Photosynthetic and Photorespiratory Carbon Metabolism in Mesophyll Protoplasts and Chloroplasts Isolated from Isogenic Diploid and Tetraploid Cultivars of Ryegrass (Lolium perenne L.)

Photosynthetic ¹⁴CO₂ fixation, [¹⁴C]glycolate formation, and the decarboxylation of [1-¹⁴C]glycolate and [1-¹⁴C]glycine by leaf mesophyll protoplasts isolated from isogenic diploid and tetraploid cultivars of ryegrass (Lolium perenne L.) were examined. The per cent O₂ inhibition of photosynthesis in protoplasts from the tetraploid cultivar was less than that of the diploid line at both 21 and 49% O₂. Kinetic studies revealed that the K_m (CO₂) for photosynthesis by the diploid protoplasts was about twice that of the tetraploid line. In contrast, the K_i (O₂) for protoplast photosynthesis was similar in both cultivars, as was the potential for oxidizing glycolate and glycine to CO₂ via the photorespiratory carbon oxidation cycle. Although the maximal rates of glycolate accumulation by the isolated protoplasts in the presence of 21% O₂ and a glycolate oxidase inhibitor were similar in the two cultivars, the percentage of total fixed ¹⁴C entering the [¹⁴C]glycolate pool and the ratio of the rate of [¹⁴C]glycolate formation to ¹⁴CO₂ fixation at 21% O₂ and low pCO₂ were about two times greater in protoplasts and intact chloroplasts isolated from the diploid line compared to the tetraploid. These results fully support the recent observation that a doubling of ploidy in various ryegrass cultivars reduced the K_m (CO₂) of purified ribulose bisphosphate carboxylase-oxygenase by about one-half without affecting the K_i (O₂) (Garrett 1978 Nature 274: 913-915).     

Enzymic and physicochemical characterization of ribulose 1,5-bisphosphate carboxylase/oxygenase from diploid and tetraploid cultivars of perennial ryegrass

Homogeneous preparations of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) were isolated from several diploid and tetraploid cultivars of perennial ryegrass (Lolium perenne L.) by three different purification protocols. The apparent K_m values for substrate CO₂ were essentially identical for the fully $\text{CO}{}_2\text{Mg}{}^{\mathrm{2+}}$-activated diploid and tetraploid enzymes, as were the kinetics for deactivation and activation of the $\text{CO}{}_2\text{Mg}{}^{\mathrm{2+}}$ activated and -depleted carboxylases, respectively. Similarly, virtually indistinguishable electrophoretic properties were observed for both the native and dissociated diploid and tetraploid ryegrass proteins, including native and subunit molecular weights and the isoelectric points of the native proteins and the large and small subunit component polypeptides. The quantity of carboxylase protein or total soluble leaf protein did not differ significantly between the diploid and tetraploid cultivars. Contrary to a previous report (M. K. Garrett, 1978, Nature (London)274, 913–915), these results indicate that increased ploidy level (i.e., nuclear gene dosage) has had essentially no effect on the quantity or enzymic and physicochemical properties of ribulosebisphosphate carboxylase/ oxygenase in perennial ryegrass.     

Ploidy Effects in Isogenic Populations of Alfalfa : II. Photosynthesis, Chloroplast Number, Ribulose-1,5-Bisphosphate Carboxylase, Chlorophyll, and DNA in Protoplasts

Photosynthetically-active protoplasts isolated from isogenic sets of diploid-tetraploid and tetraploid-octoploid alfalfa (Medicago sativa L.) leaves were used to investigate the consequences of polyploidization on several aspects related to photosynthesis at the cellular level. Protoplasts from the tetraploid population contained twice the amount of DNA, ribulose-1,5-bisphosphate carboxylase (RuBPCase), chlorophyll (Chl), and chloroplasts per cell compared to protoplasts from the diploid population. Although protoplasts from the octoploid population contained nearly twice the number of chloroplasts and amount of Chl per cell as tetraploid protoplasts, the amount of DNA and RuBPCase per octoploid cell was only 50% higher than in protoplasts from the tetraploid population. The rate of CO₂-dependent O₂ evolution in protoplasts nearly doubled with an increase in ploidy from the diploid to tetraploid level, but increased only 67% with an increase in ploidy from the tetraploid to octoploid level. Whereas leaves and protoplasts had similar increases in RuBPCase, DNA, and Chl with increase in ploidy level, it was concluded that increased cell volume rather than increased cell number per leaf is responsible for the increase in leaf size with ploidy.     

Km values of ribulose-1,5-bisphosphate carboxylase of cassava cultivars

Ribulose-1,5-bisphosphate (RuBP) carboxylase activities of two cassava cultivars increased with leaf age but their K_m(CO₂) and K_m(RuBP) values remained relatively constant. K_m(CO₂) values of 16 cassava cultivars ranged from 7.8 to 14.0 μM CO₂, while K_m(RuBP) values varied from 7.5 to 24.8 μM RuBP. Differences in the K_m values could not be attributed to different physiological ages of plant material or to intravarietal variation, and are more likely to have been inherited. The results also showed that K_m values have potential applications in cassava systematics.     

Study on the response of diploid,tetraploid and hexaploid species of wheat to the elevated CO2

Study was done to compare the response of Triticum aestivum (hexaploid), Triticum durum (tetraploid) and Triticum monococcum (diploid) wheat species to the elevated CO₂ using Free Air CO₂ Enrichment (FACE) facility. It was demonstrated that the modern cultivar of wheat Triticum aestivum (hexaploid) was largely sink limited. It appeared to have less photosynthesis per unit leaf area than Triticum monococcum (diploid wheat). While leaf size, grain weight and amylase activity increased with the ploidy level from diploid to hexaploid wheat forms, the photosynthetic rate was reduced significantly. These wheat species responded differentially to the elevated CO₂. The larger leaf area and greater seed weight and presence of 38 KDa protein band caused by elevated CO₂ had additive effect in improving the productivity of hexaploid wheat by changing the source sink ratio. Whereas, such a source sink balance was not induced by elevated CO₂ in diploid wheat. The increasing CO₂ may present opportunities to breeders and possibly allow them to select for cultivars responsive to the elevated CO₂ with better sink potential.Key words: Elevated CO₂, FACE technology, Photosynthesis, Seed weight, Source sink ratio, Triticum     

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⁺.     

Variations in K(m)(CO(2)) of Ribulose-1,5-bisphosphate Carboxylase among Grasses

A survey of the K_m(CO₂) values of ribulose-1,5-bisphosphate carboxylase from 60 grass species shows that enzyme from C₃ grasses consistently exhibits lower K_m(CO₂) than does that from C₄ grasses. Systematically ordered variation in K_m(CO₂) of ribulose-1,5-bisphosphate carboxylases from C₃ and C₄ grasses is also apparent and, among C₄ grasses, this shows some correlation with C₄ types.     

The effect of pH on the inorganic carbon source for photosynthesis in the seagrass Zostera muelleri irmisch ex aschers

The ability of the seagrass Zostera muelleri Irmisch ex Aschers. to use HCO₃⁻ as well as CO₂ for photosynthesis was investigated by measuring photosynthetic O₂ evolution over a range of pH values. It was found that the apparent K_m CO₂ fell from 0.128 mM at pH 7.9 to 0.016 mM at pH 9.1 indicating that HCO₃⁻ as well as CO₂ may act as a substrate for photosynthesis.The true K_m CO₂ could not be determined due to inhibition of photosynthesis at pHs less than 7.8 K_m CO₂ must be at least 0.128 mM, the apparent K_m at pH 7.9, and is probably of the order of 0.200 mM CO₂, the same as that reported for other marine plants. K_m HCO₃⁻¹ is about 20 mM when CO₂-dependent photosynthesis is minimal. Such a high K_m HCO₃⁻ resembles values reported for freshwater, rather than marine plants.Photosynthetic O₂ evolution is not saturated with respect to total inorganic carbon in natural seawater (pH 8.2). It is suggested that the distinctive shoulder from pH 8.1 to 8.5 in the pH profile of photosynthetic O₂ evolution at a constant concentration of inorganic carbon is caused by an effect of pH on HCO₃⁻ uptake. The effect of pH on HCO₃⁻ uptake was determined by constructing a pH profile of photosynthesis at constant HCO₃⁻ concentration, and subtracting the estimated contribution of CO₂ to photosynthesis from this rate. The resultant curve has a maximum at pH 8.4 and declines sharply at pHs less than 8.     

A possible role for C4 photosynthetic enzymes in tolerance of Zea mays to NaCl

Treatment of 14-day-old maize cultivars (Hybrid351 and Giza2) with 250 mM NaCl significantly reduced shoot fresh and dry weights and protein content during the subsequent 12 days. The magnitude of reduction was more pronounced in Giza than Hybrid. Both cultivars contained converging levels of protein for the enzymes phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), pyruvate phosphate dikinase (PPDK) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) under normal conditions; however, NaCl led to increase these levels in Hybrid and decrease them in Giza. Moreover, NaCl significantly inhibited the activities of PEPC, MDH and PPDK in both cultivars during the first 2 days, thereafter the inhibition nullified only in Hybrid; nonetheless, Rubisco was the least affected enzyme in both cultivars. In addition, NaCl slightly increased V _max of PEPC, MDH and PPDK in Hybrid with no change in K _m; nevertheless V _max dropped in Giza with an increase in K _m of only PEPC and MDH. Also K _cat, K _cat/K _m and V _max/K _m of all enzymes were lower in treated Giza than in treated Hybrid. The increased V _max of all enzymes in only Hybrid by NaCl confirms that they were synthesised more in Hybrid than in Giza. However, the decreased V _max in Giza concomitant with the increased K _m points to an interference of salinity with synthesis of enzymes and their structural integrity. This would lead to a noncompetitive inhibition for the enzymes. These findings declare that maize tolerance to NaCl was larger in Hybrid compared to Giza due to a role for C4 enzymes.     

Purification and some properties of two polyphenol oxidases from bartlett pears

Two polyphenol oxidases (enzymes A and B) from Bartlett pear (Pyrus communis) peelings were purified to electrophoretic homogeneity according to polyacrylamide gel by a combination of Sephadex gel filtration, diethylaminoethyl cellulose chromatography and hydroxyl apatite chromatography. While the two enzymes differ electrophoretically at pH 9.3, chromatographically on hydroxyl apatite, and in the effect of ionic strength on activity, they are similar with respect to chromatography on diethylaminoethyl cellulose, substrate specificity, pH activity relations, inhibition by p-coumaric and benzoic acids, and heat stability. The two enzymes are o-diphenol oxidases with no detectable monophenolase or laccase activities. Pyrocatechol, 4-methyl catechol, chlorogenic acid, and d-catechin are good substrates of the enzymes with K_m values in the range of 2 to 20 mm. Dependences of activity on oxygen and chlorogenic acid concentrations indicate a sequential mechanism for binding of these substrates to enzyme B. V_max and K_m values for oxygen and chlorogenic acid were 103 μmoles O₂ uptake per minute per milligram of enzyme, 0.11 mm and 7.2 mm, respectively, for enzyme B at pH 4.0. Both enzymes had maximum activity at pH 4.0 on chlorogenic acid. K_m values for chlorogenic acid were independent of pH from 3 to 7; the V_max values for both enzymes gave bell-shaped curves as a function of pH. p-Coumaric acid is a simple, linear noncompetitive inhibitor with respect to chlorogenic acid at pH 6.2 with K_i values of 0.38 and 0.50 mm for enzymes A and B, respectively. Benzoic acid is a linear competitive inhibitor with respect to chlorogenic acid at pH 4.0 with K_i values of 0.04 and 0.11 mm for enzymes A and B, respectively.     

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.     

Photorespiratory properties of protoplasts from C3-C4 intermediate species of moricandia

Protoplasts were isolated from leaves of the C₃-C₄ intermediate species, Moricandia arvensis (L.) DC. and Moricandia spinosa Pomel. Analysis by light and transmission electron microscopy indicated that these purified preparations contained both mesophyll protoplasts (MP) and bundle-sheath protoplasts (BSP). Conventional density gradient centrifugation procedures failed to yield separations of pure protoplasts from each cell-type. With these heterogeneous suspensions of MP and BSP, values measured for (i) the percentage inhibition of photosynthetic CO₂ fixation by O₂, (ii) the apparent K_m(CO)2 of photosynthesis, and (iii) dark/light ratios of the rate of ¹⁴CO₂ evolution during decarboxylation of exogenous [1-¹⁴C] glycine were not significantly different from those determined for protoplast preparations from related or representative C₃ plants, including M. foetida, Nicotiana tabacum, and Triticum aestivum. In contrast, previous comparisons with C₃ species, using intact leaf tissue from M. arvensis, have shown a reduced sensitivity of net photosynthesis to inhibition by O₂ [Holaday et al., Plant Sci. Lett., 27 (1982) 181] and an enhanced capacity for the photosynthetic refixation of CO₂ evolved during decarboxylation of exogenous photorespiratory substrates [Holbrook et al., Plant Physiol., 77 (1985) 578]. We conclude that these photosynthetic properties, associated with reduced photorespiration by M. arvensis and M. spinosa, are dependent upon the integrity of the anatomical and ultrastructural arrangement of bundle-sheath and mesophyll cells in these C₃-C₄ intermediate species.     

2-carboxy-d-hexitol 1,6-bisphosphate: An inhibitor of d-ribulose 1,5-bisphosphate carboxylase/oxygenase

2-Carboxy-d-hexitol 1,6-bisphosphate (CHBP) has been prepared from d-fructose 1,6-bisphosphate and cyanide. DEAE-Sephadex chromatography separated the reaction products into two fractions which were identified as CHBP and CHBP-lactone. CHBP is presumably a mixture of two diastereomers, 2-carboxy-d-glucitol 1,6-bisphosphate and 2-carboxy-d-mannitol 1,6-bisphosphate, but an attempt to separate these compounds was not successful. The material in the CHBP-lactone peak had no effect on d-ribulose 1,5-bisphosphate (RuBP) carboxylase. However, CHBP was a potent reversible inhibitor of RuBP carboxylases. This compound displayed an inhibition constant (K_i at pH 8.0 and 30 °C) of 1–2 μm with the enzymes from spinach and barley, while the K_i was 60–70 μm with bacterial RuBP carboxylases from Pseudomonas oxalaticus and Rhodospirillum rubrum. The mode of inhibition was competitive with respect to RuBP for all the carboxylases, and noncompetitive with respect to CO₂ for the enzymes from spinach, P. oxalaticus and R. rubrum. The results indicate that, in the binding of certain organic phosphates by RuBP carboxylases, there may be a fundamental difference between the enzymes isolated from microbial and from higher plant sources. RuBP oxygenase activities from spinach and P. oxalaticus were also inhibited by CHBP, with K_i values which were similar to those obtained with the carboxylase activity of the same enzymes. The mode of inhibition of the oxygenase activities was also competitive with respect to RuBP. Thus, it seems that the binding of CHBP is similar for the carboxylase and oxygenase reactions of the same enzyme.     

Relation of CO(2) Compensation Concentration to Apparent Photosynthesis in Maize

Significant differences in CO₂ compensation concentration measured in the field among varieties of the species Zea mays L. are reported for the first time. CO₂ compensation concentrations were significantly (P≤ 0.01) and negatively correlated with apparent photosynthesis at 300 μl CO₂/liter air. The Michaelis constant (as defined) for a leaf was significantly (P≤ 0.01) and positively correlated with apparent photosynthesis among varieties. While the first correlation is similar to behavior of CO₂ compensation among species of different photosynthetic efficiency, the latter correlation is the converse of the behavior of Km among species.     

Km (CO2) values of ribulose-1,5-bisphosphate carboxylase in grasses of different C4 type

Statistical analysis of K_m (CO₂) values of ribulose-1,5-bisphosphate (RuBP) carboxylase from 35 C₄ grass species shows that the mean value for PEP-carboxykinase (PCK) type C₄ species (41.4±s.e. 2.2 μM CO₂) is significantly different from that of NAD-malic enzyme (NAD-ME) type species (55.3±3.1 μM CO₂) or NADP-malic enzyme (NADP-ME type species (52.5±s.e. 2.0μM CO₂). These C₄ type differences remain detectable within both the eu-panicoid and chloridoid grass subfamilies. By contrast, no between-subfamily differences were found within C₄ types. Variation in K_m (CO₂) values of RuBP carboxylase may be related to in vivo differences in CO₂ concentration at the enzyme site, mediated perhaps by differences in CO₂-leakiness of C₄ leaf ‘photosynthetic carbon reduction’ (PCR or ‘Kranz’) tissue.     

Rat brain aspartate β-decarboxylase. A comparative study with the liver enzyme

Aspartate β-decarboxylase (AspD), which catalyses the β-decarboxylation of aspartate (Asp) to alanine (Ala), was found in significant quantities only in the brain, kidney and liver. This enzyme has an optimum pH at 7.4. Addition of exogenous pyridoxal 5′-phosphate did not increase enzyme activity presumably because of firmly bound cofactor. However, aminooxyacetic acid is a potent inhibitor.There is an apparent 8-fold variation in AspD in the seven brain regions studied, with the highest activities in the cortex and the lowest in the striatum and hippocampus. In the presence of α-ketoglutarate, the production of ¹⁴CO₂ from [¹⁴C]Asp may no longer represent AspD activity due to active transamination of Asp, presumably by aspartate aminotransferase, to oxaloacetate. Under such conditions, comparable AspD activities were observed in all seven brain regions.Kinetic analysis showed that the liver and kidney enzymes have identical affinity for Asp (K_m = 3.5 mM) while the brain enzyme has a higher affinit (K_m = 1.3 mM). The V_max values obtained indicated that the enzyme populations in liver, kidney and brain are in the ratio 18:4:1. Various amino acids were found to inhibit both brain and liver AspD. Serine, however, activated the liver enzyme but inhibited competitively the kidney and brain enzymes. These results indicate that AspD may exist as two or more isozymes.     

Hexose kinases from Rhodotorula glutinis. Identification and properties of an hexokinase and a glucokinase.

An hexokinase (EC 2.7.1.1) and a glucokinase (EC 2.7.1.2) from the red yeast Rhodotorula glutinis are described. Both enzymes have been separated and some of their properties studied. The two enzymes share many properties, the K_mfor glucose is 0.1 mm for both enzymes and the K_m values for ATP are 0.5 mm and 0.6 mm respectively for hexokinase and glucokinase. The hexokinase shows a K_m of 2 mm for fructose and 0.1 mm for mannose; the glucokinase has a K_m for mannose of 0.2 mm. Both enzymes are constitutive, show competitive inhibition by N-acetylglucosamine and xylose, have weak affinity for glucosamine and exhibit a broad pH optimum. The molecular weights determined by gel filtration are 110,000 for glucokinase and 96,000 for hexokinase. The maximal activity of both hexose kinases nearly accounts for glucose utilization by Rh. glutinis.     

Subcellular Location of NADP-Isocitrate Dehydrogenase in Pisum sativum Leaves

The subcellular location of NADP⁺-isocitrate dehydrogenase was investigated by preparing protoplasts from leaves of pea seedlings. Washed protoplasts were gently lysed and the whole lysate separated on sucrose gradients by a rate-zonal centrifugation. Organelles were located by marker enzymes and chlorophyll analysis. Most of the NADP⁺-isocitrate dehydrogenase was in the soluble fraction. About 10% of the NADP⁺-isocitrate dehydrogenase was present in the chloroplasts as a partially latent enzyme. Less than 1% of the activity was found associated with the peroxisome fraction. NADP⁺-isocitrate dehydrogenase was partially characterized from highly purified chloroplasts isolated from shoot homogenates. The enzyme exhibited apparent K_m values of 11 micromolar (NADP⁺), 35 micromolar (isocitrate), 78 micromolar (Mn²⁺), 0.3 millimolar (Mg²⁺) and showed optimum activity at pH 8 to 8.5 with Mn²⁺ and 8.8 to 9.2 with Mg²⁺. The NADP⁺-isocitrate dehydrogenase activity previously claimed in the peroxisomes by other workers is probably due to isolation procedures and/or nonspecific association. The NADP⁺-isocitrate dehydrogenase activity in the chloroplasts might help supply α-ketoglutarate for glutamate synthase action.     

The role of effector molecules in regulating ribulosebisphosphate carboxylase activity

Ribulosebisphosphate carboxylase can exist in two forms having different kinetic properties. The fraction of enzyme present in each of the two forms is determined by both the absolute and the relative amounts of substrates and other effector molecules in solution with the enzyme. High CO₂ levels induce formation of an active CO₂ form of the enzyme while high ribulosebisphosphate (RuBP) levels cause formation of a much less active form. The CO₂ form is characterized by a high K_m(RuBP), low K_m(CO2), and relatively high V. The ribulosebisphosphate form has comparatively lower K_m(RuBP) and V and higher K_m(CO2). CO₂ appears to bind before RuBP in the reaction sequence catalyzed by the CO₂ form; this catalytic binding order is apparently reversed in the RuBP form. Steady state rates of enzyme reaction reflect the contributions of both these forms. A brief model, based on the cooperative effects of binding at a small number of catalytic or activator sites in the multimeric enzyme, is presented to account for the changes in enzyme activity with varying substrate and effector molecule concentrations.