The Formation of Ribulose Diphosphate Carboxylase Protein during Chloroplast Development in Barley

Ribulose 1,5-diphosphate carboxylase is synthesized in barley leaves growing in the dark. Upon illumination there is a marked increase in the rate of synthesis of the enzyme. The specific activity of the enzyme expressed as cpm incorporated into phosphoglyceric acid per μg of fraction I protein, after isolation shows no change either during dark growth or greening. During early stages of illumination of 7 day dark grown leaves with 320 foot-candles the enzymic activity in the water soluble protein fraction of the leaf shows a short term decline after 15 min which lasts for 30 min. Leaves greening at 2 foot-candles show a similar decline which is shifted to a time between the fourth and eighth hr after the onset of illumination.     

Incorporation of Large Subunits into Ribulose Bisphosphate Carboxylase in Chloroplast Extracts : Influence of Added Small Subunits and of Conditions during Synthesis

The incorporation of newly synthesized large subunits into ribulose bisphosphate carboxylase/oxygenase (RuBisCO) in pea chloroplast extracts occurs at the expense of intermediate forms of the large subunit which are complexed with a binding protein. Most subunits of this binding protein are found in dodecameric complexes in chloroplast extracts. Addition of small subunits to these extracts results in approximately 40 to 60% increased incorporation of newly made large subunits into RuBisCO at low or zero concentrations of ATP, but is without significant effect at high concentrations of ATP, a condition in which the dodecameric binding protein complex is dissociated into subunits. Overall, these data support the assumption that the incorporation of large subunits into RuBisCO in chloroplast extracts reflects de novo assembly rather than `mere' exchange of subunits. The in vitro assembly of large subunits into RuBisCO is a function of the conditions under which the large subunits are synthesized in organello. When the large subunits are made in chloroplasts suspended in 188 millimolar sorbitol, they are approximately 2- to 3-fold better able to assemble into RuBisCO when subsequently incubated in vitro than when they are synthesized in chloroplasts suspended in 375 millimolar sorbitol. This observation indicates that mere synthesis of large subunits is not sufficient to confer maximal assembly competence on large subunits.     

The Effect of Zinc on the Formation of Ribulose Diphosphate Carboxylase in Phaseolus vulgaris

The effect of Zn on the formation of ribulose diphosphate carboxylase (RuDPCase was investigated in the leaf discs from Zn-deficient Sanilac navy bean plants (Phaseolus rulgaris L.). The incorporation of ¹⁴C-leucine into the partially purified RuDPCase was found to be a quantitative equivalent of the level and activity of the enzyme. Zn as ZnSO₄ at 10 uM stimulates the formation of RuDPCase by at least 2-fold. Neither CuSO₄ nor CdSO₄ at the same concentration substitutes for ZnSO₄. The enhancement of RuDPCase formation by added Zn is greater with increasing severity of Zn deficiency, suggesting that Zn is a limiting factor in this system. Suppression of the Zn-stimulated formation of RuDPCase by actinomycin D and cycloheximide suggests that the Zn-mediated formation of RuDPCase most likely represents de novo synthesis. Also, the possible site(s) of action of Zn is discussed.     

On the Relationship between Ribulose Diphosphate Carboxylase and Protochlorophyllide Holochrome of Phaseolus vulgaris Leaves

The relationship between ribulose diphosphate carboxylase (3-phospho-d-glycerate carboxy-lyase [dimerizing], EC 4.1.1.39, formerly known as carboxydismutase) and protochlorophyllide holochrome of etiolated Phaseolus vulgaris leaves has been studied.A procedure for partially selective extraction of the two proteins was devised using tris-HCl buffer first without and then with Triton X-100. Ribulose diphosphate carboxylase was readily extracted from etiolated bean leaves without Triton X-100, and protochlorophyllide holochrome was extracted on the addition of Triton X-100.Optimal extraction conditions for protochlorophyllide holochrome have been found to be different for tissues of different ages.     

Stimulation of Ribulose Bisphosphate Carboxylase Activity by Inorganic Orthophosphate without an Increase in Bound Activating CO2: Co-operativity between the Subunits of the Enzyme

Parry, M. A. J., Schmidt, C. N. G., Cornelius, M. J., Keys,A. J., Millard, B. N. and Gutteridge, S. 1985. Stimulation ofribulose bisphosphate carboxylase activity by inorganic orthophosphatewithout an increase in bound activating CO²: co-operativitybetween the subunits of the enzyme.—J. exp. Bot. 36: 1396–1404 Ribulose bisphosphate carboxylase/oxygenase (E.C. 4.1.1.39 [EC] )from wheat (Triticum aestivum L.), already activated by reactionwith CO² and Mg²⁺, was increased in activity on addition ofinorganic orthophosphate. This further activation took placewithout a significant increase in the amount of bound activatingCO² and the effect was relatively greater with smaller amountsof bound CO². With less than 2·0 mol of CO² bound permol holoenzyme, phosphate increased activity about five-foldwhilst with 7·0 mol of bound activating CO² per mol holoenzyme,phosphate increased activity by a factor of only 1 ·8.This decrease in the effect of orthophosphate with increasein bound activating CO² suggests negative co-operativity betweenactivated sites. The stimulation of activity by inorganic orthophosphatemust be a process distinct from activation by CO²; it was observedwith both the slow and the rapidly activating forms of ribulosebisphosphate carboxylase/oxygenase from wheat. Key words: Ribulose bisphosphate carboxylase, activation, inorganic orthophosphate CO², co-operativity     

Regulation of Ribulose Bisphosphate Carboxylase Activity in Intact Wheat Leaves by Light, CO2, and Temperature

The activity of the enzyme ribulose bisphosphate carboxylase(RuBPCase) was estimated after rapidly extracting it from intactwheat leaves pretreated under different light and CO₂ levels.No HCO₃^– was added to the extraction buffer since it isshown to inhibit RuBPCase. The activity increased as light intensityor CO₂ concentration during pretreatment was increased. Enzymeactivity increased as temperature during pretreatment was decreased.Light activation did not affect the affinity of RuBPCase forCO₂. A K_m of 30 µM CO₂ under air level O₂ was determined.CO₂, light and temperature are three main limiting factors ofphotosynthesis. It seems that the activity of RuBPCase is regulatedby these factors according to the requirements for CO₂ fixation.     

烟草核酮糖二磷酸羧化酶及其大、小亚基改进的分离纯化方法

核酮糖二磷酸羧化酶在植物叶子中含量很高,一般约占总可溶性蛋白的50％以上,是世界上最为丰富的一种蛋白质(Ellis 1979)。这种蛋白质含有高水平的必需氨基酸(Kung等1980),它作为人类的一种补充营养食品有着潜在的应用前景。因此需要研究出一种简便的、得率高的并可大规模制备的蛋白质纯化技术。核酮糖二磷酸羧化酶又是光合作用中固定二氧化     

The Activity of Ribulose Diphosphate Carboxylase in Extracts of Gonyaulax polyedra in the Day and the Night Phases of the Circadian Rhythm of Photosynthesis

The ribulose 1,5-diphosphate carboxylase from Gonyaulax polyedra Stein. has a half-life of about four hours in buffer, but can be stabilized by the addition of 50% glycerol. The optimum pH is 7.8 to 8.0 and the optimum Mg²⁺ concentration is 3 mm. Heavy metal ions (Cu²⁺, Hg²⁺, Ni²⁺, Zn²⁺), EDTA, pyrophosphate, and adenosine triphosphate were strongly inhibitory. Ribulose 1,5-diphosphate carboxylase from Gonyaulax was not cold-sensitive or activated by light activation factor from tomato or Gonyaulax. No difference in the activity of this enzyme was detected when extracts prepared at the maximum and the minimum of the circadian rhythm of photosynthesis were compared. The Km of HCO₃⁻ was also the same (16 to 19 mm).     

Effects of Photosynthetic Intermediates on the Activation State of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Euglena gracilis Z

The half-saturating concentration of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from Euglena gracilis Z for CO₂ in its activation by CO₂ in the presence of a saturating concentration of MgCl₂ (KJ was measured by analyzing the partial reversible inactivation of the fully activated enzyme in the medium with dilute CO₂. The K_d of the Euglena enzyme was 12.5 μm. The K,_d values were 6.3/im for the enzyme from soybean, 10.8 fiM from maize, 23.3 jiM from Scenedesmus obliquus, and 20.8 μm from Anabaena 7120. The activated state of Euglena RuBisCO was stabilized by 6-phosphogluconate, fructose 1,6-bisphosphate, and 3-phosphoglycerate in the medium containing low concentrations of CO₂. Both fructose 6-phosphate and ATP stimulated inactivation in the medium. NADPH not only stabilized the activated state of the enzyme, but also enhanced the enzyme activity over the full activity measured in the absence of NADPH. NADP⁺ did not nullify the effects of NADPH on the activation at all. The physiological significance of the effects of these photosynthetic metabolites on the activated state of Euglena RuBisCO is discussed.     

Protein Synthesis Linked to Respiration and Phosphorylation in Intact Euglena Mitochondria

Highly purified condensed mitochondria obtained from bleachedmutant. W₁₀BSmL of Euglena gracilis Klebs var bacillaris Coriincorporate [³⁵S]methionine into protein when fortified withmalate, ADP, Mg²⁺, phosphate and a sucrose osmoticum. Twentyto twenty-five polypeptide bands were found to be labeled inorganello when the labeled protein was subjected to sodium dodecylsulfatepolyacrylamide gel electrophoresis. Methionine incorporation,but not respiration or oxidative phosphorylation, was blockedby chloramphenicol and other 70S ribosomal translation inhibitorsbut cycloheximide and ribonuclease were without effect. Inhibitorsof electron transport and uncouplers of oxidative phosphorylationwere excellent inhibitors of protein synthesis. Thus, thesemitochondrial preparations carry out protein synthesis in organellothat is linked to respiration and oxidative phosphorylation. ¹Present address: VA Hospital Outpatient Clinic, 17 Court St.,Boston, MA 02115, U.S.A. ²Present address: Laboratories de Microbiologia e Inmunologia,Universidad Catolica de Chile, Casilla 114-D, Santiago, Chile. ³Present address: Botany Department, University of Massachusetts,Amherst, MA 01003, U.S.A. (Received June 17, 1985; Accepted October 28, 1985)     

Changes in the Ability of Photophosphorylation and Activities of Surface-Bound Adenosine Triphosphatase and Ribulose Diphosphate Carboxylase of Chloroplasts Isolated from the Barley Leaves Senescing in Darkness

Dark-induced aging of detached primary leaves of 11-day-old barley seedlings brings about a significant decline in the rates of ferricyanide [Fe(CN)₆]^3? reduction and photophosphorylations of isolated chloroplasts. Ferricyanide-supported noncyclic photophosphorylation is somewhat more susceptible to leaf aging than phenazine methosulfate (PMS)-supported cyclic phosphorylation. Non-latent membrane-bound adenosine triphosphatase (ATPase) and ribulosediphosphate carboxylase (RuDPCase) lose about half of their initial activities after 24 h, while during this period the electron transport and photophosphorylation activities are much less affected. Also, the loss of RuDPCase is almost complete, while chloroplasts still exhibit a significant level of [Fe(CN)₆]^3? reduction and photophosphorylations after 7 days of dark incubation. This would suggest that the enzymatic dark reactions are more sensitive to aging stress than the primary photochemical reactions of chloroplasts. Studies on the effect of divalent cations such as Mg²⁺ and Ca²⁺ on non-latent ATPase activity revealed that the dark stressed aging of detached leaves brings about a time dependent alteration in the response of this enzyme to Mg²⁺, but not to Ca²⁺. The former showed inhibitory as well as stimulatory response, whereas the latter always caused the usual stimulation. Addition of kinetin (50 μM) ensured retention of [Fe(CN)₆]^3? reduction, photophosphorylations and RuDPCase activity in chloroplasts during leaf aging, but it failed to preserve the initial loss in the activity of the non-activated membrane-bound ATPase.     

Photosynthetic Rates, Gross Patterns of Carbon Dioxide Assimilation and Activities of Ribulose Diphosphate Carboxylase in Marine Algae Grown at Different Temperatures

Studies of the marine green flagellate Dunaliella tertiolecta have confirmed and extended previous observations of Steemann Nielsen and his colleagues. Algae, grown at 12°C, assimilated carbon dioxide under light-saturated conditions more rapidly than did those grown at 20°C; for both, the assimilation rate being higher at 20°C than at 12°C. Cells grown at the lower temperature contained higher concentrations of soluble protein, higher activities of ribulose diphosphate carboxylase and showed an enhanced relative rate of protein synthesis during the photosynthetic assimilation of carbon dioxide. This appears to represent true adaptation since it allowed the growth rate at 12°C to be almost the same as that at 20°C. Studies of the marine diatom Phaeodactylum tricornutum have not revealed the same picture of temperature adaptation. Cultures grown at 5°C had significantly higher rates of photosynthesis than did those grown at 10°C, but the same was not true when algae grown at 10°C were compared with those grown at 20°C. In this organism, growth at the lower temperatures reduced its ability to photosynthesize at 20°C. Cells grown at the lower temperatures contained more protein than did those grown at 20°C; this was particularly marked in cells growing at 5°C, a temperature which reduced the growth rate. The relative rate of protein synthesis was higher in Phaeodactylum grown at lower temperatures; but this difference was most marked when the measurements were made at 20°C.     

Light-induced de Novo Synthesis of Ribulose 1,5-Diphosphate Carboxylase in Greening Leaves of Barley

An antibody specific for ribulose 1,5-diphosphate carboxylase was used to isolate the enzyme from greening barley (Hordeum vulgare L.) leaves. The increase in enzymatic activity during greening was due to de novo synthesis of the enzyme. Increases in enzymatic activity were accompanied by corresponding increases in enzyme protein and by incorporation of radioactive leucine, all of which were inhibited by low concentrations of cycloheximide. ¹⁴C-Labeled amino acids were incorporated into the enzyme by covalent peptide bonding.     

Linearity and Functioning Forms in the Carboxylase Reaction of Spinach Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase

The reaction of spinach RuBisCO activated with CO₂ and Mg²⁺proceeded in two phases, an initial burst for a few minutesand the subsequent linear phase, in the presence of saturatingconcentrations of CO₂, ribulose 1,5-bisphosphate (RuBP), andMg²⁺. The percentage of the activity in the linear phase tothat in the initial burst was 55% with RuBisCO prepared withpolyethylene glycol, and very close to the value with the enzymereleased immediately from isolated chloro-plasts. RuBisCO preparedwith ammonium sulfate had a much larger decrease of the activityin the linear phase. The Euglena enzyme had a linear courseof reaction with time for up to 20 minutes. The K_m for CO₂ of spinach RuBisCO activated beforehand was 20µM in the initial burst, and 28 µM in the linearphase. In the carboxylase reaction initiated with inactive enzyme,the activity was initially negligible, but in 5 minutes increasedto the level observed in the linear phase of the activated enzyme.The K_m for CO₂ in the linear phase of the pre-inactivated enzymewas 70 µM. The concentration of RuBP was the immediate cause of the two-phasiccourse of the carboxylase reaction of spinach RuBisCO. The curvatureof the time course was not observed below 35 µM RuBP.The enzyme required over 88 µM RuBP for the conventionaltwo-phasic course. Further increase of the concentration ofRuBP increased the extent of the curvature, but did not startthe curvature sooner after the start of the reaction. Even ifspinach RuBisCO was in the linear phase, dilution of RuBP orits consumption by the enzymatic reaction to less than 30 µMcaused the enzyme to show the resumed biphasic reaction courseafter addition of a high concentration of RuBP. ¹This paper is the twenty-fourth in a series on PhotosyntheticCarbon Metabolism in Euglena gracilis. (Received September 19, 1988; Accepted November 25, 1988)     

Light Regulatilon of the Synthesis of Ribulose 1,5-Bisphosphate Carboxylase and Fructose 1, 6-Bisphosphatase

The activity of ribulose 1,5-bisphosphate carboxylase (RuBPCase, E. C. 4. 1. 1. 395, fructose 1,6-bisphosphatase (FBPase, E. C. 3. 1. 3. 11) and sedoheptulose 1,7-bisphosphatase (SBPase, E. C. 3. 1. 3. 37) was assayed in the etiolated cotyledons of Brassica juncea after red light or far- red light stimulation. There seemed to be a light-sensitive phase in the course of germination as indicated by the response of leaves to light. During this phase red light stimulated the synthesis of RuBPCase and FBPase, but not SBPase. This effect of red light could be reversed by farred light. Therefore, the initiation of the synthesis of the two enzymes was mediated by phytochrome. The amount of enzyme synthesized was not concerned with the number of light quanta. Phytochrome is only involved in the initiation of the synthesis of certain enzymes, but whether the synthesis will proceed continuosely ro not depends on many other factors, e. g. the availability of substrate and energy.     

Ribulose Bisphosphate Carboxylase Activity in Anther-Derived Plants of Saintpaulia ionantha Wendl. Shag

Plants obtained from anther culture of the African violet, Saintpaulia ionantha Wendl. `Shag' and vegetatively cloned copies of the parent anther donor plant were examined for their ploidy and ribulose-1,5-biphosphate carboxylase (RuBPcase) activity. The cloned parent plants were all diploid and did not vary much in their nuclear DNA, chlorophyll, and RuBPcase activity. Some of the anther-derived plants were similar to the parent plants while others were not. Different levels of ploidy were observed among the androgenetic plants. RuBPcase activities higher than that of the parent plants were found in some anther-derived plants. However, there was no direct correlation between ploidy and RuBPcase activity. Expression of nuclear genes from a single parent in the anther-derived plants and it's diploidization or plastid changes during early stages of microsporogenesis or androgenesis are suggested as possible reasons for the variations observed among them. This could be a useful technique to obtain physiological variants which could be agronomically desirable.     

Ribulose Bisphosphate Carboxylase Activity and Photosynthesis during Leaf Development in the Tomato

Besford, R. T., Withers, A. C. and Ludwig, L. J. 1985. Ribulosebisphosphate carboxylase activity and photosynthesis duringleaf development in the tomato.—J. exp Bot. 36: 1530–1541. The carboxylase activity of ribulose-1,5-bisphosphate carboxylase/oxygenaseand of phosphoenolpyruvate carboxylase, and the light saturatedrate of net photosynthesis were measured in the developing 5thleaf of tomato plants. Values for light saturated net photosynthesiswere also calculated from the measured carboxylase activitiesand estimates of internal CO₂ and oxygen concentrations. Thecalculated rate using the activity of ribulose bisphosphatecarboxylase alone for net CO₂ assimilation in 300 mm³ dm^–3CO₂ was greater than the measured rate at 80% and full expansionbut less than the measured rate in younger leaves. When theactivities of both the carboxylases were taken into accountbetter agreement was evident for young leaves but the rate wasfurther overestimated for older leaves The calculated rate forphotosynthesis in 1200 mm³ dm^–3 CO², assuming saturationof ribulose bisphosphate carboxylase with RuBP, was an overestimatefor young leaves but was close to the observed values for leavesnear full expansion. The results are discussed in terms of measuredconductances for CO₂ and the availability of RuBP in the leaf Key words: Tomato, leaf development, photosynthesis, RuBP carboxylase, oxygenase     

The Effect of and Ions on the Reactions of Ribulose Bisphosphate Carboxylase

The effects of sulphite ion () and sulphate ion () on both the activation and the catalytic activities of ribulose- 1, 5-bisphosphate carboxylase (EC 4.1.1.39 [EC] )were studied and compared to those of other effectors of theenzyme, particularly inorganic phosphate (P₁). The activationby CO₂ and Mg²⁺ of a slow activating form of the carboxylasein the presence of the two anions produced high specific activitieswith significant lower concentrations of CO₂ than normally required.This was due to stabilization of the ternary complex betweenthe enzyme, CO₂ and Mg²⁺. With a rapidly activating speciesof enzyme, and caused only a small increase in activation with subsaturating CO₂. , and P₁, with saturatingconcentrations of CO₂ also enhanced the catalytic activity abovethat achieved with CO₂ and Mg²⁺ alone; P₁ was the most effectiveof the anions, producing a 50% increase in the specific activity,both with the slow and rapidly activating species. and were potent inhibitors of the carboxylase and oxygenase reactions of the enzyme. was a non-competitive inhibitor with respect to CO₂, and competitive/mixedwith respect to ribulose-1, 5-bisphosphate. The time courseof the carboxylase and oxygenase reactions in the presence of were biphasic with inhibition apparent only in the second phase. Key words: Ribulose bisphosphate carboxylase, Activation, SO₃^2-, SO₄^2-