, M. (1975) Plant Physiol. 56, 320-322 481-485 Lilley, R. McC. & Walker, D. A. (1974) Biochim. Biophys. Bassham, J. A. & Calvin, M. (1957) The Path ofCarbon in Acta 368, 269-278 Photosynthesis, pp. 1-107, Prentice-Hall, Englewood Lilley, R. McC., Holborow, K. & Walker, D. A. (1974) Cliffs New Phytol. 73, 657-662 de la Haba, G., Leder, I. G. & Racker, E. (1955) J. Biol. Stokes, D. M. & Walker, D. A. (1972) Biochem. J. 128, Chem. 214, 409-426 1147-1157 Delieu, T. & Walker, D. A. (1972)NewPhytol. 71,201-225 Tung, T. C., Ling, K. H., Byrne, W. L. & Lardy, H. A. Horecker, B. L., Smyrniotis, P. Z. & Klenow, H. (1953) (1954) Biochim. Biophys. Acta 14, 488-494 J. Biol. Chem. 205, 661-682 Walker, D. A. & Slabas, A. R. (1976) Plant Physiol. 57, Hurwitz, J., Weissbach, A., Horecker, B. L. & Smyrniotis, in the press P. Z. (1955) J. Biol. Chem. 218, 769-783 Wood, T. (1970) Anal. Biochem. 33, 297-306

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