Crassulacean acid metabolism (CAM) in Kalanchoë daigremontiana: Temperature response of phosphoenolpyruvate (PEP)-carboxylase in relation to allosteric effectors

Net CO₂ dark fixation of Kalanchoë daigremontiana varies with night temperature. We found an optimum of fixation at about 15° C; with increasing night temperature fixation decreased. We studied the temperature dependence of the activity of phosphoenolpyruvate (PEP)-carboxylase, the key enzyme for CO₂ dark fixation. We varied the pH, the substrate concentration (PEP), and the L-malate and glucose-6-phosphate (G-6-P) concentration in the assay. Generally, lowering the pH and reducing the amount of substrate resulted in an increase in activation by G-6-P and in an increase in malate inhibition of the enzyme. Furthermore, malate inhibition and G-6-P activation increased with increasing temperature. Activity measurements between 10° C and 45°C at a given concentration of the effectors revealed that the temperature optimum and maximum activities at that optimum varied with the effector applied. Under the influence of 5 mol m^-3 L-malate the temperature optimum and maximum activity dropped drastically, especially when the substrate level was low (at 0.5 mol m^-3 PEP from 32° C to 20° C). G-6-P raised the temperature optimum and maximum activity when the substrate level was low. If both malate and G-6-P were present, intermediate values were measured. We suggest that changes in metabolite levels in K. daigremontiana leaves can alter the temperature features of PEP-carboxylase so that the observed in vivo CO₂ dark fixation can be explained on the basis of PEP-carboxylase activity.Abbreviations PEP-c phosphoenolpyruvate carboxylase - CAM crassulacean acid metabolism - PEP phosphoenolpyruvate - G-6-P glucose-6-phosphate     

Fructose-and sedoheptulosebisphosphatase. The sites of a possible control of CO2 fixation by light-dependent changes of the stromal Mg2+ concentration

1. The enzymatic steps of the CO₂ fixation cycle responsible for the overall inhibition of CO₂ fixation caused by the lowering of the Mg²⁺ concentration in the stroma were investigated. For this the Mg²⁺ concentration in the stroma was decreased by addition of the ionophore A 23187, and the levels of the intermediates of the CO₂ fixation cycle in the stroma of intact chloroplasts were assayed by ion exchange chromatography.2. The addition of the ionophore caused an increase of NADPH, ATP, fructose- and sedoheptulosebisphosphate and a dramatic decrease of phosphoglycerate in the stroma. These changes were reversed by the addition of Mg²⁺ and again affected by a subsequent addition of Ca²⁺. Ribulosebisphosphate and pentosemonophosphate levels in the stroma were only a little affected under these different conditions.3. The increase of the NADPH and ATP reflects the decreased utilization of these compounds due to the overall inhibition of CO₂ fixation. As phosphoglycerate and triosephosphate appear to be in near equilibrium with NADPH and ATP, the decrease of phosphoglycerate seems to be a consequence of the changes in the nucleotide levels.4. The rapid increase of fructose- and sedoheptulosebisphosphate after the addition of the ionophore A 23187 clearly demonstrates that the overall inhibition of CO₂ fixation caused by lowering the stromal Mg²⁺ is due to the inhibition of the hydrolysis of these sugar bisphosphates. It is concluded that the activities of fructose- and sedoheptulosebisphosphatase can be controlled by light dependent changes of the stromal Mg²⁺ concentration.     

Regulation of fructose-1,6-bisphosphatase activity in leaves

Fructose-1,6-bisphosphatase (EC 3.1.3.11) activity increased markedly (greater than 10-fold) upon illumination of wheat leaves. Darkening caused a relatively slow but complete reversal of light activation. The effects of O₂ and CO₂ concentration and light intensity on fructose-bisphosphatase activation were measured. In ratelimiting light, 2% O₂ stimulated enzyme activity, whereas varying the CO₂ concentration had little effect. In saturating light, lowering the oxygen tension had no effect, but CO₂ at near-saturating concentrations for photosynthesis inhibited enzyme activity. Dark inactivation of the enzyme was completely prevented by incubation of leaves in N₂, but was facilitated by O₂, indicating that O₂ is the major oxidant in darkened leaves. It is argued that while fructose bisphosphatase is redox-regulated in leaves, modulation of enzyme activity by this mechanism is unlikely to contribute to the regulation of CO₂ fixation in leaves.     

Growth,14CO2 fixation,activites of photosystems,ribulose 1,5-bisphosphate carboxylase and nitrate reductase in trees as affected by simulated acid rain

In seedlings of the tropical tree speciesErythrina variegata Lam. andHardwickia binata Roxb. exposed to different acidic mist (H₂SO₄, pH 5, 3 and 2) for 5 d significant reduction in seedling growth, biomass accumulation and¹⁴CO₂ fixation were determined. In isolated chloroplasts a decrease in the activities of photosystem 2 and whole electron transport chain was observed only at pH 3 and 2, but no significant change in photosystem 1 activity was observed. SDS-PAGE analysis of crude leaf extracts of ribulose 1,5-bisphosphate carboxylase (RuBPC) indicated a significant loss of 55 and 15 kDa polypeptides at pH 2 inErythrina. The reduction in the RuBPC activity in seedlings grown under acidic mists correlated well with CO₂ fixation.     

Effect of molecular hydrogen and carbon dioxide on chemo-organotrophic growth of Acetobacterium woodii and Clostridium aceticum

During growth of Acetobacterium woodii on fructose, glucose or lactate in a medium containing less than 0.04% bicarbonate, molecular hydrogen was evolved up to 0.1 mol per mol of substrate. Under an H₂-atmosphere growth of A. woodii with organic substrates was completely inhibited whereas under an H₂/CO₂-atmosphere rapid growth occurred. Under these conditions H₂+CO₂ and the organic substrate were utilized simultaneously indicating that A. woodii was able to grow mixotrophically. Clostridium aceticum differed from A. woodii in that H₂ was only evolved in the stationary phase, that the inhibition by H₂ was observed at pH 8.5 but not at pH 7.5, anf that in the presence of fructose and H₂+CO₂ only fructose was utilized.The hydrogenase activity of fructose-grown cells of C. aceticum amounted to only 12% of that of H₂+CO₂-grown cells. With A. woodii a corresponding decrease of the activity of this enzyme was not observed.     

Pyruvate fermentation in light-grown cells ofRhodospirillum rubrum during adaptation to anaerobic dark conditions

Pyruvate fermentation inRhodospirillum rubrum (strains F₁, S₁, and Ha) was investigated using cells precultured on different substrates anaerobically in the light and than transferred to anaerobic dark conditions. Pyruvate formate lyase was always the key enzyme in pyruvate fermentation but its activity was lower than in cells which have been precultured aerobically in darkness. The preculture substrate also had a clear influence on the pyruvate formate lyase activity. Strains F₁ and S₁ metabolized the produced formate further to H₂ and CO₂. A slight production of CO₂ from pyruvate, without additional H₂-production, could also be detected. It was concluded from this that under anaerobic dark conditions a pyruvate dehydrogenase was also functioning. On inhibition of pyruvate formate lyase the main part of pyruvate breakdown was taken over by pyruvate dehydrogenase.When enzyme synthesis was inhibited by chloramphenicol, propionate production in contrast to formate production was not affected. Protein synthesis was not significant during anaerobic dark culture. Bacteriochlorophyll. however, showed, after a lag phase, a clear rise.Abbreviations Bchl Bacteriochlorophyll - CoA Coenzyme A - DSM Deutsche Sammlung von Mikroorganismen (Göttingen) - OD optical density - PHBA poly--hydroxybutyric acid - R Rhodospirillum     

Inhibited light activation of fructose and sedoheptulose bisphosphatase in spinach chloroplasts exposed to osmotic stress

The light activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) and sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37) was inhibited in isolated intact spinach (Spinacia oleracea L.) chloroplasts exposed to reduced osmotic potentials. Decreases in the velocity and magnitude of light activation correlated with the overall reduction in CO₂ fixation rates. Responses of osmotically stressed chloroplasts to both varying pH and exogeous dihydroxyacetone phosphate (DHAP) or 3-phosphoglycerete (PGA) were examined. In the presence of DHAP, the absolute rate of CO₂ fixation was increased and this increase was most pronounced at alkaline pH. Enhanced light activation of these enzymes was also observed under these conditions. However, in the presence of PGA, similar increases in photosynthetic rate and enzyme activation were not evident. Light-dependent stromal alkalization was unaffected by the stress treatments. Inhibition of light activation under hypertonic conditions is discussed in terms of substrate availability, possible alterations of the redox state of ferredoxin and associated electron carriers, and inhibited enzyme-enzyme or enzyme-substrate interactions involved in the light activation process.Abbreviations and symbols DHAP dihydroxyacetone phosphate - PGA 3-phosphoglycerate - _s osmotic potential     

On the Participation of Phosphoribulokinase in the Light Regulation of CO(2) Fixation

CO₂ fixation by a suspension of isolated spinach chloroplasts was terminated by turning off the light, and changes of metabolite levels in the chloroplast stroma and the surrounding medium were assayed. Whereas CO₂ fixation comes to a total stop within 15 seconds, a conversion of triose phosphates to heptose, hexose, and pentose monophosphates is found to occur for 1 to 2 minutes afterwards. It seems from these data that an inactivation of fructose and sedoheptulose bisphosphatases proceeds with a lag period. In contrast, the conversion of pentose monophosphates to ribulose 1,5-bisphosphate is inhibited immediately after the stop of illumination. As the stromal level of freely available ATP was not depleted under this condition, these data demonstrate that ribulose 5-phosphate kinase was very rapidly inactivated after darkening of the chloroplasts. Essentially, the same effect is also observed when CO₂ fixation is partially inhibited by addition of moderate concentrations of m-chlorocarbonyl phenylhydrazone, partially uncoupling photophosphorylation. It appears from these results, that the activity of ribulose 5-phosphate kinase is not only regulated by light through the mediation of reduced carriers like thioredoxin but also by alternative parameters, e.g. stromal metabolite levels.     

The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO2 fixation in the light and dark

1. The pH in the stroma and in the thylakoid space has been measured in a number of chloroplast preparations in the dark and in the light at 20 °C. Illumination causes a decrease of the pH in the thylakoid space by 1.5 and an increase of the pH in the stroma by almost 1 pH unit.2. CO₂ fixation is shown to be strongly dependent on the pH in the stroma. The pH optimum was 8.1, with almost zero activity below pH 7.3. Phosphoglycerate reduction, which is a partial reaction of CO₂ fixation, shows very little pH dependency.3. Low concentrations of the uncoupler m-chlorocarbonylcyanide phenylhydrazone (CCCP) inhibit CO₂ fixation without affecting phosophoglycerate reduction. This inhibition of CO₂ fixation appears to be caused by reversal of light induced alkalisation in the stroma by CCCP.4. Methylamine has a very different effect compared to CCCP. Increasing concentrations of methylamine inhibit CO₂ fixation and phosphoglycerate reduction to the same extent. The light induced alkalisation of the stroma appears not to be significantly inhibited by methylamine, but the protons in the thylakoid space are neutralized. The inhibition of CO₂ fixation by higher concentrations of methylamine is explained by an inhibition of photophosphorylation. It appears that methylamine does not abolish proton transport.5. It is shown that intact chloroplasts are able to fix CO₂ in the dark, yielding 3-phosphoglycerate. This requires the addition of dihydroxyacetone phosphate as precursor of ribulosemonophosphate and also to supply ATP, and the addition of oxaloacetate for reoxidation of the NADPH in the stroma.6. Dark CO₂ fixation in the presence of dihydroxyacetone phosphate and oxaloacetate has the same pH dependency as CO₂ fixation in the light. This demonstrates that CO₂ fixation in the dark is not possible, unless the pH in the medium is artificially raised to pH 8.8.7. It is shown that pH changes occurring in the stroma after illumination are sufficient to switch CO₂ fixation from zero to maximal activity. This offers a mechanism for light control of CO₂ fixation, avoiding wasteful CO₂ fixation in the dark.     

Stomatal responses to carbon dioxide of isolated epidermis from a C3 plant,the Argenteum mutant of Pisum sativum L., and a crassulacean-acid-metabolism plant Kalanchoë daigremontiana Hamet et Perr

The response of stomata in isolated epidermis to the concentration of CO₂ in the gaseous phase was examined in a C₃ species, the Argenteum mutant of Pisum sativum, and a crassulacean-acid-metabolism (CAM) species, Kalanchoë daigremontiana. Epidermis from leaves of both species was incubated on buffer solutions in the presence of air containing various volume fractions of CO₂ (0 to 10000·10^–6). In both species and in the light and in darkness, the effect of CO₂ was to inhibit stomatal opening, the maximum inhibition of opening occurring in the range 0 to 360·10^–6. The inhibition of opening per unit change in concentration was greatest between volume fractions of 0 and 240·10^–6. There was little further closure above the volume fraction of 360·10^–6, i.e. approximately ambient concentration of CO₂. Thus, although leaves of CAM species may experience much higher internal concentrations of CO₂ in the light than those of C₃ plants, this does not affect the sensitivity of their stomata to CO₂ concentration or the range over which they respond. Stomatal responses to CO₂ were similar in both the light and the dark, indicating that effects of CO₂ on stomata occur via mechanisms which are independent of light. The responses of stomata to CO₂ in the gaseous phase took place without the treatments changing the pH of the buffered solutions. Thus it is unlikely that CO₂ elicited stomatal movement by changing either the pH or the HCO ₃ ^– /CO ₃ ^2- equilibria. It is suggested that the concentration of dissolved unhydrated CO₂ may be the effector of stomatal movement and that its activity is related to its reactivity with amines.     

Energetics and regulations of formate and hydrogen metabolism by Methanobacterium formicicum

Accumulation of formate to millimolar levels was observed during the growth of Methanobacterium formicicum species on H₂–CO₂. Hydrogen was also produced during formate metabolism by M. formicicum. The amount of formate accumulated in the medium or the amount H₂ released in gas phase was influenced by the bicarbonate concentration. The formate hydrogenlyase system was constitutive but regulated by formate. When methanogenesis was inhibited by addition of 2-bromoethane sulfonate, M. formicicum synthesized formate from H₂ plus HCO _inf3 ^sup- or produced H₂ from formate to a steady-state level at which point the Gibbs free energy (G) available for formate synthesis or H₂ production was approximately -2 to -3 kJ/reaction. Formate conversion to methane was inhibited in the presence of high H₂ pressure. The relative rates of conversion of formate and H₂ were apparently controlled by the G available for formate synthesis, hydrogen production, methane production from formate and methane production from H₂. Results from ¹⁴C-tracer tests indicated that a rapid isotopic exchange between HCOO^- and HCO _inf3 ^sup- occurred during the growth of M. formicicum on H₂–CO₂. Data from metabolism of ¹⁴C-labelled formate to methane suggested that formate was initially split to H₂ and HCO _inf3 ^sup- and then subsequently converted to methane. When molybdate was replaced with tungstate in the growth media, the growth of M. formicicum strain MF on H₂–CO₂ was inhibited although production of methane was not Formate synthesis from H₂ was also inhibited.     

A CO2-Flux Mechanism Operating via pH-Polarity in Hydrilla Verticillata Leaves With C3 and C4 Photosynthesis

The aquatic angiosperm Hydrilla verticillata lacks Kranz anatomy, but has an inducible, C₄-based, CO₂ concentrating mechanism (CCM) that concentrates CO₂ in the chloroplasts. Both C₃ and C₄ Hydrilla leaves showed light-dependent pH polarity that was suppressed by high dissolved inorganic carbon (DIC). At low DIC (0.25 mol m⁻³), pH values in the unstirred water layer on the abaxial and adaxial sides of the leaf were 4.2 and10.3, respectively. Abaxial apoplastic acidification served as a CO₂ flux mechanism (CFM), making HCO₃ ⁻ available for photosynthesis by conversion to CO₂. DIC at 10 mol m⁻³ completely suppressed acidification and alkalization. The data, along with previous results, indicated that inhibition was specific to DIC, and not a buffer effect. Acidification and alkalization did not necessarily show 1:1 stoichiometry; their kinetics for the apolar induction phase differed, and alkalization was less inhibited by 2.5 mol m⁻³ DIC. At low irradiance (50 μmol photons m⁻² s⁻¹), where CCM activity in C₄ leaves is minimized, both leaf types had similar DIC inhibition of pH polarity. However, as irradiance increased, DIC inhibition of C₃ leaves decreased. In C₄ leaves the CFM and CCM seemed to compete for photosynthetic ATP and/or reducing power. The CFM may require less, as at low irradiance it still operated maximally, if [DIC] was low. Iodoacetamide (IA), which inhibits CO₂ fixation in Hydrilla, also suppressed acidification and alkalization, especially in C₄ leaves. IA does not inhibit the C₄ CCM, which suggests that the CFM and CCM can operate independently. It has been hypothesized that irradiance and DIC regulate pH polarity by altering the chloroplastic [DIC], which effects the chloroplast redox state and subsequently redox regulation of a plasma-membrane H⁺-ATPase. The results lend partial support to a down-regulatory role for high chloroplastic [DIC], but do not exclude other sites of DIC action. IA inhibition of pH polarity seems inconsistent with the chloroplast NADPH/NADP⁺ ratio being the redox transducer. The possibility that malate and oxaloacetate shuttling plays a role in CFM regulation requires further investigation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Light inhibition of the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in leaves is mediated through carbon dioxide

The mechanism of light-inhibited ethylene production in excised rice (Oryza sativa L.) and tobacco (Nicotiana tabacum L.) leaves was examined. In segments of rice leaves light substantially inhibited the endogenous ethylene production, but when CO₂ was added into the incubation flask, the rate of endogenous ethylene production in the light increased markedly, to a level which was even higher than that produced in the dark. Carbon dioxide, however, had no appreciable effect of leaf segments incubated in the dark. The endogenous level of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, was not significantly affected by lightdark or CO₂ treatment, indicating that dark treatment or CO₂exerted its effect by promoting the conversion of ACC to ethylene. This conclusion was supported by the observations that the rate of conversion of exogenously applied ACC to ethylene was similarly inhibited by light, and this inhibition was relieved in the presence of CO₂. Similar results were obtained with tobacco leaf discs. The concentrations of CO₂ giving half-maximal activity was about 0.06%, which was only slightly above the ambient level of 0.03%. The modulation of ACC conversion to ethylene by CO₂ or light in detached leaves of both rice and tobacco was rapid and fully reversible, indicating that CO₂ regulates the activity, but not the synthesis, of the enzyme converting ACC to ethylene. Our results indicate that light inhibition of ethylene production in detached leaves is mediated through the internal level of CO₂, which directly modulates the activity of the enzyme converting ACC to ethylene.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid Recipient of a Republic of China National Science Council Fellowship     

Regulation of sedoheptulose-1,7-bisphosphatase by sedoheptulose-7-phosphate and glycerate,and of fructose-1,6-bisphosphatase by glycerate in spinach chloroplasts

Using partially purified sedoheptulose-1,7-bisphosphatase from spinach (Spinacia oleracea L.) chloroplasts the effects of metabolites on the dithiothreitoland Mg²⁺-activated enzyme were investigated. A screening of most of the intermediates of the Calvin cycle and the photorespiratory pathway showed that physiological concentrations of sedoheptulose-7-phosphate and glycerate specifically inhibited the enzyme by decreasing its maximal velocity. An inhibition by ribulose-1,5-bisphosphate was also found. The inhibitory effect of sedoheptulose-7-phosphate on the enzyme is discussed in terms of allowing a control of sedoheptulose-1,7-bisphosphate hydrolysis by the demand of the product of this reaction. Subsequent studies with partially purified fructose-1,6-bisphosphatase from spinach chloroplasts showed that glycerate also inhibited this enzyme. With isolated chloroplasts, glycerate was found to inhibit CO₂ fixation by blocking the stromal fructose-1,6-bisphosphatase. It is therefore possible that the inhibition of the two phosphatases by glycerate is an important regulatory factor for adjusting the activity of the Calvin cycle to the ATP supply by the light reaction.Abbreviations DTT dithiothreitol - FBPase fructose-1,6-bisphosphatase - Fru-1,6-P₂ fructose-1,6-bisphosphate - Fru-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate - Ru-1,5-P₂ ribulose-1,5-bisphosphate - Ru-5-P ribulose-5-phosphate - SBPase sedoheptulose-1,7-bisphosphatase - Sed-1,7-P₂ sedoheptulose-1,7-bisphosphate - Sed-7-P sedoheptulose-7-phosphate This work was supported by the Deutsche Forschungsgemein-schaft.     

Regulation of nitrogenase activity by light in the azolla-anabaena symbiosis

Nitrogenase activity and the rate of photosynthesis were measured simultaneously in Azolla by a continuous gas flow system. The mode of interaction between light, photosynthesis and nitrogenase activity was analysed.Nitrogenase activity dropped off when either Azolla plants or the cyanobiont Anabaena were transferred from light to dark. This decline was immediate and was independent of length or intensity of the prior light phase. Reillumination restored nitrogenase activity.Nitrogenase activity did not depend on the rate of photosynthesis at light intensities below 10 μE m⁻² s⁻¹. Its activity was saturated at 200 μE m⁻² s⁻¹ while CO₂ fixation was saturated at a light intensity of 850 μE m⁻² s⁻¹. Azolla photosynthetic activity followed the absorption spectrum of chlorophyll a, while nitrogenase activity markedly increased between 690 and 710 nm. Inhibition of photosynthesis by DCMU was accompanied by an increase in nitrogenase activity. These results suggest direct light regulation of nitrogenase activity in Azolla independent of CO₂ fixation, and a possible inhibition of nitrogenase activity by the oxygen produced in photosynthesis.     

Control of CO2 fixation regulation of stromal fructose-1,6-bisphosphatase in spinach by pH and Mg2+ concentration

The effect of pH and of Mg²⁺ concentration on the light activated form of stromal fructose-1,6-bisphosphatase (FBPase) was studied using the enzyme rapidly extracted from illuminated spinach chloroplasts. The (fructose-1,6-bisphosphate^4-)(Mg²⁺) complex has been identified as the substrate of the enzyme. Therefore, changes of pH and Mg²⁺ concentrations have an immediate effect on the activity of FBPase by shifting the pH and Mg²⁺ dependent equilibrium concentration of the substrate. In addition, changes of pH and Mg²⁺ concentration in the assay medium have a delayed effect on FBPase activity. A correlation of the activities observed using different pH and Mg²⁺ concentrations indicates, that the effect is not a consequence of the pH and Mg²⁺ concentration as such, but is caused by a shift in the equilibrium concentration of a hypothetical inhibitor fructose-1,6-bisphosphate^3- (uncomplexed), resulting in a change of the activation state of the enzyme. The interplay between a rapid effect on the concentration of the substrate and a delayed effect on the activation state enables a rigid control of stromal FBPase by stromal Mg²⁺ concentrations and pH. Fructose-1,6-bisphosphatase is allosterically inhibited by fructose-6-phosphate in a sigmoidal fashion, allowing a fine control of the enzyme by its product.Abbreviations Fru1,6 bis P fructose-1,6-bisphosphate - Fru6P fructose-6-phosphate - FBPase fructose-1,6-bisphosphatase Some of these results have been included in a preliminary report (Heldt et al. 1984)     

The effects of low pH on the respiration of Daphnia magna Straus.

The effect of low pH values on Daphnia magna is shown to severely depress the O₂ uptake rates. The reason for this is discussed with respect to a decrease in the ability of the gills to exchange CO₂ with the surrounding medium due to a reduction in the CO₂ diffusion gradient as a result of increasing acidity. Respiration in CO₂ free water at acid pH values is shown to be apparently unaffected.     

Metabolic regulation of carbon and electron flow as a function of pH during growth of Sarcina ventriculi

The physiology and biochemistry of Sarcina ventriculi was studied in order to determine adaptations made by the organism to changes in environmental pH. The organism altered carbon and electron flow from acetate, formate and ethanol production at neutral pH, to predominantly ethanol production at pH 3.0. Increased levels of pyruvate dehydrogenase (relative to pyruvate decarboxylase) and acetaldehyde dehydrogenase occurred when the organism was grown at neutral pH, indicating the predominance of carbon flux through the oxidative branch of the pathway for pyruvate metabolism. When the organism was grown at acid pH, there was a significant increase in pyruvate decarboxylase levels and a decrease in acetaldehyde dehydrogenase, causing flux through the non-oxidative branch of the pathway. CO₂ reductase and formate dehydrogenase were not regulated as a function of growth pH. Pyruvate dehydrogenase possessed Michaelis-Menten kinetics for pyruvate with an apparent K _m of 2.5 mM, whereas pyruvate decarboxylase exhibited sigmoidal kinetics, with a S_0.5 of 12.0 mM. Differences in total protein banding patterns from cells grown at pH extremes suggested that synthesis of pyruvate decarboxylase and other enzymes was in part responsible for metabolic regulation of the fermentation products formed.     

Inhibition of the photosynthetic activities of isolated spinach chloroplasts by phosphonate compounds

The effects of three closely related phosphonate compounds on several photosynthetic activities of isolated chloroplasts were investigated. Phosphonoformic and phosphonopropionic acid were found to inhibit both CO₂ fixation and the reduction of 3-phosphoglyceric acid, with CO₂ fixation being more sensitive. In contrast, phosphonoacetic acid was only slightly inhibitory. The lack of inhibition appeared to be due to its inability to enter the stroma via the phosphate translocator. Measurements of changes in stromal metabolite levels following the inhibition of CO₂ fixation by either phosphonoformic or phosphonopropionic acid indicated that the activity of ribulose bisphosphate carboxylase/oxygenase was reduced. Studies with the isolated enzyme confirmed that both of these compounds were effective competitive inhibitors of the carboxylase activity of the enzyme.