Photoassimilation of acetate and metabolism of carbohydrate in Chlorobium thiosulfatophilum

1. Washed cell suspensions of Chlorobium thiosulfatophilum form large amounts of a polyglucose in the light. Addition of acetate to the cells increases the formation of polysaccharide considerably. During incubation in the dark, polysaccharide decreases with time, and organic acids such as succinic and propionic acid are excreted into the medium. 2. Glucose isolated from cells which had photoassimilated 1-, 2-, and U-¹⁴C-acetate had a specific activity which lay between 1 and 2 times that of the acetate substrates. 3. To analyse the distribution of radioactivity in the glucose units formed during photoassimilation of ¹⁴C-acetate, 2 microbial degradations, with bakers' yeast and Zymomonas mobilis respectively, were used. The results show that acetate gives rise to carbon atoms 1+2 and 5+6 of glucose, whereas carbon atomes 3+4 are not labelled. Further, the results indicate that glucose is not formed via the reductive pentose phosphate cycle when acetate is present.     

Autotrophic CO2 fixation in Chlorobium limicola. Evidence against the operation of the Calvin cycle in growing cells

Chlorobium limicola has been proposed to assimilate CO₂ autotrophically via a reductive tricarboxylic acid cycle rather than via the Calvin cycle. This proposal has been a matter of considerable controversy. In order to determine which pathway is operative, the bacterium was grown on a mineral salts medium with CO₂ as the main carbon source supplemented with specifically labeled ¹⁴C-pyruvate, and the incorporation of ¹⁴C into alanine (intracellular pyruvate), aspartate (oxaloacetate), glutamate (-ketoglutarate), and glucose (hexosephosphate) was measured in exponentially growing cells in long term labeling experiments. During growth in presence of pyruvate, 20% of the cell carbon were derived from pyruvate in the medium, 80% from CO₂. Since pyruvate was not oxidized to CO₂, only those compounds should become labeled which were synthesized from CO₂ via pyruvate.The three amino acids and glucose were found to be labeled. Alanine had one fifth the specific radioactivity of the extracellular pyruvate, indicating that 20% of the intracellular pyruvate pool were derived from pyruvate in the medium, 80% were synthesized from CO₂. Glucose had twice the specific radioactivity of alanine, showing that hexosephosphate synthesis from CO₂ proceeded via the pyruvate pool. The latter finding is not consistent with the operation of the Calvin cycle, in which pyruvate is not an intermediate. The specific radioactivities of aspartate (oxaloacetate) and of glutamate (-ketoglutarate) were practically identical but considerably lower than that of alanine ( intracellular pyruvate). These findings are compatible with the operation of a reductive tricarboxylic acid cycle as mechanism of autotrophic CO₂ fixation. Degradation studies of the cell components support this interpretation. Offprint requests to: G. Fuchs     

ATP-linked citrate lyase activity in the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum

Cell-free extracts of the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum strains 1C and L have been shown to cleave citrate with the formation of oxaloacetate and acetyl-CoA. This capacity was found in autotrophically grown cells as well as in the cells grown on media with acetate or L-glutamate. Citrate lyase activity in cell-free extracts is only measurable in the presence of citrate, adenosine-5-triphosphate, coenzyme A and Mg²⁺ or Mn²⁺. It is concluded on the basis of the obtained data that C. limicola f. thiosulfatophilum contains adenosine-5-triphosphate-linked citrate lyase (E.C.4.1.3.8). In contrast to green bacteria in the purple bacteria Ectothiorhodospira shaposhnikovii, Rhodospirillum rubrum and Thiocapsa roseopersicina citrate lyase activity was not found.     

Some properties of glutamine synthetase and glutamate synthase from Chlorobium vibrioforme f. thiosulfatophilum

The phototrophic green sulphur bacterium Chlorobium vibrioforme f. thiosulfatophilum assimilated ammonia via glutamine synthetase and glutamate synthase when grown with ammonia up to 30 mM, but above this level glutamate dehydrogenase was the key enzyme. Glutamine synthetase purified 42-fold was found to be adenylylated. The -glutamyltransferase activity of the enzyme was markedly inhibited by alanine, glycine, serine and lysine, and these amino acids in various combinations showed cumulative inhibition. Adenine nucleotides also inhibited enzyme activity, especially ATP. Glutamate synthase purified 222-fold had a maximum absorption at 440 nm which was reduced by sodium dithionite, and the enzyme was inhibited by atebrin indicating the presence of a flavin component. The enzyme had specific requirements for NADH, -ketoglutarate and l-glutamine, the K _m values for these were 13.5, 270 and 769 M respectively. Glutamate synthase was sensitive to feedback inhibition by amino acids, adenine nucleotides and other metabolites and the combined effects of these inhibitors was cumulative.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamic dehydrogenase     

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     

A novel aminoglycosphingolipid found in Chlorobium limicola f. thiosulfatophilum 6230

An aminolipid from Chlorobium limicola f. thiosulfatophilum has been purified and characterized by thin-layer chromatography, infrared specroscopy, ¹H-NMR, ¹³C-NMR, plasma desorption mass spectrometry, and fast atom bombardment mass spectrometry. The structure is that of an aminosugar (neuraminic acid) attached to a sphingosine backbone with one myristic acid linked to the sphingosine by an amide bond. Related glycosphingolipids and capnoids are found in the Bacterioides/Flavobacteria which are related to the green sulfur bacteria by the criterion of 16S rRNA structure. No aminoglycosphingolipid was found in Chloroflexus aurantiacus.     

Ribulose 1,5-bisphosphate carboxylase and polyhedral bodies of Chlorogloeopsis fritschii

Ribulose 1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) activity was approximately equally distributed between supernatant and pellet fractions produced by differential centrifugation of disrupted cells of Chlorogloeopsis fritschii. Low ionic strength buffer favoured the recovery of particulate RuBP carboxylase. Density gradient centrifugation of resuspended cell-free particulate material produced a single band of RuBP carboxylase activity, which was associated with the polyhedral body fraction, rather than with the thylakoids or other observable particles. Isolated polyhedral body stability was improved by density gradient centrifugation through gradients of Percoll plus sucrose in buffer, which yielded apparently intact polyhedral bodies. These were 100 to 150 nm in diameter and contained ring-shaped, 12 nm diameter particles. It is inferred that the C. fritschii polyhedral bodies are carboxysomes. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of SDS-dissociated polyhedral bodies revealed 8 major polypeptides. The most abundant, with molecular weights of 52,000 and 13,000, correspond with the large and small subunits, respectively, of RuBP carboxylase.Abbreviations RuBP ribulose 1,5-bisphosphate - Ru5P ribulose 5-phosphate - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - EDTA ethylenediamine tetraacetic acid - Tris tris (hydroxymethyl) methylamine - IB isolation buffer - TCA trichloroacetic acid     

A Comparative Study of the Isocitrate Dehydrogenases of Chlorobium limicola forma thiosulfatophilum and Rhodopseudomonas palustris

The carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle in the obligate photolithotroph Chlorobium limicola forma thiosulfatophilum and the oxidation of isocitrate in the tricarboxylic acid cycle in the photoheterotroph Rhodopseudomonas palustris are catalyzed by isocitrate dehydrogenases. A comparative study of these enzymes isolated from the two bacteria showed that they virtually do not differ in enzymatic and kinetic properties.     

Ribulose bisphosphate carboxylase from Thiobacillus A2. Its purification and properties

Ribulose bisphosphate carboxylase (EC 4.1.1.39) from Thiobacillus A2 has been purified to homogeneity on the basis of polyacrylamide gel electrophoresis and U.V. analysis during sedimentation velocity studies. The enzyme had an optimum pH of about 8.2 with Tris-HCl buffers. The molecular weight was about 521000 with an S _rel. of 16.9. K _m for RuBP was 122 M, for total CO₂ it was 4.17 mM, and for Mg²⁺ 20.0 M. The absolute requirement for a divalent cation was satisfied by Mg²⁺ which was replaceable to a certain extent by Mn²⁺. Activity was not significantly affected by SO ₄ ^2- , SO ₃ ^2- , or S₂O ₃ ^2- at 1.0 mM. At this concentration S^2- caused a 27% stimulation. All mercurials tested were inhibitory. pHMB was the most potent causing about 60% inhibition at 0.01 mM. This inhibition was reversible by low concentrations of cysteine. Cyanide was also inhibitory. Its mode of inhibition with respect to RuBP was un-competitive and with a K _i of 20 M. Lost activity could be restored partially by GSH or Cu²⁺. Although azide at the concentration tested had no significant effect on enzyme activity, 2,4-dinitrophenol at 1.0 mM caused 91% inhibition. Finally, activity was also affected by energy charge.Abbreviations ATP adenosine-5-triphosphate - GAPDH glyceraldehyde phosphate dehydrogenase - GSH (reduced) glutathione - G6P glucose-6-phosphate - NAD⁺ nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - pHMB parahydroxymercuribenzoate - 6PG 6-phosphogluconate - 3-PGA 3-phosphoglycerate - PGK phosphoglyceratekinase - RuBP ribulose-1,5-bisphosphate     

The in-vivo response of the ribulose-1,5-bisphosphate carboxylase activation state and the pool sizes of photosynthetic metabolites to elevated CO2 inPhaseolus vulgaris L.

The short-term, in-vivo response to elevated CO₂ of ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39) activity, and the pool sizes of ribulose 1,5-bisphosphate, 3-phosphoglyceric acid, triose phosphates, fructose 1,6-bisphosphate, glucose 6-phosphate and fructose 6-phosphate in bean were studied. Increasing CO₂ from an ambient partial pressure of 360–1600 bar induced a substantial deactivation of RuBPCase at both saturating and subsaturating photon flux densities. Activation of RuBPCase declined for 30 min following the CO₂ increase. However, the rate of photosynthesis re-equilibrated within 6 min of the switch to high CO₂, indicating that RuBPCase activity did not limit photosynthesis at high CO₂. Following a return to low CO₂, RuBPCase activation increased to control levels within 10 min. The photosynthetic rate fell immediately after the return to low CO₂, and then increased in parallel with the increase in RuBPCase activation to the initial rate observed prior to the CO₂ increase. This indicated that RuBPCase activity limited photosynthesis while RuBPCase activation increased. Metabolite pools were temporarily affected during the first 10 min after either a CO₂ increase or decrease. However, they returned to their original level as the change in the activation state of RuBPCase neared completion. This result indicates that one role for changes in the activation state of RuBPCase is to regulate the pool sizes of photosynthetic intermediates.Abbreviations and symbols A net CO₂ assimilation rate - C_a ambient CO₂ partial pressure - C_i intercellular CO₂ partial pressure - CABP 2-carboxyarabinitol 1,5-bisphosphate - k_cat catalytic turnover rate per RuBPCase molecule - PFD photon flux density (400 to 700 nm on an area basis) - PGA 3-phosphoglyceric acid - P_i orthophosphate - RuBP ribulose 1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39)     

Cytochromes of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum. Purification,characterization and sulfur metabolism

Three cytochromes of the thiosulfate-utilizing green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum were highly purified by ion exchange column chromatography and ammonium sulfate fractionation. All three cytochromes are located in the soluble fraction. Cytochrome c-551 (highest purity index obtained: A₂₈₀/A₄₁₆=0.39) shows maxima at 551 nm (-band), 521 nm (-band), and 416 nm (-band) for the reduced form. This cytochrome is an acidic protein with a molecular weight of 32,000, a redox potential of 150 mV, and an isoelectric point at pH 6.0. Cytochrome c-553 (highest purity index obtained: A₂₈₀/A₄₁₇=0.8) is also an acidic protein with maxima at 553,5 nm, 523,5 nm and 417 nm for the reduced form, a molecular weight of 63,000, a redox potential of 90 mV, an isoelectric point at pH 6.3, and it contains FAD as flavin component. It is autoxidizable and participates in sulfide oxidation, but cannot catalyze the reverse reaction. The cytochrome c-555 (highest purity index obtained: A₂₈₀/A₄₁₈=0.16) is a small basic protein with maxima at 555 nm, 523 nm and 418 nm (reduced form), a molecular weight of 12,500, an isoelectric point between pH 10 and 10.5, and a redox potential of 155 mV. The ratio of the cytochrome contents to each other is constant and does not change when the organism has only thiosulfate or sulfide as the main electron donor in the medium.The soluble fraction further contains the non-heme ironcontaining proteins rubredoxin and ferredoxin. The anaerobic sulfide oxidation in a growing culture of Chlorobium vibrioforme f. thiosulfatophilum is accompanied by a rapid formation of thiosulfate, which is only utilized when sulfide is no longer available, while the elemental sulfur concentration increases constantly until thiosulfate is consumed.Non-common abbreviations C Chlorobium - SDS sodium dodecylsulfate - HIPIP high-potential-iron-sulfur-protein     

Purification,some properties and quaternary structure of thed-ribulose 1,5-diphosphate carboxylase ofAlcaligenes eutrophus

d-Ribulose 1,5-diphosphate carboxylase has been purified from autotrophically grown cells of the facultative chemolithotrophic hydrogen bacteriumAlcaligenes eutrophus. The enzyme was homogeneous by the criteria of polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 505000 determined by gel filtration and sucrose density gradient centrifugation, and a sedimentation coefficient of 18.2 S was obtained. It was demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis that the enzyme consists of two types of subunits of molecular weight 52000 and 13000.Electron microscopy on the intact and the partially dissociated enzyme lead to the construction of a model for the quaternary structure of the enzyme which is composed of 8 large and 8 small subunits. The most probable symmetry of the enzyme molecule is 4:2:2.Michaelis constant (K _m ) values for ribulose 1,5-diphosphate, Mg^2-, and CO₂ were 0.59 mM, 0.33 mM, and 0.066 mM measured under air. Oxygen was a competitive inhibitor with respect to CO₂ suggesting that the enzyme also exhibits an oxygenase activity. The oxygenolytic cleavage of ribulose 1,5-diphosphate was shown and a 1:1 stoichiometry between oxygen consumption and 3-phosphoglycerate formation observed.Abbreviations DTE dithioerythritol - EDTA ethylenediamine tetraacetate - RuDP d-ribulose 1,5-diphosphate     

Effect of temperature on the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the rate of respiration in the light

Responses of the rate of net CO₂ assimilation (A) to the intercellular partial pressure of CO₂ (p _i ) were measured on intact spinach (Spinacia oleracea L.) leaves at different irradiances. These responses were analysed to find the value of p _i at which the rate of photosynthetic CO₂ uptake equalled that of photorespiratory CO₂ evolution. At this CO₂ partial pressure (denoted ), net rate of CO₂ assimilation was negative, indicating that there was non-photorespiratory CO₂ evolution in the light. Hence was lower than the CO₂ compensation point, . Estimates of were obtained at leaf temperatures from 15 to 30°C, and the CO₂/O₂ specificity of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (E.C. 4.1.1.39) was calculated from these data, taking into account changes in CO₂ and O₂ solubilities with temperature. The CO₂/O₂ specificity decreased with increasing temperature. Therefore we concluded that temperature effects on the ratio of photorespiration to photosynthesis were not solely the consequence of differential effects of temperature on the solubilities of CO₂ and O₂. Our estimates of the CO₂/O₂ specificity of RuBP carboxylase/oxygenase are compared with in-vitro measurements by other authors. The rate of nonphotorespiratory CO₂ evolution in the light (R _d ) was obtained from the value of A at . At this low CO₂ partial pressure, R _d was always less than the rate of CO₂ evolution in darkness and appeared to decrease with increasing irradiance. The decline was most marked up to about 100 mol quanta m^-2 s^-1 and less marked at higher irradiances. At one particular irradiance, however, R _d as a proportion of the rate of CO₂ evolution in darkness was similar in different leaves and this proportion was unaffected by leaf temperature or by [O₂] (ambient and greater). After conditions of high [CO₂] and high irradiance for several hours, the rate of CO₂ evolution in darkness increased and R _d also increased.Abbreviations and symbols A rate of net CO₂-assimilation - CO₂ compensation point - CO₂ compensation point in the absence of R _d - p _i intercellular partial pressure of CO₂ - R _d (day respiration) rate of non-photorespiratory CO₂ evolution in the light - R _n (night respiration) rate of CO₂ evolution in darkness - RuBP ribulose-1,5-bisphosphate - Rubisco RuBP carboxylase/oxygenase     

Species specific release of sulfate from adenylyl sulfate by ATP sulfurylase or ADP sulfurylase in the green sulfur bacteria Chlorobium limicola and Chlorobium vibrioforme

High activities of ATP sulfurylase were found in the soluble protein fraction of two Chlorobium limicola strains, whereas ADP sulfurylase was absent. ATP sulfurylase was partially purified and characterized. It was a stable soluble enzyme with a molecular weight of 230,000, buffer-dependent pH optima at 8.6 and 7.2 and an isoelectric point at pH 4.8. No physiological inhibitor was found. Inhibition was observed with p-CMB and heavy metals. Sulfur compounds had no effect on enzyme activity. The stoichiometry of the reaction was proven. In contrast, an ADP sulfurylase, but no ATP sulfurylase, was found in Chlorobium vibrioforme. This enzyme was very labile with a molecular weight of about 120,000 and buffer-dependent pH optima at 9.0 and 8.5. Under test conditions the apparent K _m value was determined to be 0.28 mM for adenylyl sulfate and 8.0 mM for phosphate.Abbreviations APS adenylyl sulfate - p-CMB parachloromercuribenzoate - PP_i inorganic pyrophosphate     

Purification and properties of ribulose 1,5-bisphosphate carboxylase from sunflower leaves

Extracts from sunflower leaves possess a high ribulose-1,5-bisphosphate (RuBP) carboxylase capacity but this enzyme activity is not stable. A purification procedure, developed with preservation of carboxylase activity by MgSO₄, yielded purified RuBP carboxylase with high specific activity (40 nkat mg^-1 protein). Measurement of kinetic parameters showed high Km values (RuBP, HCO ₃ ^- ) and high Vmax of the reaction catalyzed by this sunflower enzyme; the results are compared with those obtained for soybean carboxylase. Enzyme characteristics are discussed in relation to stabilization and activation procedures and to the high photosynthesis rates of this C₃ species.     

One large subunit of ribulose 1,5-bisphosphate carboxylase oxygenase in Medicago,Spinacia and Nicotiana

Summary Isoelectric focusing of subunits of ribulose 1,5-bisphosphate carboxylase oxygenase of Medicago, Spinacia and Nicotiana were investigated, using a rapid isolation technique, without S-carboxymethylation. RuBPC-ase and its subunits were isolated by gel electrophoresis. Isoelectric focusing of RuBPC-ase of M. sativa and M. falcata showed that this enzyme consists of one large subunit (LSU) polypeptide and two or three small subunits (SSU), depending on the genotype. The pl of the LSU's was identical, but the pl of SSU's of the two genotypes was different. Amino acid composition and tryptic peptide maps further supported the concept of a conserved nature of LSU and heterogeneity of SSU polypeptides in Medicago. It was also found that S. oleracea, N. tabacum, N. glutinosa and N. excelsior have a single LSU polypeptide, but they differ in respect of pl values. The SSU polypeptides appeared to be variable. S-carboxymethylation affected the number as well as the pl values of LSU and SSU polypeptides. It is suggested that one LSU polypeptide is probably the general rule in higher plants, rather than the three LSU polypeptides demonstrated by Chen et al. (1977) and Wildman (1979).     

Thioredoxin: an unexpected meeting place

For much of the latter part of the 20th century, photosynthesis research at Berkeley was dominated by Daniel Arnon and Melvin Calvin. In this article, I have briefly described how their contributions jointly provided the foundation for our work on thioredoxin and how important Andrew Benson was to this effort.     

Variations in ribulose 1,5-bisphosphate carboxylase protein levels,activities and subcellular distribution during photoautotrophic batch culture of Chlorogloeopsis fritschii

Ribulose 1,5-bisphosphate (RuBP) carboxylase is present in the cytoplasm and carboxysomes (polyhedral bodies) of the cyanobacterium Chlorogloeopsis fritschii. In vitro enzyme activities have been measured throughout photoautotrophic batch culture, together with RuBP carboxylase protein concentrations, determined by rocket immunoelectrophoresis. Enzyme activities and protein levels in the cytoplasmic and carboxysomal fractions varied in an apparently inverse manner during growth. The RuBP carboxylase activities per unit enzyme protein were maximal in late lag phase/early exponential phase for both cellular enzyme pools. Both rates per unit enzyme protein declined during exponential phase, cytoplasmic enzyme activity remaining consistently higher than that of the carboxysomal enzyme. Activities per unit cytoplasmic and carboxysomal enzyme protein showed very low, similar rates in late stationary phase and death phase. Dialysis experiments indicated that such changes were not due to interference in activity assays by soluble endogenous effectors. Major shifts in the subcellular distribution of RuBP carboxylase protein were found versus culture age, enzyme protein levels being predominantly carboxysomal in lag phase, mainly soluble in exponential phase and then mainly carboxysomal again in stationary/death phase. The data are discussed in terms of carboxysome function and the question of control of RuBP carboxylase synthesis in cyanobacteria.Abbreviations RuBP D-ribulose 1,5-bisphosphate - LTIB low Tris isolation buffer - HTIB high Tris isolation buffer - RIE rocket immunoelectrophoresis     

Nuclear DNA regulates the level of ribulose 1,5-bisphosphate carboxylase oxygenase in Medicago sativa L.

Summary The response to selection for leaf proteins was studied during three selection cycles. Selection for high total nitrogen content showed 75% heritability, and the levels of both ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco) and cytoplasmic protein were strongly under nuclear DNA control. High and low protein content were correlated with chloroplast area. Although the amounts of nuclear DNA were similar, the ratio of Rubisco/DNA and chlorophyll/DNA changed during the selection process. It can be concluded that the levels of Rubisco achieved in mature plants of M. sativa are under nuclear DNA control. The possible involvement of small subunit (SSU) genes in controlling these levels is discussed.     

The CO2/O2 specificity of ribulose 1,5-bisphosphate carboxylase/oxygenase

The substrate specificity factor, V _cK_o/V_oK_c, of spinach (Spinacia oleracea L.) ribulose 1,5-bisphosphate carboxylase/oxygenase was determined at ribulosebisphosphate concentrations between 0.63 and 200 M, at pH values between 7.4 and 8.9, and at temperatures in the range of 5° C to 40° C. The CO₂/O₂ specificity was the same at all ribulosebisphosphate concentrations and largely independent of pH. With increasing temperature, the specificity decreased from values of about 160 at 5° C to about 50 at 40° C. The primary effects of temperature were on K _c [Km(CO₂)] and V _c [Vmax (CO₂)], which increased by factors of about 10 and 20, respectively, over the temperature range examined. In contrast, K _o [Ki (O₂)] was unchanged and V _o [Vmax (O₂)] increased by a factor of 5 over these temperatures. The CO₂ compensation concentrations () were calculated from specificity values obtained at temperatures between 5° C and 40° C, and were compared with literature values of . Quantitative agreement was found for the calculated and measured values. The observations reported here indicate that the temperature response of ribulose 1,5-bisphosphate carboxylase/oxygenase kinetic parameters accounts for two-thirds of the temperature dependence of the photorespiration/photosynthesis ratio in C₃ plants, with the remaining one-third the consequence of differential temperature effects on the solubilities of CO₂ and O₂.Abbreviations RuBPC/O(ase) ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - CO₂ compensation concentration