Biochem. J. (1976) 153, 613-619 613 Printed in Great Britain Inhibition of Spinach Phosphoribulokinase 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 chloroplast phosphoribulokinase is inhibited by DL-glyceraldehyde. The in- hibition is non-competitive with respect to ribulose 5-phosphate (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 chloroplasts 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 substrate 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 ethanol (4vol.) was tor of photosynthesis 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 product (1.45g) contained 0.795mmol action was the reaction catalysed by phosphoribulo- of ribulose 5-phosphate when assayed by the carb- kinase (ATP-D-ribulose 5-phosphate 1-phospho- azole method (see below). TheBa2+ salt was converted transferase, EC 2.7.1.19), but no direct evidence was into the Na+ form by ion 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. Pyruvate kinase (ATP-pyruvate phosphotransferase, Experimental EC 2.7.1.40) from rabbit skeletal muscle (type II) Plant material and lactate dehydrogenase (L-lactate-NAD oxido- reductase EC 1.1.1.27) 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 enzyme 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 isomerase, EC 5.3.1.6) 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 protein 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 cysteine 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-dithiothreitol. 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 proteins 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.