Carbonyl sulfide: an inhibitor of inorganic carbon transport in cyanobacteria

Cells of a high CO₂-requiring mutant (E₁) and wild type of Synechococcus PCC7942 were incubated with COS in the light, then suspended in COS-free medium and their CO₂ exchange was measured using an open gas-analysis system under the conditions where photosynthetic CO₂ fixation is inhibited. When the suspension of cells untreated with COS was illuminated, the rate of CO₂ uptake was high and addition of carbonic anhydrase during illumination released a large amount of CO₂ from the medium into the gas phase. The COS treatment in the light markedly reduced the rate of CO₂ uptake by the cells and the amount of CO₂ released by carbonic anhydrase. Incubation of cells with COS in the dark had no effect on the CO₂-exchange profile. The COS concentration required for 50% inhibition of CO₂ uptake was about 25 micromolar when the concentration of inorganic carbon (C_i) in the medium was 60 micromolar; higher C_i concentrations reduced the inhibitory effect of COS. Measurement of C_i uptake in E₁ cells by a silicone oil centrifugation method also indicated marked reduction of the activities of ¹⁴CO₂ and H¹⁴CO₃⁻ uptake in the cells treated with COS in the light. The results demonstrated that COS is a potent inhibitor of C_i transport.     

Use of Carbon Oxysulfide, a Structural Analog of CO(2), to Study Active CO(2) Transport in the Cyanobacterium Synechococcus UTEX 625

Carbon oxysulfide (carbonyl sulfide, COS) is a close structural analog of CO₂. Although hydrolysis of COS (to CO₂ and H₂S) does occur at alkaline pH (>9), at pH 8.0 the rate of hydrolysis is slow enough to allow investigation of COS as a possible substrate and inhibitor of the active CO₂ transport system of Synechococcus UTEX 625. A light-dependent uptake of COS was observed that was inhibited by CO₂ and the ATPase inhibitor diethylstilbestrol. The COS taken up by the cells could not be recovered when the lights were turned off or when acid was added. It was concluded that most of the COS taken up was hydrolyzed by intracellular carbonic anhydrase. The production of H₂S was observed and COS removal from the medium was inhibited by ethoxyzolamide. Bovine erythrocyte carbonic anhydrase catalysed the stoichiometric hydrolysis of COS to H₂S. The active transport of CO₂ was inhibited by COS in an apparently competitive manner. When Na⁺-dependent HCO₃⁻ transport was allowed in the presence of COS, the extracellular [CO₂] rose considerably above the equilibrium level. This CO₂ appearing in the medium was derived from the dehydration of transported HCO₃⁻ and was leaked from the cells. In the presence of COS the return to the cells of this leaked CO₂ was inhibited. These results showed that the Na⁺-dependent HCO₃⁻ transport was not inhibited by COS, whereas active CO₂ transport was inhibited. When COS was removed by gassing with N₂, a normal pattern of CO₂ uptake was observed. The silicone fluid centrifugation method showed that COS (100 micromolar) had little effect upon the initial rate of HCO₃⁻ transport or CO₂ fixation. The steady state rate of CO₂ fixation was, however, inhibited about 50% in the presence of COS. This inhibition can be at least partially explained by the significant leakage of CO₂ from the cells that occurred when CO₂ uptake was inhibited by COS. Neither CS₂ nor N₂O acted like COS. It is concluded that COS is an effective and selective inhibitor of active CO₂ transport.     

Consumption of Carbonyl Sulphide by Chlamydomonas reinhardtii with Different Activities of Carbonic Anhydrase (CA) Induced by Different CO2 Growing Regimes

The green alga, Chlamydomonas reinhardtii, was grown under high and low CO₂ regimes inducing significantly different activities of the extracellular carbonic anhydrase (CA). In close relation to the CA activities, the algae exhibited different consumption rates of the climatically relevant atmospheric trace gas, carbonyl sulphide (COS), thus indicating that CA is responsible for uptake of COS from the medium.     

Carbon Oxysulfide Inhibition of the CO(2)-Concentrating Process of Unicellular Green Algae

Carbonyl sulfide (COS), a substrate for carbonic anhydrase, inhibited alkalization of the medium, O₂ evolution, dissolved inorganic carbon accumulation, and photosynthetic CO₂ fixation at pH 7 or higher by five species of unicellular green algae that had been air-adapted for forming a CO₂-concentrating process. This COS inhibition can be attributed to inhibition of external HCO₃⁻ conversion to CO₂ and OH⁻ by the carbonic anhydrase component of an active CO₂ pump. At a low pH of 5 to 6, COS stimulated O₂ evolution during photosynthesis by algae with low CO₂ in the media without alkalization of the media. This is attributed to some COS hydrolysis by carbonic anhydrase to CO₂. Although COS had less effect on HCO₃⁻ accumulation at pH 9 by a HCO₃⁻ pump in Scenedesmus, COS reduced O₂ evolution probably by inhibiting internal carbonic anhydrases. Because COS is hydrolyzed to CO₂ and H₂S, its inhibition of the CO₂ pump activity and photosynthesis is not accurate, when measured by O₂ evolution, by NaH¹⁴CO₃ accumulation, or by ¹⁴CO₂ fixation.     

Effect of external pH on inorganic carbon assimilation in unicellular marine green algae

Carbonic anhydrase (CA) induction has been studied in three marine green algae under acidic (pH 4.5) or alkaline (pH 8.0) conditions. An inhibition of the induction of the external CA in acidic conditions, similar to that observed in some freshwater green algae, could be observed in only Chlorella saccharophila. In the two other species, Chlorococcum littorale and Stichococcus bacillaris, no significant difference in CA induction was found under two pH conditions. The exact function of the external CA of C. saccharophila remains unclear, since cells grown under acidic conditions (under which this enzyme is repressed) possess the same abilities to use inorganic carbon (Ci) as alkaline‐grown cells. Internal pH values were not modified by the pH of the medium used to cultivate C. saccharophila. Regardless of the growth conditions, activities related to carbon fixation, that is, photosynthetic oxygen evolution, Ci uptake and assimilation were enhanced when the measurements were performed at acidic pH. This indicates that this marine alga is able to use CO₂ more efficiently than HCO₃^?. No evidence could be found for a specific Ci uptake and assimilation system in the acid‐grown cells.     

Oxygen Dependence of Elongation Growth and Oxygen Uptake in Maize Coleoptiles

Auxin-mediated elongation growth of maize coleoptile segments is inhibited by reducing the O₂ concentration in the incubation medium to GT 100 μmol . 1^?1. The half-maximal elongation rate is reached at 40 μmol . 1^?1 O₂, i.e. about two orders of magnitude higher than with mitochondrial respiration. O₂ uptake of the segments measured under similar conditions with an O₂ electrode shows a very similar dependence on O₂ concentration. Auxin increases O₂ uptake by 5–10% when it induces growth. About 40% of the O₂ uptake is insensitive to inhibition by KCN. Auxin has no effect on O₂ uptake in the presence of KCN. The possibility that auxin-mediated elongation growth depends on a KCN-sensitive oxidative process, other than cytochrome c oxidase-catalyzed respiration, is discussed.     

KINETICS OF NUTRIENT UPTAKE AND GROWTH IN PHYTOPLANKTON1

Microscopic algae can grow rapidly in natural waters that are extremely low in essential macro and micro nutrients. Yet, their nutrient uptake systems exhibit only mediocre nutrient affinities, the saturation constants being often 10–1000 times the (estimated) ambient concentrations. The large difference which exists between the saturation constants for growth (K_u) and short term uptake (K_p) are due to the acclimation capabilities of the organisms. Over the acclimation range, K_u, to K_p the algae can maintain maximum growth rate by modulating both their internal nutrient quotas (Q) and their maximum short term nutrient uptake rates (P_max) in response to variations in external nutrient concentrations. The commonly assumed hyperbolic relationships for steady growth and uptake (viz “chemostat theory”) are coherent with a hyperbolic expression for short term uptake including a variable maximum (P_max). The ratio of the saturation constants for growth and uptake is then directly related to the extreme in quotas and maximum uptake rates: K^μ/K^ρ= Q^min/Q^maxρ^max/ρQ^max. This result is applicable even when the exact hyperbolic laws are not. Published data on Fe, Mn, P and N limitation in algae are generally in accord with the theory and demonstrate a wider acclimation range for trace than for major nutrients.     

Studies on selected corallinaceae (Rhodophyta) and other algae in a defined marine culture medium

A marine culture medium (MCM) has been developed and shown to have the unique ability to support the growth of several coralline algae. The results of experiments designed to determine the effects of varying certain ionic concentrations and buffers are presented for this defined medium. Optima of 5 mM Ca²⁺, 1 mM SO^2?₄ and 1 μM BO^3?₃ (lower than the respective sea-water levels) were found for growth or oxygen evolution in Corallina. No organic buffer was needed for growth of Corallinaceae, but growth stimulation was observed for a strain of Callithamnion (Ceramiaceae) when Tris—(hydroxymethyl)aminomethane buffer was added. This stimulation could not be duplicated with other similar buffers. Results of growth studies with a diverse selection of marine macrophytes have indicated that MCM generally supports growth better than sea water alone but often not as well as enriched sea water. The best MCM growth results were observed with members of the Rhodophyceae and certain Chlorophyceae.     

KINETICS OF NUTRIENT UPTAKE AND GROWTH IN PHYTOPLANKTON1

Microscopic algae ran grow rapidly in natural waters that are extremely low in essential macro and micro nutrients. Yet, their nutrient uptake systems exhibit only mediocre nutrient affinities, the saturation constants being often 10–1000 times the (estimated) ambient concentrations. The large difference which exists between the saturation constants for growth (K_μ) and short term uptake (K_ρ) are due to the acclimation capabilities of the organisms. Over the acclimation range, K_μ to K_ρ, the algae can maintain maximum growth rate by modulating both their internal nutrient quotas (Q) and their maximum short term nutrient uptake rates (ρ_max) in response to variations in external nutrient concentrations. The commonly assumed hyperbolic relationships for steady growth and uptake (viz “chemostat theory”) are coherent with a hyperbolic expression for short term uptake including a variable maximum (ρ_max). The ratio of the saturation constants for growth and uptake is then directly related to the extreme in quotas and maximum uptake rates: K_μ/K_ρ= Q_min/Q_max·ρ^lo_max/ρ^hi_max. This result is applicable even when the exact hyperbolic laws are not. Published data on Fe, Mn, P and N limitation in algae are generally in accord with the theory and demonstrate a wider acclimation range for trace than for major nutrients.     

LIVE AND DEAD SPIRULINA SP. TO REMOVE ARSENIC (V) FROM WATER

Arsenic (As)-contaminated water is a grave health hazard and its removal from water poses a great challenge. Conventional methods are associated with many shortcomings. Biosorption of arsenic using blue-green algae is an interesting alternative to conventional methods. In this article, the results of the biosorption of As(V) as AsO₄ ^{? 3} by live and dead Spirulina sp. are reported. The sorption of arsenic could be explained satisfactorily both by the Freundlich and the Langmuir isotherms. The maximum sorption capacities of live and dead Spirulina were estimated to be 525 and 402mg/g, respectively. These values are high in comparison with those reported for other arsenic sorbents. The sorption kinetics of arsenic by both live and dead Spirulina sp. could be well modeled by Lagergrens pseudosecond order-rate equation. Infrared spectra have been employed to understand how Spirulina sp. binds with arsenate. Scanning electron micrography and fluorescent microscopic images are used to discuss the extent of uptake. Preferential uptake of Cu(II), Ni(II), Cd(II), and AsO₄ ^?3 by live Spirulina sp. was investigated and explained with the help of rate constants for sorption.     

Photosynthetic inorganic carbon utilization and growth of Porphyra linearis (Rhodophyta)

Photosynthetic (oxygen evolution) and growth (biomass increase) responses to ambient pH and inorganic carbon (Ci) supply were determined for Porphyralinearis grown in 0.5 L glass cylinders in the laboratory, or in 40 L fibreglass outdoor tanks with running seawater. While net photosynthetic rates were uniform at pH 6.0–8.0, dropping only at pH 8.7, growth rates were significantly affected by pH levels other than that of seawater (c. pH 8.3). In glass cylinders, weekly growth rates averaged 76% at external pH 8.0, 13% at pH 8.7 and 26% at pH 7.0. Photosynthetic O₂ evolution on a daily basis(i.e. total O₂ evolved during day time less total O₂ consumed during night time) was similar to the growth responses at all experimental pH levels, apparently due to high dark respiration rates measured at acidic pH. Weekly growth rates averaged 53% in algae grown in fibreglass tanks aerated with regular air (360 mg L_-1 CO₂) and 28% in algae grown in tanks aerated with CO₂-enriched air (750 mg L^-1 CO₂). The pH of the seawater medium in which P. linear is was grown increased slightly during the day and only rarely reached 9.0. The pH at the boundary layer of algae submerged in seawater increased in response to light reaching, about pH 8.9 within minutes, or remained unchanged for algae submerged in a CO₂-free artificial sea water medium. Photosynthesis of P. linearissaturated at Ci concentrations of seawater (K_0.5560 μM at pH 8.2) and showed low photosynthetic affinity for CO₂(K_0.5 61 μM) at pH 6.0. It is therefore concluded that P. linearisuses primarily CO₂ with HCO₃ ^- being an alternative source of Ci for photosynthesis. Its fast growth could be related to the enzyme carbonic anhydrase whose activity was detected intra- and extracellularly. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of culture medium on hydrogen production by sulfur-deprived marine green algae Platymonas subcordiformis

To study the effect of culture medium on hydrogen production by the marine green algae, Platymonas subcordiformis under sulfur deprivation, cell growth, hydrogen production, and starch and protein catabolism was investigated in the work. Algae cells cultured only in optimized medium required 6～8 days to reach the late logarithmic at the approximate density of (2.00 ± 0.18) × 10⁶ cells/mL, which in traditional medium needed 18～22 days to reach (1.85 ± 0.20) × 10⁶ cells/mL. Increased levels of Chlorophyll (10.74 ± 0.20 μg/mL), starch (149.50 ± 6.15 μg/mL), and protein (213.00 ± 7.36 μg/mL) were accumulated in optimized medium, which were 1.06, 1.47, and 1.87-fold of the algae cells cultured in traditional medium, respectively. The sealed culture of algae cells in sulfur-deprived optimized medium shifted to anaerobic conditions after 96 h of light illumination and produced 0.45 ± 0.12 mL H₂, but in traditional medium maintained aerobic condition and no hydrogen was produced. In addition, changes in starch and protein content during continuous light illumination indicated that more endogenous substrate was consumed in the sulfur-deprived optimized medium than that in the sulfur-deprived traditional medium.     

Na+-Ca2+ exchange activity in rabbit lymphocyte plasma membranes

Plasma membranes of rabbit thymus lymphocytes accumulated Ca²⁺ when a Na⁺ gradient (intravesicular > extravesicular) was formed across the membranes. Dissipation of the Na⁺ gradient by the addition of Na⁺ to the external medium decreased Ca²⁺ uptake. Ca²⁺ preloaded into the lymphocytes was extruded when Na⁺ was added to the external medium. The Ca²⁺ uptake decreased at acidic pH but increased at alkaline pH (above 8) and the activity was saturable for Ca²⁺ (apparent K_m for Ca²⁺ was 61 μM and apparent V_max was 11.5 nmol/mg protein per min). Na⁺-dependent uptake of Ca²⁺ was inhibited by tetracaine and verapamil, and partially inhibited by La³⁺. The uptake was not influenced by orthovanadate.     

土壤氮水交互对马尾松和杉木COS和CO2通量的影响

羰基硫(COS)和CO_2化学结构相似,且植物对COS和CO_2具有共吸收特性,因此可利用COS作为示踪物来估算生态系统总初级生产力,而不同植物吸收COS和CO_2对环境因子变化的响应差异较大。以南亚热带典型树种马尾松(Pinus massoniana)和杉木(Cunninghamia lanceolata)为研究对象,设置2个氮水平及3个土壤水分梯度处理。采取顶空套袋法采集气体样品,用预浓缩—气质联用仪分析样品COS浓度,同时测量植物光合参数。结果表明:马尾松和杉木吸收COS,吸收速率均值分别为39.58—127.27 pmol m~(-2) s~(-1)和0.81—66.92 pmol m~(-2) s~(-1)。整体而言,施氮可促进植物吸收COS,但除施氮对马尾松COS通量有显著影响外(P0.05),施氮、土壤水分和两者交互作用对马尾松和杉木的COS和CO_2通量及其比值均无显著性影响。施氮情况下,高土壤水分处理促进马尾松COS吸收而低土壤水分处理促进杉木COS吸收。中等土壤水分和高土壤水分条件下马尾松和杉木COS通量与气孔导度呈正相关关系。线性拟合结果表明,植物COS通量(F_(COS))与CO_2通量(F_(CO_2))呈极显著正相关(P0.01),马尾松和杉木F_(COS)/F_(CO_2)值分别为1.48×10~(-6)和1.01×10~(-6)。中等土壤水分条件均可提高马尾松F_(COS)/F_(CO_2)比值,而低土壤水分条件下施氮增加杉木F_(COS)/F_(CO_2)比值,高土壤水分条件下施氮降低杉木F_(COS)/F_(CO_2)比值。低土壤水分和高土壤水分使马尾松蒸汽压亏缺增大,促使气孔导度减小从而降低净光合速率。低土壤水分和高土壤水分下施氮导致杉木气孔导度增加从而增强净光合速率。研究结果不仅对进一步了解区域氮沉降和降水对树木COS通量及F_(COS)/F_(CO_2)的影响有重要意义,而且可为模型估算总初级生产力提供区域性数据支持。     

Role of external carbonic anhydrase in light-dependent alkalization byFucus serratus L. andLaminaria saccharina (L.) Lamour. (Phaeophyta)

It has been proposed that many marine macroalgae are able to utilize HCO ₃ ^– for photosynthesis and growth, and that energy-dependent ion pumping is involved in this process. We have therefore studied the light-dependent alkalization of the surrounding medium by two species of marine macroscopic brown algae,Fucus serratus L. andLaminaria saccharina (L.) Lamour. with the aim of investigating the role of extracellular carbonic anhydrase (EC 4.2.1.1.) in the assimilation of inorganic carbon from the seawater medium. In particular, the influence of membrane-impermeable or slowly permeable carbonic-anhydrase inhibitors on the rate of alkalization of the seawater has been investigated. Inhibition of the alkalization rate occurred in both species at an alkaline pH (pH 8.0) but no inhibition was observed at an acidic pH (pH 6.0). The alkalization was found to be light-dependent and inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and, thus, correlated with photosynthesis. Alkalization by macroalgae has previously been shown to be proportional to inorganiccarbon uptake. We suggest that alkalization of the medium at alkaline pH in both of the species examined is mainly the consequence of an extracellular reaction. The reaction is catalyzed by extracellular carbonic anhydrase which converts HCO ₃ ^– to OH^– and CO₂; CO₂ is then taken up through the plasmalemma. However, we do not exclude the involvement of other mechanisms of inorganic-carbon uptake.Abbreviations AZ acetazolamide - CA carbonic anhydrase - CAext extracellular carbonic anhydrase - C_i inorganic carbon - DBS dextran-bound sulfonamide - DCMU 3-(3,4-dichloro-phenyl)-1,1-dimethylurea - PPFD photosynthetic photon flux density This study was carried out with financial support by SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Trygger's Fund for Scientific Research (Sweden), SJFR (Swedish Council for Forestry and Agricultural Research) and CICYT (Spain). Z. Ramazanov is an invited professor of Ministerio de Educación y Ciencia, Spain.     

Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca²⁺ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37°C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 μM CaCl₂, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca²⁺ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca²⁺ uptake, a second phase of Ca²⁺ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca²⁺ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca²⁺ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca²⁺ influx of sarcoplasmic reticulum near steady state of Ca²⁺ uptake was measured by pulse labeling with ⁴⁵Ca²⁺. The Ca²⁺ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca²⁺ uptake in normal medium, Ca²⁺ influx was balanced by Ca²⁺ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca²⁺ exchange rate at the first plateau of Ca²⁺ uptake was about half of that in normal medium. When the second phase of Ca²⁺ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca²⁺ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 μg/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca²⁺ uptake was also observed. These data suggest that the Ca²⁺ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca²⁺ efflux, which subsequently stimulates Ca²⁺ exchange.     

Photosynthesis and photorespiration in algae

The CO₂ exchange of several species of fresh water and marine algae was measured in the laboratory to determine whether photorespiration occurs in these organisms. The algae were positioned as thin layers on filter paper and the CO₂ exchange determined in an open gas exchange system. In either 21 or 1% O₂ there was little difference between ¹⁴CO₂ and ¹²CO₂ uptake. Apparent photosynthesis was the same in 2, 21, or 50% O₂. The compensation points of all algae were less than 10 μl 1⁻¹. CO₂ or ¹⁴CO₂ evolution into CO₂-free air in the light was always less than the corresponding evolution in darkness. These observations are inconsistent with the proposal that photorespiration exists in these algae.     

Oxygen inhibition of dissolved inorganic carbon uptake in unicellular green algae

Unicellular green algae have a dissolved inorganic carbon (DIC) concentrating mechanism, commonly known as the DIC pump, to concentrate inorganic carbon into cells and chloroplasts. The DIC pump activity is normally measured as the K_0.5(DIC) that equals the CO₂ plus HCO₃‐ concentration at a cited pH at which the rate of DIC‐dependent photosynthetic O₂ evolution is half‐maximal, or by the amount of intra‐cellular DIC accumulation in 15–60 s, using a limited amount of NaH¹⁴CO₃, measured by the silicone oil cen‐trifugation technique. The dissolved oxygen in the assay inhibits or reduces the DIC uptake by the cells of unicellular green algae Chlamydomonas reinhardtii Dangeard, strain 137 and in a cell wall‐less marine algae Dunaliella tertiolecta Butcher. The algal cells concentrated the highest amount of DIC when little or no oxygen was present in the assay medium. The results suggest that the amount of O₂ and DIC must be carefully monitored before DIC‐pump assay.     

Cytosolic pyruvate kinase: subunit composition,activity, and amount in developing castor and soybean seeds,and biochemical characterization of the purified castor seed enzyme

Antibodies against Brassica napus cytosolic pyruvate kinase (PK_c) (EC 2.7.1.40) were employed to examine PK_c subunit composition and developmental profiles in castor and soybean seeds. A 56-kDa immunoreactive polypeptide was uniformly detected on immunoblots of clarified extracts from developing castor endosperm or soybean embryos. Maximal PK_c activities occurred early in castor oil seed (COS) and soybean development (7.1 and 5.5 (μmol of pyruvate produced/min) g⁻¹ FW, respectively) and were up to 25-fold greater than those of fully mature seeds. Time-course studies revealed a close correlation between extractable PK_c activity and the relative amount of the immunoreactive 56-kDa PK_c polypeptide. PK_c from developing COS was purified 1,874-fold to homogeneity and a final specific activity of 73.1 (μmol of pyruvate produced/min) mg⁻¹ protein. Gel filtration and SDS-PAGE indicated that this PK_c exists as a 230-kDa homotetramer composed of 56-kDa subunits. The mass fingerprint of tryptic peptides of the 56-kDa COS PK_c subunit best matched three putative PK_cs from Arabidopsis thaliana. The purified enzyme was relatively heat-stable and displayed a broad pH optimum of 6.4. However, more efficient substrate utilization (in terms of V _max /K _m for phosphoenolpyruvate or ADP) was observed at pH 7.4. Glutamate was the most effective inhibitor, whereas aspartate functioned as an activator by partially relieving glutamate inhibition. Together with our previous studies, the results: (1) allow a model to be formulated regarding the coordinate allosteric control of PKc and phosphoenolpyruvate carboxylase by aspartate and glutamate in developing COS, and (2) provide further biochemical evidence that castor plant PK_c exists as tissue-specific isozymes that exhibit substantial differences in their respective physical and regulatory properties.