Ribulose bisphosphate carboxylase and glycollate oxidase from jack pine: Effects of sulphur dioxide fumigation

Ribulose bisphosphate (RuBP) carboxylase and glycollate oxidase were partially purified from jack pine (Pinus banksiana Lamb.) needles. Preincubation of RuBP carboxylase with HCO₃^? and Mg²⁺ markedly stimulated its activity. RuBP carboxylase showed hyperbolic reaction kinetics with respect to HCO₃^?, Mg²⁺, and RuBP. Both SO₃^2- and SO₄^2- inhibited RuBP carboxylase, but SO₃^2- was more inhibitory than SO₄^2-. The SO₃^2- inhibition was competitive with respect to HCO₃^? (whether SO₃^2- was present during activation or was added to the activated enzyme), while the SO₄^2- inhibition was non-competitive with respect to HCO₃^?. Glycollate oxidase was inhibited more severely by low concentrations of SO₃^2- than by SO₄^2-. Fumigation of jack pine seedlings with 0.34 ppm sulphur dioxide for 24 and 48 hr produced a considerable decline in the activities of these enzymes, but 1 hr of fumigation produced no effect. During the longer exposures the sulphur content of the needles increased considerably, although the needles showed no visible injury. It is suggested that the accumulation of SO₃^2- and SO₄^2- in the needles following sulphur dioxide exposure influenced the enzyme activities.     

Bicarbonate-stimulated ATPase in the renal proximal tubule luminal (brush border) memebrane.

Brush border membranes of the rabbit renal tubule have an ATPase which was stimulated 60% by 50 mm HCO₃^?. The K_a for HCO₃^? was 36 mm. Kinetic studies of the “HCO₃^?-ATPase” indicate that HCO₃^? had no effect on the K_m for ATP and ATP did not alter the K_a for HCO₃^?. Several anions, notably SO₃^2?, also accelerated the rate of dephosphorylation of ATP. The V for “SO₃^2?-ATPase” was fivefold greater than that for “HCO₃^?-ATPase.” The K_a for SO₃^2? was 0.78 mm. Other anions including Cl^? and phosphates, did not enhance ATPase activity. Thus, of the anions present in the glomerular filtrate in appreciable concentrations only HCO₃^? stimulated the luminal membrane enzyme. The anion-stimulated ATPase activity increased sharply from pH 6.1 to 7.1 and moderately with higher pH. The renal ATPase was not inhibited by SCN^? nor methyl sulfonyl chloride and was relatively insensitive to oligomycin and quercetin. Carbonyl cyanide p-trifluoromethoxy phenylhydrazone increased the basal rate of the membranal ATPase, suggesting that the ATPase activity is limited by transmembrane H⁺ flux. Carbonic anhydrase significantly increased the HCO₃^?-stimulated ATPase activity. This increment was blocked by Diamox. These findings provide evidence consistent with the hypothesis that the brush border membrane ATPase is involved in the extrusion of H⁺ from tubular cell to lumen and support suggested interrelationships between HCO₃^?-stimulated ATPase, H⁺ secretion, and bicarbonate transport in the kidney.     

Effect of SO32- on the activity of ribulose bisphosphate carboxylase from seedlings of Pinus silvestris

Abstract The effect of sulphite on ribulose bisphosphate carboxylase, extracted from needles of Pinus silvestris L., was studied in vitro at pH 8.15 and 25°C. 1 mM and higher concentrations of SO₃^2- inhibited the enzyme. The enzyme was activated either in the assay medium (2.5 – 20 mM HCO_3, 20 mM MgCl₂) or in 10 or 20 mM HCO₃^- and 20–25 mM MgCl₂. Linear reciprocal plots of the activity versus the substrate concentration were obtained, when the HCO₃^- concentration during activation was 4 mM or higher. When the enzyme was activated at high HCO₃^- and Mg²⁺ concentrations, the K_m(CO₂) was c. 27 μM. With respect to HCO₃^-. SO₃^2- inhibited the enzyme in a non-competitive fashion. The inhibition was similar, whether SO₃^2- was present during activation or not. Apparently. SO₃^2- did not interfere with the binding of CO₂ and Mg²⁺ at the activating site. The K₁ was 11–13 mM SO₃^2-. With respect to ribulose bisphosphate the inhibition was also noncompetitive. Similar results with respect to HCO₃^- were obtained for spinach, Spinacia oleracea L., which is contrary to earlier reports.     

Effects of CO2, O2 and temperature on a high-affinity form of ribulose diphosphate carboxylase-oxygenase from spinach

A high-affinity form of ribulose diphosphate carboxylase, observed transiently in spinach-leaf extracts soon after extraction, was inhibited by O₂ competitively with respect to CO₂. Analogously, the ribulose diphosphate oxygenase activity for this form was inhibited by CO₂, competitively with respect to O₂. For each gas, the K_m for the reaction in which it was a substrate was similar to its K_i for the reaction it inhibited. The Arrhenius activation energy for the oxygenase reaction was 1.5 times that of the carboxylase. These characteristics are consistent with ribulose diphosphate oxygenase being the enzymatic reaction responsible for synthesizing the substrate for photorespiration and with the concept that the balance between photosynthesis and photorespiration of leaves is a reflection of the ratio between the two activities of this bi-functional enzyme.     

Properties of NADP-malic enzyme from glumes of developing wheat grains

Properties of partially purified NADP-malic enzyme (EC 1.1.1.40) from glumes of developing wheat grains were examined. The pH optimum for enzyme activity was influenced by malate and shifted from 7.3 to 7.6 when the concentration of malate was increased from 2 to 10 mM. The K_m values, at pH 7.3, for various substrates were: malate, 0.76 mM; NADP, 20 μM and Mn²⁺, 0.06 mM. The requirement of Mn²⁺ cation for enzyme activity could be partially replaced by Mg²⁺ or Co²⁺. Mn²⁺ dependent enzyme activity was inhibited by Pb²⁺, Ni²⁺, Hg²⁺, Zn²⁺, Cd²⁺, Al³⁺ and Fe³⁺. During the reaction, substrate molecules (malate and NADP) reacted with enzyme sequentially. Activity of malic enzyme was inhibited by products of the reaction viz pyruvate, HCO₃^? and NADPH₂. At a limiting fixed concentration of NADP, these products induced a positive cooperative response to increasing concentrations of malate.     

Development and Characterization of Ribulose-1,5-bisphosphate Carboxylase : Evidence Indicating a Lack of Carboxylase Function in CO(2) Fixation in Endosperm of Germinating Castor Bean Seedlings

Ribulose-1,5-bisphosphate carboxylase activity was found in endosperm of germinating castor bean seed Ricinus communis and was localized in proplastids. The endosperm carboxylase has been extensively purified and is composed of two different subunits. The molecular weights of the native carboxylase and its subunits were 560,000, 55,000, and 15,000 daltons, respectively. The Michaelis-Menten constants, K_m, for the endosperm carboxylase with respect to ribulose 1,5-bisphosphate, bicarbonate, CO₂, and magnesium in millimolar are 0.54, 13.60, 0.92, and 0.57, respectively. The endosperm carboxylase was activated by Mg²⁺ and HCO₃⁻. The preincubation of the carboxylase with 1 millimolar HCO₃⁻ and 5 millimolar MgCl₂ resulted in activation by low and inhibition by high concentrations of 6-phosphogluconate.

In studies of dark ¹⁴CO₂ fixation by endosperm slices, [¹⁴C]malate and [¹⁴C]citrate were the predominantly labeled products after 30 seconds of exposure of the tissue to H¹⁴CO₃⁻. In pulse-chase experiments, 87% of the label is malate, and citrate was transferred to sugars after a 60-minute chase with a small amount of the label appearing in the incubation medium as ¹⁴CO₂. The minimal incorporation of the label from ¹⁴CO₂ into phosphoglyceric acid indicated a lack of the endosperm ribulose-1,5-bisphosphate carboxylase participation in the endosperm's CO₂ fixation system. The activities of key Calvin cycle enzymes were examined in the endosperms and cotyledons of dark-grown castor bean seedlings. Many of these autotrophic enzymes develop in the dark in these tissues. The synthesis of ribulose-1,5-bisphosphate carboxylase in the nonphotosynthetic endosperms is not repressed in the dark, and high levels of enzymic activity appear with germination. All of the Calvin cycle enzymes are present in the castor bean endosperm except NADP-linked glyceraldehyde 3-P dehydrogenase, and the absence of this dehydrogenase probably prevents the functioning of these series of reactions in dark CO₂ fixation.

     

Effect of glycidate, an inhibitor of glycolate synthesis in leaves, on the activity of some enzymes of the glycolate pathway

Under conditions where glycolate synthesis was inhibited at least 50% in tobacco (Nicotiana tabacum L.) leaf discs treated with glycidate (2,3-epoxypropionate), the ribulose diphosphate carboxylase activity in extracts and the inhibition of the activity by 100% oxygen were unaffected by the glycidate treatment. [1-¹⁴C]Glycidate was readily taken into leaf discs and was bound to leaf proteins, but the binding occurred preferentially with proteins of molecular weight lower than ribulose diphosphate carboxylase. Glycidate added to the isolated enzyme did not inhibit ribulose diphosphate carboxylase activity or affect its inhibition by 100% O₂. Thus, glycidate did not inhibit glycolate synthesis by a direct effect on ribulose diphosphate carboxylase/oxygenase.     

Author index for volume 179, number 1

Sulfite ion, the hydrated form of SO₂ which is an air pollutant, was found to be an inhibitor of phosphoenolpyruvate carboxylase(s) isolated from corn leaves. The inhibition was partial even in the presence of excess SO₃^2?. It inhibited the enzyme competitively with respect to HCO₃^?, noncompetitively with respect to phosphoenolpyruvate, and uncompetitively with respect to Mg²⁺. The kinetics of inhibition suggest that an alternate pathway is operative in the presence of SO₃^2?. The enzyme(s) were activated by glucose 6-phosphate which affected primarily the affinity of the enzyme for phosphoenolpyruvate. The binding site of glucose 6-phosphate was apparently distinct from the catalytic site of the enzyme since partial destruction of the catalytic site by heat had no effect on the inhibition by SO₃^2?, but glucose 6-phosphate lost its activating effect. The inhibition due to SO₃^2? was relieved by glucose 6-phosphate.     

Quantitation of Rates of Transport, Metabolic Fluxes, and Cytoplasmic Levels of Inorganic Carbon in Maize Root Tips during K Ion Uptake

Our aim was to determine whether fixation of inorganic carbon (C_i), due to phosphoenolpyruvate carboxylase activity, is limited by the availability of C_i in the cytoplasm of maize (Zea mays L.) root tips. Rates of C_i uptake and metabolism were measured during K₂SO₄ treatment, which stimulates dark C_i fixation. ¹³C_i uptake was followed by ¹³C-nuclear magnetic resonance (NMR); 5 millimolar K₂SO₄ had no significant effect on ¹³C_i influx. The contribution of respiratory CO₂ production to cytoplasmic HCO₃⁻ was measured using in vivo ¹³C-NMR and ¹H-NMR of cell extracts; K₂SO₄ treatment had no effect on respiratory CO₂ production. The concentration of cytoplasmic HCO₃⁻ was estimated to be approximately 11 millimolar, again with K₂SO₄ having no significant effect. These experiments allowed us to determine the extent to which extracellularly supplied ¹⁴C_i was diluted in the cytoplasm by respiratory CO₂ and thereby measure phosphoenolpyruvate (PEP) carboxylase activity in vivo using ¹⁴C_i. PEP carboxylase activity in root tips was enhanced approximately 70% over controls within 12 minutes of the addition of 5 millimolar K₂SO₄. The activity of carbonic anhydrase, which provides PEP carboxylase with C_i, was determined by saturation transfer ¹³C-NMR to be more than 200 times that of PEP carboxylase in vivo. The regulation of PEP carboxylase in K₂SO₄-treated roots is discussed.     

Potential difference responses due to K+, Na+ and Cl− changes in Bullfrog antrum with and without HCO3−

The effect of changing [K⁺], [Na⁺] and [Cl^?] in nutrient solution was studied in bullfrog antrum with and without HCO₃^? in nutrient. In 25 mM HCO₃^? (95% O₂/5% CO₂) and in zero HCO₃^? (100% O₂), nutrient pH was maintained at 7.3. Changing from 4 to 40 mM K⁺ or from 81 to 8.1 mM Cl^? gave a decrease 10 min later in transmucosal PD (nutrient became more negative) — a normal response. These responses were less in zero than in 25 mM HCO₃^?. A decrease from 102 to 8 mM Na⁺ decreased PD (anomalous response of electrogenic NaCl symport). This effect was attenuated or eliminated in zero HCO₃^?. In contrast, change from 4 to 40 mM K⁺ gave initial anomalous PD response and change from 102 to 8 mM Na⁺, initial normal PD response with either zero or 25 mM HCO₃^?. Both responses were associated with (Na⁺ + K⁺)-ATPase pump and were greater in zero than in 25 mM HCO₃^?. Initial PD increases in zero HCO₃^? are explained as due to increase in the resistance of passive conductance and/or NaCl symport pathways. Thus, removal of HCO₃^? modifies conductance pathways of nutrient membrane.     

The response to ribulose bisphosphate4− (RuBP4−) and RuBP-Mg2− in catalysis by structurally divergent RuBP carboxylase/oxygenases

Free ribulose bisphosphate (RuBP^4?) rather than its magnesium complex (RuBP-Mg^2?) was the apparent substrate for spinach ribulose bisphosphate carboxylase/oxygenase. The apparent K_m for total RuBP (pH 8.0 at 30° C) increased with increasing Mg²⁺ concentrations from 11.6 μM at 13.33 mM Mg²⁺ to 32.6 μM at 40.33 mM Mg²⁺. Similarly the apparent K_m for RuBP-Mg^2? complex increased with increasing Mg²⁺ from 9.4 μM at 13.33 mM Mg²⁺ to 29.7 μM at 40.33 mM Mg²⁺. However, the K_m values for uncomplexed RuBP^4? were independent of the (saturating) concentration of Mg²⁺ (K_m=2.2 μM). The V_max did not vary with the changing concentrations of Mg²⁺. In contrast, the K_m for total RuBP remained constant with varying Mg²⁺ concentrations (K_m=59.5 μM) for the enzyme from R. rubrum. The apparent K_m for the RuBP-Mg^2? complex decreased with increasing Mg²⁺ concentrations from 16.0 μM at 7.5 mM Mg²⁺ to 5.9 μM at 27.5 mM Mg²⁺. The initial velocity for the C. vinosum enzyme was also found to be independent of the (saturating) concentration of Mg²⁺ when total RuBP was varied in the assay. Thus the response to total RuBP by these two bacterial enzymes, which markedly differ in structure, was closely similar.     

The response to ribulose bisphosphate4− (RuBP4−) and RuBP-Mg2− in catalysis by structurally divergent RuBP carboxylase/oxygenases

Free ribulose hisphosphate (RuBP^4?) rather than its magnesium complex (RuBP-Mg^2?) was the apparent substrate for spinach ribulose bisphosphate carboxylase/oxygenase. The apparent K_m for total RuBP (pH 8.0 at 30° C) increased with increasing Mg²⁺ concentrations from 11.6 μM at 13.33 mM Mg²⁺ to 32.6 μM at 40.33 mM Mg²⁺. Similarly the apparent K_m for RuBP-Mg^2? complex increased with increasing Mg²⁺ from 9.4 μM at 13.33 mM Mg²⁺ to 29.7 μM at 40.33 mM Mg²⁺. However, the K_m values for uncomplexed RuBP^4? were independent of the (saturating) concentration of Mg²⁺ (K_m=2.2 μM). The V_max did not vary with the changing concentrations of Mg²⁺. In contrast, the K_m for total RuBP remained constant with varying Mg²⁺ concentrations (K_m=59.5 μM) for the enzyme from R. rubrum. The apparent K_m for the RuBP-Mg^2? complex decreased with increasing Mg²⁺ concentrations from 16.0 μM at 7.5 mM Mg²⁺ to 5.9 μM at 27.5 mM Mg²⁺. The initial velocity for the C. vinosum enzyme was also found to be independent of the (saturating) concentration of Mg²⁺ when total RuBP was varied in the assay. Thus the response to total RuBP by these two bacterial enzymes, which markedly differ in structure, was closely similar.     

Phosphoenolpyruvate carboxylase in Hydrilla plants with varying CO2 compensation points

Incubation of the submersed aquatic macrophyte, Hydrilla verticillata Royle, for up to 4 weeks in growth chambers under winter-like or summer-like conditions produced high (130 to 150 μl CO₂/1) and low (6 to 8 μl CO₂/l) CO₂ compensation points (Γ), respectively. The activities of both ribulose bisphosphate (RuBP) and phosphoenolpyruvate (PEP) carboxylases increased upon incubation but the major increase was in the activity of PEP carboxylase under the summer-like conditions. This reduced the ratio of RuBP/PEP carboxylases from 2.6 in high Γ plants to 0.2 in low Γ plants. These ratios resemble the values in terrestrial C₃ and C₄ species, respectively. Kinetic measurements of the PEP carboxylase activity in high and low Γ plants indicated the Vmax was up to 3-fold greater in the low Γ plants. The Km (HCO₃ ^?) values were 0.33 and 0.22 mM for the high and low Γ plants, respectively. The Km (PEP) values for the high and low Γ plants were 0.23 and 0.40 mM, respectively; and PEP exhibited cooperative effects. Estimated Km (Mg²⁺) values were 0.10 and 0.22 mM for the high and low Γ plants, respectively. Malate inhibited both PEP carboxylase types similarly. The enzyme from low Γ plants was protected by malate from heat inactivation to a greater extent than the enzyme from high Γ plants. The results indicated that C₄ acid inhibition and protection were not reliable methods to distinguish C₃ and C₄ PEP carboxylases. The PEP carboxylase from low Γ plants was inhibited more by NaCl than that from hight Γ plants. These analyses indicated that Hydrilla PEP carboxylases had intermediate characteristics between those of terrestrial C₃ and C₄ species with the low Γ enzyme being different from the high Γ enzyme, and closer to a C₄ type.     

Factors affecting interconversion between kinetic forms of ribulose diphosphate carboxylase-oxygenase from spinach

Ribulose diphosphate carboxylase was found to exist in two distinct kinetic forms in spinach leaf extracts. One form displayed an apparent K_m for CO₂ in excess of 200 μm and is likely to be the form purified and studied by many previous workers. However, if leaf extracts were prepared in the presence of Mg²⁺ and atmospheric levels of CO₂, the recently described high-affinity form was obtained. It had a K_m for CO₂ of about 20 μm, was quite stable even at 25 °C, and its properties were consistent with it being the form which operates in photosynthesis in vivo. Mg²⁺ was also able to convert the high-K_m (CO₂) form to the low-K_m (CO₂) form when it was added to an extract which had been prepared in its absence. Mg²⁺ was more effective in causing this conversion if bicarbonate was added as well. This activating effect of bicarbonate is a probable cause of previously reported apparent homotropic effects of bicarbonate on ribulose diphosphate carboxylase activity. It is possible that the apparently high-K_m (CO₂) form is not intrinsically active and appears to have activity only by virtue of the low-K_m (CO₂) form produced by contact with Mg²⁺ and bicarbonate (or CO₂) during the course of the assay. Extracts prepared with ribose 5-phosphate in the absence of Mg₂₊ also showed low-K_m (CO₂) carboxylase activity initially, but the presence of this sugar phosphate was deleterious during storage at 25 °C, where it promoted conversion to the apparently high-K_m (CO₂) form.Effects on the affinity of ribulose diphosphate carboxylase for CO₂ were paralleled by effects on the activity of the associated ribulose diphosphate oxygenase. Treatments which produced the low-K_m (CO₂) form of the carboxylase also resulted in high oxygenase activity, and it is possible that the apparently high-K_m (CO₂) form of the carboxylase has little, if any, oxygenase activity associated with it.The carboxylase and oxygenase activities of the low-K_m (CO₂) form showed broad and quite similar responses to pH variation, and the oxygenase had a K_m for O₂ of 0.22 mm.The stability of the low-K_m (CO₂) form in the presence of Mg²⁺ and bicarbonate was quite sufficient for it to be partially purified by Sepharose chromatography. The significance of the low-K_m (CO₂) form is discussed with respect to activation of photosynthesis by Mg²⁺.     

Uptake of bicarbonate ion in darkness by isolated chloroplast envelope membranes and intact chloroplasts of spinach

Bicarbonate uptake by isolated chloroplast envelope membranes and intact chloroplasts of spinach (Spinacia oleracea L. var. Viroflay) in darkness exhibited a similar dependency upon temperature, pH, time, and concentrations of isolated or attached envelope membranes. This similarity in uptake properties demonstrates the usefulness of the envelope membranes for the study of chloroplast permeability. Maximal rates for dark HCO₃^- uptake by isolated envelope membranes and intact chloroplasts were more than sufficient to account for the maximal rates of photosynthetic CO₂ fixation observed with intact chloroplasts. The active species involved in the uptake process was found to be HCO₃^- and not CO₂. The significance of HCO₃^- uptake and its relationship to carbonic anhydrase and ribulose diphosphate carboxylase is discussed. Conditions for maximal HCO₃^- uptake in darkness by intact chloroplasts were found to be similar to those required for maximal photosynthetic CO₂ fixation, suggesting that HCO₃^- uptake by the envelope membrane may regulate photosynthetic CO₂ fixation.     

Inhibition of the circadian rhythm of CO2 metabolism in Bryophyllum leaves by cycloheximide and dinitrophenol

The circadian rhythm of CO₂ output in darkened leaves of Bryophyllum fedtschenkoi R. Hamet and Perrier can be inhibited by cycloheximide (10^-6 mol) and 2,4-dinitrophenol (10^-5 mol) applied via the transpiration stream. After having been suppressed by 10^-6 M cycloheximide, the rhythm can be reinitiated with a 12-h exposure to light. Experiments using ¹⁴CO₂ show that cycloheximide abolishes the rhythm by inhibiting the dark fixation of CO₂. Cycloheximide inhibits malate accumulation and acidification of the leaves, but does not affect the amount of the CO₂-fixing enzyme phosphoenol-pyruvate carboxylase (PEP-C, EC 4.1.1.31) which can be extracted from the leaves during the 45 h of the experiment. Cycloheximide has no direct effect on the activity of the enzyme as measured in the assay. PEP-C from desalted leaf extracts was inhibited by L-malate (K_i=0.4 mmol). The most likely explanation for the inhibitory effect of cycloheximide and dinitrophenol is that they cause changes in tonoplast properties which result in a redistribution of malate from the vacuole to the cytoplasm. An increase in malate concentration in the cytoplasm will lead to inhibition of PEP-carboxylase, and hence the suppression of the rhythm of CO₂ output.Abbreviations CAM crassulacean acid metabolism - PEP-C phosphoenol-pyruvate carboxylase - MDH malate dehydrogenase - CHM cycloheximide - DNP 2,4-dinitrophenol - LD light-dark-cycle - DD continuous darkness     

Properties of phosphoenolpyruvate carboxylase in desalted extracts from isolated guard-cell protoplasts

Properties of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) obtained from isolated guard-cell protoplasts of Vicia faba L. were determined following rapidly desalting of the extract on a Sephadex G 25 column. The activity of PEP carboxylase was measured as a function of PEP and malate concentration, pH and K⁺ concentration within 2–3 min after homogenization of the guard-cell protoplasts. The activity of this enzyme was stimulated by PEP concentrations of 0.1 to 0.75 mM and by K⁺ ions (12 mM), but inhibited by PEP concentrations above 1 mM and by malate. Changes in the K_m(PEP) and V_max values with increasing malate concentrations (2.5 and 5 mM) indicate that the malate level, varying in relation to the physiological state of guard cells, plays an important role in regulating the properties of phosphoenolpyruvate carboxylase.Abbreviations CAM Crassulacean acid metabolism - GCP guard-cell protoplast - PEP phosphoenolpyruvate Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Veränderliche Markierungsmuster bei14CO2-Fütterung vonBryophyllum tubiflorum zu verschiedenen Zeitpunkten der Hell/Dunkelperiode

Summary Detached phyllodia ofBryophyllum tubiflorum were fed under illumination with¹⁴CO₂ at different times during the light/dark period (12:12 hours). After photosynthesis in presence of¹⁴CO₂ during the intrinsic dark period the greatest part of soluble radioactivity was found in malate. When the same experiment was repeated during the light period, radioactivity was incorporated mainly into sucrose in the first hours while malate was labelled rather weakly. In the late afternoon (last third of the light period), malate became most heavily labelled again during photosynthesis with¹⁴CO₂.Our results indicate that the synthesis of malate by PEP-carboxylase/malate dehydrogenase is inhibited at certain times during the night/day period by end product inhibition of PEP-carboxylase, as was demonstrated byQueiroz (1967, 1968) andTing (1968) in vitro.During inhibition of the PEP-carboxylase there is no competition between the synthesis of malate and CO₂-fixation by the Calvin cycle. Thus radioactivity can flow into sucrose via the Calvin cycle during this time. When the malate content of the phyllodia is low, CO₂-fixation by PEP-carboxylase is not inhibited. Now this pathway dominates over photosynthesis via the Calvin cycle, for PEP-carboxylase has a higher affinity for CO₂ than carboxydismutase. Therefore malate now becomes more labelled than sucrose.     

d-Ribulose 1,5-diphosphate carboxylase from the blue-green alga Aphanocapsa 6308

d-Ribulose 1,5-diphosphate carboxylase from extracts of the unicellular blue-green alga Aphanocapsa 6308 has been purified by ammonium sulphate precipitation and linear sucrose density gradient centrifugation. The molecular weight was estimated to be 525 000 and the enzyme consisted of two types of sub-unit of molecular weights 51 000 and 15 000. The small sub-units were not detected after purification involving acid precipitation but were observed if the acid precipitation step was omitted. The Michaelis constants for Mg²⁺ and CO₂, when tested under air, were 0.35 mM and 0.071 mM respectively. Oxygen acted as a competitive inhibitor with respect to CO₂, suggesting that the enzyme also acts as an oxygenase. This was confirmed by measuring ribulose diphosphate-dependent O₂ uptake. A 1:1 stoichiometry between ribulose diphosphate utilization and O₂ consumption was observed. 6-Phosphogluconate inhibited carboxylase activity both at high (20 mM) and low (1 mM) bicarbonate concentrations. The data are compared with the properties of ribulose diphosphate carboxylase from other autotrophic prokaryotes and from chloroplasts.Abbreviations RuDP d-Ribulose 1,5-diphosphate - EDTA ethylene diamine tetraacetic acid - GSH reduced glutathione - SDS sodium dodecyl sulphate - 6PGluc 6-phosphogluconate - STB supplemented Tris buffer     

Malate inhibition of phosphoenolpyruvate carboxylase from crassula

Phosphoenolpyruvate carboxylase partially purified from leaves of Crassula and rendered insensitive to malate by storage without adjuvants can be altered to the form sensitive to malate inhibition by brief, 5-minute preincubation with 5 millimolar malate. The induction of malate sensitivity is reversible by lowering the malate²⁻ concentration. Of the reaction components only HCO₃⁻ increases the sensitivity to malate in subsequent assay. Phosphoenolpyruvate (PEP), which itself tends to lower sensitivity to subsequent malate inhibition, also reduces the effect of malate in the assay, as does glucose-6-phosphate. PEP isotherms showed that the insensitive or unpreincubated enzyme, responds to the presence of 5 millimolar malate during assay with a 3-fold increase in K_m, but no effect on V_max. Enzyme preincubated with malate shows the same effect of malate on K_m, but in addition V_max is inhibited 72%. It thus appears that both sensitive and insensitive forms of PEP carboxylase are subject to K-type inhibition by malate, but only the sensitive form also shows V-type inhibition. Preincubation with malate at different pH values showed that at pH 6.15, the inhibition by malate in subsequent assay at pH 7 was much lower than at pH 7 or 8. When the reaction is prerun for 30 minutes with increasing concentrations of PEP, subsequent assay with malate shows progressively less inhibition due to malate. When 0.3 millimolar PEP either alone or with 0.1 millimolar ATP and 0.3 millimolar NaF is present during preincubation, the effect of malate in a following assay is to activate the reaction. These results may indicate an effect of phosphorylation of the enzyme on sensitivity to malate.