The Association of d-Ribulose- 1,5-Bisphosphate Carboxylase/Oxygenase with Phosphoribulokinase

When Ribulose- 1,5-bisphosphate carboxylase/oxygenase was purified from spinach leaves (Spinacia oleracea) using precipitation with polyethylene glycol and MgCl₂ followed by DEAE cellulose chromatography, 75% of phosphoribulokinase and 7% of phosphoriboisomerase activities copurified with ribulose- 1,5-bisphosphate carboxylase/oxygenase. This enzyme preparation showed ribose-5-phosphate and ribulose-5-phosphate dependent carboxylase and oxygenase activities which were nearly equivalent to its corresponding ribulose- 1,5-bisphosphate dependent activity. The ribose-5-phosphate and ribulose-5-phosphate dependent reaction rates were stable and linear for much longer time periods than the ribulose- 1,5-bisphosphate dependent rates. When sucrose gradients were used to purify ribulose- 1,5-bisphosphate carboxylase/oxygenase from crude stromal extracts, phosphoribulokinase was found to cosediment with ribulose- 1,5-bisphosphate carboxylase. Under these conditions most of the phosphoriboisomerase activity remained with the slower sedimenting proteins. Ammonium sulfate precipitation resulted in separation of the ribulose- 1,5-bisphosphate carboxylase peak from phosphoribulokinase peak. Crude extracts of peas Pisum sativum and spinach contained 0.725 to 0.730 milligram of phosphoribulokinase per milligram of chlorophyll, respectively, based on an enzyme-linked immunosorbent assay.     

Regulation of Synthesis of Chlorophyll, Carotene, Ribulosel,5-diP Carboxylase and Phosphoribulokinase in a Temperature-sensitive Chlorophyll Mutant of Medicago sativa

The activities of several enzymes were studied in a temperature-sensitive chlorophyll mutant of alfalfa (Medicago saliva). In leaves grown at 10°C photosynthetic capacity was essentially nil with ribulose-1,5-diP carboxylase, chlorophyll, and carolene present in greatly limiting concentrations. The activity of phosphoribulokinase was 3.5 times lower at 10°C than at 27°C, but was still sufficiently high at 10°C to not limit the rate of CO₂ fixation. Activities of phosphoriboisomerase, phosphoenolpyruvate carboxylase, glucose-6-P dehydrogenase and malate dehydrogenase were not different at 10°C and 27°C. The low fraction I protein content (which also accounts for the ribulose-1,5-dip carboxylase activity in alfalfa) indicated that synthesis of the carboxylase was effectively blocked at 10°C. A large, comparable increase in carboxylase activity and in concentration of fraction I protein in alfalfa leaves grown at 27°C indicated that the carboxylase was synthesized de novo. The initial induction of the carboxylase, chlorophyll, and carotene may be related, but after induction the carboxylase was not linearly correlated with the other two and had a different temperature optima. Nevertheless, the synthesis of each appeared to be regulated by the temperature-sensitive gene of this mutant.     

Influence of age and illumination on distribution of several calvin cycle enzymes in greening barley leaves

Activities of phosphoriboisomerase, phosphoribulokinase, and ribulose 1,5-diphosphate carboxylase, protein content, and chlorophyll accumulation in dark-grown barley seedlings were measured before and after illumination. Enzymatic activities, levels of soluble protein, and accumulation (upon illumination) of chlorophyll in leaves declined from tips toward the base. In response to increasing time of illumination, chlorophyll accumulation and activities of phosphoribulokinase and ribulose 1,5-diphosphate carboxylase (enzymes located in chloroplasts) increased most in tip portions whereas activity of phosphoriboisomerase and levels of soluble protein (constituents not confined to chloroplasts) increased similarly in all sections of the leaf. Maximum activity of phosphoribulokinase and maximum accumulation of chlorophyll shifted toward median portions of the leaf blade with increased age of seedling before illumination. Maximum activity of ribulose 1,5-diphosphate carboxylase and maximum level of soluble protein occurred in all leaf sections when the seedlings were 7 days of age before illumination.     

Development of Enzymes Involved in Photosynthetic Carbon Assimilation in Greening Seedlings of Maize (Zea mays)

Upon illumination of dark-grown maize seedlings (5 days old) with incandescent light, there occurred a nearly simultaneous increase, after a certain lag period, in the activities of enzymes engaged in the C₄ pathway and the Calvin-Benson cycle. The light-induced biosynthesis of chlorophyll (a and b) precedes the increase in enzyme activities and proceeds without lag phase. A diphasic feature in the elevation of enzyme activities as a function of the intensities of light provided was observed; the increase in enzyme activities was enhanced by light intensities greater than 10³ ergs per square centimeter per second in comparison with light of lower intensities. Under light intensities greater than 10³ ergs per square centimeter per second, the simultaneous addition of levulinic acid, which inhibited chlorophyll formation, markedly reduced the increase of enzyme activities. However, neither the diphasic light effect nor the inhibitory effect of levulinic acid was observed with ribulose-1,5-bisphosphate carboxylase. The enzyme activities in the dark-grown maize seedlings were enhanced by a brief irradiation with the red light and the red light effect was reversed by the following far red light treatment. The red light-induced increase in the enzyme activities did not accompany chlorophyll synthesis, and was completely inhibited by cycloheximide, indicating that enzyme synthesis rather than activation might be involved. Light may play a dual role in enzyme induction; one is as an energy source through the photosystems at high intensities and the other is presumably as a signal mediated by phytochrome at low intensities.     

Elimination of the lag period in chloroplast development in a chlorophyll mutant of peanuts

The mutation of a nuclear gene in peanut (Arachis hypogaea L.) plants results in a reduced light-dependent development of chloroplast fine structure, soluble protein, ribulose-1, 5-diP carboxylase, NADP-glyceraldehyde-3-P dehydrogenase, fructose-1, 6-diP aldolase, glycerate-3-P kinase, phosphoenolpyruvate carboxylase, malate dehydrogenase, and dark respiration during the 72-hour lag period of chlorophyll synthesis in dark-grown leaves exposed to continuous light. The mutation has pleiotropic affects. Kinetic analysis shows there is also a 72-hour lag period in the light-dependent development of NADP-glyceraldehyde-3-P dehydrogenase and fructose-1, 6-diP aldolase in the mutant leaves, whereas there is no lag in the development of NAD-malate dehydrogenase and dark respiration. There is minimal development of the chloroplast during the 72-hour mutationally induced lag period, but there is pronounced cytoplasmic and mitochondrial activity during this phase. There is a 24-hour lag period in the light-dependent enlargement of the mutant leaves. At the completion of leaf enlargement, chloroplast differentiation is initiated. The mutation does not result in any chloroplast deletions, it only affects the timing of the synthesis of these components.     

Evidence for in vivo Light-induced Synthesis of Ribulose-1,5-diP Carboxylase and Phosphoribulokinase in Greening Barley Leaves

When actinomycin D, puromycin, streptomycin, chloramphenicol, and cycloheximide, known inhibitors of protein synthesis, were applied to leaves of intact seedlings or detached leaves of barley prior to their greening, the same general response resulted: the light-induced increase in activity of ribulose 1,5-diphosphate carboxylase was prevented while that of phosphoribulokinase was only partially suppressed; synthesis of chlorophyll was arrested. This is taken as preliminary evidence that de novo synthesis of protein may be responsible for the observed increase in ribulose-1,5-diphosphate carboxylase activity during greening. However, other factors may be involved with the light-induced stimulation of phosphoribulokinase.

Carbohydrate metabolites and substrates of the enzymes failed to induce the formation of ribulose-1,5-diphosphate carboxylase and phosphoribulokinase in the dark. No evidence was found for the presence of inhibitors in etiolated seedlings or activators in illuminated leaves of barley. Carboxylase activity almost equal to that of the illuminated water control was stimulated by MgCl₂ in the dark; MgCl₂ had no effect on the activity of the kinase.

     

Early effects of illumination on the activity of some photosynthetic enzymes

Leaves of dark-grown corn (Zea mays) were illuminated for periods ranging from 3 minutes to 12 hours. The changes in the activities of ribose-5-phosphate isomerase, ribulose-5-phosphate kinase, and ribulose-1,5-diphosphate carboxylase were followed.

The activity of ribose-5-phosphate isomerase did not change significantly until between 12 and 24 hours of illumination. An increase in ribulose-5-phosphate kinase activity occurred after a lag of about 6 hours. The increase in carboxylase activity began after 3 minutes of illumination and increased until after 3 to 6 hours in the light, after which it began to decline. The increases in these enzymes appear to be the result of protein synthesis.

     

The presence of ribulose 1,5-diphosphate carboxylase in the nonphotosynthetic endosperm of germinating castor beans

Ribulose 1,5-diphosphate carboxylase was detected in extracts of germinating castor bean (Ricinus communis var. Hale) endosperms. This is the first report of this enzyme in a nonphotosynthetic (no chlorophyll) plant tissue. Radioactive 3-phosphoglyceric acid has been identified as the principle product resulting from the enzymatic condensation of ¹⁴C-bicarbonate and ribulose-1,5-diP in endosperm extracts. The Km values of bicarbonate and ribulose-1,5-diP for the endosperm carboxylase are 1.14 × 10⁻²m and 7.5 × 10⁻⁵m, respectively. The carboxylase activity peaks at 4 days in endosperms of castor beans germinated in the dark. The specific activity of the carboxylase at this stage of germination is 4.3 μmoles of 3-phosphoglycerate formed/mg protein·hr. The presence of ribulose-1,5-diP carboxylase and other enzymes of the reductive pentose phosphate pathway show the potential of this pathway in castor bean endosperms.     

Phytochrome-mediated plastid development in etiolated pea stem apices

The effects of red and far-red light on growth and plastid development in the stem apices of etiolated pea seedlings have been examined. Changes were determined in various growth parameters (DNA, soluble protein and fresh weight) and also in the activities of the plastid-localized enzymes ribulose-1,5-bisphosphate carboxylase, NADP-glyceraldehyde-3-phosphate dehydrogenase and alkaline-1,6-bisphosphatase and the non-photosynthetic (cytoplasmic) enzymes NADP-isocitrate dehydrogenase, enolase and NAD-malate dehydrogenase. Changes in the amounts of Fraction I protein were also measured. Brief daily irradiation with low intensity red light increased growth 5·1–7·6-fold which was correlated with increases of about 3·5-fold in activities of the non-photosynthetic enzymes. The chloroplast enzymes, however, showed much greater increases in activity ranging from 15- to 91-fold. Fraction I protein increased 11·7-fold. These increases approached the levels attained in fully green leaves. All these responses were largely prevented by far-red light indicating that they were mediated by phytochrome. In experiments with red light given at daily intervals there was a lag of 24 hr before the initially very low activity of ribulose-1,5-bisphosphate carboxylase increased. Fraction I protein which was initially present in significant amounts showed a similar lag in its synthesis. However, for 3 days after the initial irradiation, the rate of increase of the enzymic activity was much greater than the rate of net synthesis of Fraction I protein. A single initial red irradiation was as effective as 3 daily irradiations in increasing the activity of ribulose-1,5-bisphosphate carboxylase. A fourth irradiation, however, gave an additional response which exceeded that of the single initial irradiation. It was shown that there was a rapid activation of ribulose-1,5-bisphosphate carboxylase by either continuous white or 3 min of red light. The red light response was slowly reversed in the dark. These results are discussed with particular emphasis on the relation between growth and plastid development in a phytochrome-mediated system.     

Changes in enzymatic activities in etiolated bean seedling leaves after a brief illumination

The phytochrome controlled increase in total protein in the primary leaf pair of etiolated bean (Phaseolus vulgaris var. Black Valentine) seedlings, which occurs during growth in the dark subsequent to a brief illumination, was investigated. Enzymes from the chloroplasts, the mitochondria, and the soluble cytoplasm all increase in total activity after the illumination.

The total protein and the ribulose carboxylase increases are not inhibited by FUdR, an inhibitor of DNA synthesis. Cycloheximide, an inhibitor of protein synthesis, applied at a time when the ribulose carboxylase activity increase has already commenced, blocks further increase. It was concluded that the total protein and the enzyme increases in the leaf are the result of increases in the per cell levels.

The initial brief illumination is saturating, but 40 minutes later the seedlings have acquired the ability to respond to a second brief illumination. The rate of increase in ribulose carboxylase activity in seedlings that have been illuminated twice is greater than the rate in seedlings that have been illuminated only once.

Far-red light prevents further increase in enzyme activity 48 hours after the initial illumination. There is a lag period interposed between the time of illumination with far-red light and the time at which the seedlings show the greatest effect of far-red light. It was concluded that the phytochrome influence on protein synthesis is not at the terminal steps.

     

Effect of growth conditions on carboxylating enzymes of Zea mays plants

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) and ribulose-1,5-bisphospate (RuBP) carboxylase (EC 4.1.1.39) activities in leaves of different maize hybrids grown under field conditions (high light intensity) and in a growth chamber (low light intensity) were determined. Light intensity and leaf age affected PEP carboxylase activity, whereas RuBP carboxylase was affected by leaf age only at low light intensity. PEP carboxylase/RuBP carboxylase activity ratio decreased according to light intensity and leaf age. Results demonstrate that Zea mays grown under field conditions is a typical C₄ species in all leaves independently from their position on the stem, whereas it may be a C₃ plant when it is grown in a growth chamber at low light intensityAbbreviations PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

NITRATE REDUCTASE ACTIVITY OF AMPHIDINIUM CARTERI AND CACHONINA NIEI (DINOPHYCEAE) IN BATCH CULTURE: DIEL PERIODICITY AND EFFECTS OF LIGHT INTENSITY AND AMMONIA1,2

The activity of extracted NADH-NO₃^? reductase was measured in the marine dinoflagellates Amphidinium carteri Hulburt and Cachonina niei Loeblich. Its activity showed a diel periodicity and was ca. twice as great at midday as at midnight. The enzyme activity was unstable, with an in vitro half-life of 2–3 h. Values of enzyme activity were low or undetectable during lag phase but paralleled the instantaneous growth rate value during log phase. Nitrate reductase activity was not found in the stationary phase of growth, but additions of NO₃^? resulted in enzyme activity after 24h. When A. carteri was exposed to a series of light intensities for several weeks, the division rate and enzyme activity increased with increasing light intensity up to saturating intensities. In 6 h exposures, enzyme activity decreased with decreasing light intensities below light intensities saturating division rate. Additions of NH₄⁺ (0.5–50 μm) to A. carteri cultures decreased the amount of extractable enzyme. The in vitro activity was not inhibited by similar NH⁺₄ concentrations.     

Purification and characterization of pea chloroplastic phosphoriboisomerase

Pea (Pisum sativum L.) chloroplastic phosphoriboisomerase (EC 5.3.1.6) can be purified to apparent homogeneity in less than 2 days time with a 53% yield. Important steps in the purification include heat treatment and pseudoaffinity chromatography on Red H-3BN Sepharose. The purified isomerase has a subunit molecular mass of 26.4 kD. The N-terminal sequence has been determined through 34 residues. pH optima are 7.8 (ribose-5-phosphate) and 7.7 (ribulose-5-phosphate); K_m values are 0.9 millimolar (ribose-5-phosphate) and 0.6 millimolar (ribulose-5-phosphate). The enzyme is inhibited by erythrose-4-phosphate, sedoheptulosebisphosphate, glyceraldehyde-3-phosphate, and 3-phosphoglycerate at concentrations close to those found in photosynthesizing chloroplasts. Countercurrent phase partitioning experiments indicate that the pea chloroplastic phosphoriboisomerase interacts physically with phosphoribulokinase.     

Development of Photosynthetic Activity following Anaerobic Germination in Rice-Mimic Grass (Echinochloa crus-galli var oryzicola)

Shoots of anaerobically germinated Echinochloa crus-galli var oryzicola are nonpigmented whether germinated in light or dark, and chlorophyll synthesis is minimal for the first 12 to 18 hours of greening after exposure to ambient conditions. When chlorophyll development is compared between greening anoxic and etiolated shoots, there is a 100-fold difference in chlorophyll levels at 8 hours, an 8-fold difference at 24 hours, but roughly equal amounts at 60 hours. The chlorophyll a/b ratio approaches 3 earlier in greening anoxic shoots than in greening etiolated shoots, relative to total chlorophyll. The long lag in chlorophyll synthesis can be shortened by giving dark-grown anoxic shoots a 24-hour midtreatment of air before light.

Development of photosynthetic activity in etiolated shoots, determined by CO₂ gas exchange, ¹⁴CO₂ uptake, and activity of carboxylating enzymes closely parallels development of chlorophylls. However, development of photosynthetic capability in greening anoxic shoots does not parallel chlorophyll development; ability to fix carbon lags behind chlorophyll synthesis. A reason for this lag is the very low activity of RuBP carboxylase during the first 36 hours of greening in anoxic shoots. The activity of phosphoenolpyruvate carboxylase is also delayed, but its kinetics more closely match those of chlorophyll development.

     

光强对两种硅藻光合作用、碳酸酐酶和RubisCO活性的影响

为研究海洋浮游硅藻光合固碳能力与光强的关系, 以三角褐指藻和威氏海链藻为实验材料, 测定了不同光强培养下三角褐指藻和威氏海链藻生长、光合特性、碳酸酐酶和核酮糖-1, 5-二磷酸羧化/氧化酶活性(RubisCO)的变化, 结果显示高光强促进两种硅藻的生长, 但对威氏海链藻的影响更明显。高光强导致两种硅藻叶绿素a、c含量、光系统Ⅱ的最大光化学效率和实际光化学效率明显下降, 非光化学淬灭系数明显升高, 但对光化学淬灭系数并没有明显影响。在高光下威氏海链藻和三角褐指藻胞内外碳酸酐酶活性明显升高。在高光强下培养的威氏海链藻RubisCO活性明显高于低光下培养, 但三角褐指藻正好相反, 不管高光还是低光培养威氏海链藻RubisCO活性始终高于三角褐指藻。以上结果表明不同硅藻对光强变化的响应存在差异, 它们可以通过调节光合生理特征、光合固碳关键酶和CO2供应以适应光强的变化。       

Effects of Light on Degradation of Chlorophyll and Proteins during Senescence of Detached Rice Leaves

Regulatory effects of light on senescence of rice leaves wereinvestigated by measuring degradation of chlorophyll and proteinsin leaf segments which had been kept in the dark or under illuminationwith light of different intensities and colors. When leaveshad been left in total darkness for three days at 30°C,there was an initial long lag that lasted for one whole dayand then chlorophyll was rapidly degraded in the second andthird days. Breakdown of chlorophyll was strongly retarded bycontinuous illumination with white light of intensity as lowas 0.5 µmol photons m^–2 s^–1 but the effectof light decreased at intensities above 10 µmol photonsm^–2 s^–2. The initial lag and subsequent degradationof chlorophyll in the dark were little affected by illuminationwith red or far red light at the beginning of dark treatment.However, a brief illumination with red light at the end of thefirst and/or second day significantly suppressed degradationof chlorophyll during subsequent dark periods and the effectof red light was nullified by a short irradiation with far redlight. Thus, degradation of chlorophyll is regulated by phytochrome.Thylakoid membrane proteins and soluble proteins were also largelydegraded during three days in the dark. Degradation of membraneproteins such as the apoproteins of light-harvesting chlorophylla/b proteins of photosystem II and chlorophyll a-binding proteinsof reaction center complexes showed a long lag and was stronglysuppressed by illumination with weak white light. Thus, theloss of chlorophyll can be correlated with degradation of chlorophyll-carryingmembrane proteins. By contrast, light had only a weak protectingeffect on soluble proteins and ribulose-1,5-bisphosphate carboxylase/oxygenaserapidly disappeared under illumination with weak white light.Thus, breakdown of thylakoid membrane and soluble proteins aredifferently regulated by light. Artifacts which would be introducedby detachment of leaves were also discussed. ¹ Present address: Department of Applied Biology, Faculty ofScience and Technology, Science University of Tokyo, Yamazaki,Noda-shi, Chiba, 278 Japan. ² Present address: Department of Life Science, Faculty of Science,Himeji Institute of Technology, Harima Science Park City, Hyogo,678-12 Japan.     

Current views on the regulation of autotrophic carbon dioxide fixation via the Calvin cycle in bacteria

The Calvin cycle of carbon dioxide fixation constitutes a biosynthetic pathway for the generation of (multi-carbon) intermediates of central metabolism from the one-carbon compound carbon dioxide. The product of this cycle can be used as a precursor for the synthesis of all components of cell material. Autotrophic carbon dioxide fixation is energetically expensive and it is therefore not surprising that in the various groups of autotrophic bacteria the operation of the cycle is under strict metabolic control. Synthesis of phosphoribulokinase and ribulose-1,5-bisphosphate carboxylase, the two enzymes specifically involved in the Calvin cycle, is regulated via end-product repression. In this control phosphoenolpyruvate most likely has an alarmone function. Studies of the enzymes isolated from various sources have indicated that phosphoribulokinase is the target enzyme for the control of the rate of carbon dioxide fixation via the Calvin cycle through modulation of existing enzyme activity. In general, this enzyme is strongly activated by NADH, whereas AMP and phosphoenol-pyruvate are effective inhibitors. Recent studies of phosphoribulokinase inAlcaligenes eutrophus suggest that this enzyme may also be regulated via covalent modification.     

Development of the photosynthetic apparatus during light-dependent greening of a mutant of Chlorella fusca

The formation of chlorophyll, cytochrome f, P-700, ribulose bisphosphate carboxylase as well as photosynthesis and Hill reaction activities were tested during the light-dependent greening process of the Chlorella fusca mutant G 10. Neither chlorophyll nor protochlorophyllide was detected in the darkgrown cells. When transferred to light the mutant cells developed chlorophyll and established its photosynthetic capacity after a short lag phase. In the in vivo absorption spectra a spectral shift of the red absorption peak position from 674 to 680 nm was indicated during the first 3 h of greening. Cytochrome f was already present in the dark-grown cells, but during the greening phase a threefold increase in the cytochrome f content could be seen. At the early stages of greening a characteristic primary oscillation in the content of cytochrome f was observed. P-700 was lacking in the dark and during the first 30 min of illumination. From the first to the second h of light a forced synthesis of P-700 took place and the time-course curve for the ratios of P-700/chlorophyll rose to a sharp maximum. The synthesis of P-700 started together with photosystem I activity and showed similar kinetics. We found the simultaneous appearance of photosystem II, photosystem I, and photosynthetic activities 30 min after the beginning of the illumination. Based on chlorophyll content they attained maximum activity after 2 h of light, but at this time photosystem I capacity proved to be remarkably higher than photosynthetic and photosystem II activities. Highest carboxylase activity existed in darkgrown cells. During the greening process the activity of the enzyme decreased continuously. After 2 h of illumination chlorophyll synthesis partially served to increase the size of the photosynthetic unit, which consequently led to a decrease in the light energy needed to saturate photosynthesis and also to a decrease of photosynthetic rate based on chlorophyll content.Abbreviations Chl chlorophyll - Cyt f cytochrome f - DPIP 2,6-dichlorophenolindophenol - EDTA ethylenediaminetetraacetic acid - GSH glutathione - LH light-harvesting - PS photosystem - RuBP ribulose bisphosphate     

Enzyme levels in relation to obligate phototrophy in chlamydobotrys

During the transition from photoheterotrophic growth on acetate to phototrophic growth on carbon dioxide, there is a decrease in isocitrate lyase and increase in ribulose-1,5-diphosphate carboxylase activity in Chlamydobotrys stellata cultures. The increase in ribulose-1,5-diphosphate carboxylase activity is the result of protein synthesis, there being a close correlation between increase in enzyme activity and protein precipitated by antibody to ribulose-1,5-diphosphate carboxylase. The purified ribulose-1,5-diphosphate carboxylase was similar to the constitutive enzyme from other green algae having a molecular weight of 530,000 and composed of two types of subunit of molecular weight 53,000 and 14,000.     

Evidence for lack of turnover of ribulose 1,5-diphosphate carboxylase in barley leaves

Turnover of ribulose 1,5-diphosphate carboxylase in barley leaves (Hordeum vulgare L.) was followed over time in light and dark. The enzyme was degraded in prolonged darkness and was resynthesized after the plants were returned to light. Labeling with ¹⁴C showed that simultaneous synthesis and degradation (turnover) did not occur in light. In contrast, the remaining soluble protein was turned over rapidly in light. Although ribulose 1,5-diP carboxylase can be both degraded and synthesized, these processes seem not to occur simultaneously, but can be induced independently by changing environmental conditions.