Ribulose-1,5-biphosphate carboxylase-deficient plastome mutants of Oenothera.

In spite of only slightly subnormal pigment contents, two plastome mutants of Oenothera (Valpha, Isigma) were practically incapable of photosynthetic CO2 fixation and another one exhibited considerably reduced photosynthesis (IVbeta). While other photosynthetic enzymes were present as far as investigated, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) activity was very low or missing altogether. As shown by gel electrophoresis, mutant IVbeta contained some, though little, fraction I protein. In the other two mutants fraction I protein could not be detected. Also, neither the small nor the large subunit of ribulose-1,5-biphosphate carboxylase could be found in these mutants. In immunodiffusion experiments with a monospecific antiserum against rye ribulose-1,5-bisphosphate carboxylase, only extracts from wild-type Oenothera produced visible precipitation lines. Still, the presence of very low levels of immunochemically reactive antigen was indicated for all three mutants. The highest level was observed in mutant IVbeta. The behaviour of the mutant extracts suggested that the antigens of mutant and wild type leaves reacting with the antiserum were not identical. All mutants appeared to have a coupled electron transport system as shown by ATP measurements, light scattering and 515 nm absorption changes. Linear electron transport was possible in the mutants. Still, the photoresponse of cytochrome f and fluorescence measurements suggested altered electron transport properties in the mutants. These are interpreted to be secondary lesions of the photosynthetic apparatus caused by primary deficiency in ribulose-1,5-bisphosphate carboxylase activity. From the absence in two mutants (Valpha, Isigna) of the small subunit of ribulose-1,5-bisphosphate carboxylase, which is known to be coded for by nuclear DNA and to be synthesized on cytoplasmic ribosomes, it appears that the genetic system of the plastids is capable of interfering with the genome-controlled synthesis of plastid components.     

Cyanide-resistant electron transport in sporulating Bacillus megaterium KM

The NADH oxidase activity of stage V mother-cell membranes, isolated from sporulating Bacillus megaterium KM, shows a greater inhibition by cyanide and displays this response at lower concentrations of cyanide than the stage V forespore inner membrane. Comparison of the effects of various respiratory inhibitors reveals that the difference in cyanide sensitivity between these membranes is located on the oxidase side of the 2-heptyl-4-hydroxyquinoline N-oxide-sensitive step. Both membranes contain cytochromes a+a₃, b-562, b-555, c and d, with three potential oxidases: cytochromes a+a₃, o and d. Cyanide difference spectra suggest that cytochromes b-562 and d may be the components involved in the cyanide-resistant electron transport pathway. Membrane ascorbate-N,N,N′,N′-tetramethylphenylenediamine and ascorbate 2,6-dichlorophenolindophenol oxidase activities are highly sensitive to cyanide. Evidence is presented for terminal branching of the respiratory chain with branches differing in cyanide sensitivity. The cyanide sensitivity of the NADH oxidase of membranes prepared from various stages of sporulation is compared. Morphogenesis of the mother-cell plasma membrane to a cyanide-sensitive form during stages II and III of sporulation is postulated.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase from parsley.

Ribulose-1,5-bisphosphate carboxylase/oxygenase from parsley leaves was purified by Sepharose 6B gel filtration at pH 8.3 as a single, colorless peak containing both activities. Approximately 0.2 g atom copper per mole enzyme was detected by atomic absorption spectroscopy, but this copper was not detectable by EPR spectrometry.     

核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)

文章就核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)的分布、结构、性质、分类与功能的研究进展作了介绍。     

Ribulose-1,5-bisphosphate carboxylase/oxygenase from thermophilic cyanobacterium Thermosynechococcus elongatus

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) can be divided into two branches: the “red-like type” of marine algae and the “green-like type” of cyanobacteria, green algae, and higher plants. We found that the “green-like type” rubisco from the thermophilic cyanobacterium Thermosynechococcus elongatus has an almost 2-fold higher specificity factor compared with rubiscos of mesophilic cyanobacteria, reaching the values of higher plants, and simultaneously revealing an improvement in enzyme thermostability. The difference in the activation energies at the transition stages between the oxygenase and carboxylase reactions for Thermosynechococcus elongatus rubisco is very close to that of Galdieria partita and significantly higher than that of spinach. This is the first characterization of a “green-like type” rubisco from thermophilic organism.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase protein prevents the in vitro decline in activity of ribulose-1,5-bisphosphate carboxylase/oxygenase

The rate of CO₂ fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) following addition of ribulose 1,5-bisphosphate (RuBP) to fully activated enzyme, declined with first-order kinetics, resulting in 50% loss of rubisco activity after 10 to 12 minutes. This in vitro decline in rubisco activity, termed fall-over, was prevented if purified rubisco activase protein and ATP were added, allowing linear rates of CO₂ fixation for up to 20 minutes. Rubisco activase could also stimulate rubisco activity if added after fallover had occurred. Gel filtration of the RuBP-rubisco complex to remove unbound RuBP allowed full activation of the enzyme, but the inhibition of activated rubisco during fallover was only partially reversed by gel filtration. Addition of alkaline phosphatase completely restored rubisco activity following fallover. The results suggest that fallover is not caused by binding of RuBP to decarbamylated enzyme, but results from binding of a phosphorylated inhibitor to the active site of rubisco. The inhibitor may be a contaminant in preparations of RuBP or may be formed on the active site but is apparently removed from the enzyme in the presence of the rubisco activase protein.     

Energy-dependent accumulation of iron by isolated rat liver mitochondria V. Effect on factors controlling respiration and oxidative phosphorylation

1. Depending on the metabolic state, the addition of iron(III)-sucrose induces an inhibition or a stimulation of the respiration rate when added to isolated rat liver mitochondria.2. Under conditions identical to those used in the accumulation studies (Romslo, I. and Flatmark, T. (1973) Biochim. Biophys. Acta 305, 29?40), the ferric complex induces a decrease in the oxygen uptake concomitant to an oxidation of cytochromes c (+c₁) and a (+a₃). These results suggest that ferric iron is reduced to ferrous iron by the respiratory chain prior to or simultaneously with its energy-dependent accumulation.3. On the other hand, the addition of iron(III)-sucrose induces a stimulation of respiration in State 4 and State 3 provided Mg²⁺ is present in the suspending medium. In contrast to Ca²⁺, iron stimulates State 4 respiration in a cyclic process only within narrow concentration limits; at concentrations of iron above 100 μM the respiration remains in the activated state until anaerobiosis. The stimulation of State 4 respiration is more pronounced with succinate than with NAD-linked substrates, a difference which partly may be attributed to a stimulation of the succinate dehydrogenase complex.4. The stimulation of respiration by iron is approx. 3 times higher in State 3 than in State 4 and this difference can be attributed to a stimulation of the adenine nucleotide exchange reaction in State 3 with a concomitant increase in the rate of oxidative phosphorylation, although the $\text{P}\text{O}$ ratio is slightly diminished.     

Interaction of Hydrogen Peroxide with Ribulose-1,5-bisphosphate Carboxylase/Oxygenase from Rice

The properties of rice-derived ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) in different concentrations of hydrogen peroxide (H2O2) solutions have been studied. The results indicate that at low H2O2 concentrations (0.2-10 mM), the properties of rubisco (e.g., carboxylase activities, structure, and susceptibility to heat denaturation) change slightly. However, at higher H2O2 concentrations (10-200 mM), rubisco undergoes an unfolding process, including the loss of secondary and tertiary structure, forming extended hydrophobic interface, and leading to cross-links between large subunits. High concentrations of H2O2 can also result in an increase in susceptibility of rubisco to heat denaturation. Further pre-treatments with or without reductive reagents to rubisco show that the disulfide bonds in rubisco help to protect the enzyme from damage by H2O2 as well as other reactive oxygen species.     

Enzymic Properties of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Purified from Rice Leaves

The enzymic properties of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase purified from rice (Oryza sativa L.) leaves were studied. Rice RuBPcarboxylase, activated by preincubation with CO₂ and Mg²⁺ like other higher plant carboxylases, had an activation equilibrium constant (K_cK_Mg) of 1.90 × 10⁵ to 2.41 × 10⁵ micromolar² (pH 8.2 and 25°C). Kinetic parameters of carboxylation and oxygenation catalyzed by the completely activated enzyme were examined at 25°C and the respective optimal pHs. The K_m(CO₂), K_m(RuBP), and V_max values for carboxylation were 8 micromolar, 31 micromolar, and 1.79 units milligram⁻¹, respectively. The K_m(O₂), K_m(RuBP), and V_max values for oxygenation were 370 micromolar, 29 micromolar, and 0.60 units milligram⁻¹, respectively.

Comparison of rice leaf RuBP carboxylase with other C₃ plant carboxylases showed that it had a relatively high affinity for CO₂ but the lowest catalytic turnover number (V_max) among the species examined.

     

Defective ion regulation in a class of membrane-excitation mutants in Paramecium

The “paranoiac” mutants of Paramecium aurelia show prolonged backward swimming in solutions containing Na⁺, unlike wild-type paramecia, which jerk back and forth in Na⁺ solutions. The paranoiac mutants in Na⁺ solutions also show large losses of cellular K⁺ and large influxes of Na⁺. Three different paranoiac mutants all show similar defects in ion regulation but to different degrees. Wild-type Paramecium, in contrast, shows no Na⁺-dependent loss of cellular K⁺ and a much smaller Na⁺ influx. In K⁺-containing solutions, there is no difference between wild-type and paranoiac paramecia with respect to their cellular K⁺ content.The Na⁺ influx, the K⁺ loss, and the duration of backward swimming are all proportional to the extracellular Na⁺ concentration. Electrophysiologically, the backward swimming of the paranoiac mutants corresponds to a prolonged depolarization of the membrane potential, while the backward jerks of wild-type Paramecium correspond to a series of transient depolarizations. We propose that the large Na⁺ influxes and the large K⁺ effluxes in paranoiacs occur during the periods of backward swimming, while the membrane is depolarized.     

Ribulose-1,5-bisphosphate carboxylase as a nuclear and chloroplast marker

Summary The data on the primary structure of ribulose-1,5-bisphosphate carboxylase/oxygenase are reviewed. Examples of their use as markers and in the elucidation of the evolution, adaptation and function of this key enzyme are given.     

Partial Purification and Characterization of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large Subunit epsilonN-Methyltransferase

The large subunit (LS) of tobacco (Nicotiana rustica) ribulose-1,5-bisphosphate carboxylase/oxygenase (ribulose-P₂ carboxylase) contains a trimethyllysyl residue at position 14, whereas this position is unmodified in spinach ribulose-P₂ carboxylase. A protein fraction was isolated from tobacco chloroplasts by rate-zonal centrifugation and anion-exchange fast protein liquid chromatography that catalyzed transfer of methyl groups from S-adenosyl-[methyl-³H]-l-methionine to spinach ribulose-P₂ carboxylase. ³H-Methyl groups incorporated into spinach ribulose-P₂ carboxylase were alkaline stable but could be removed by limited tryptic proteolysis. Reverse-phase high-performance liquid chromatography of the tryptic peptides released after proteolysis showed that the penultimate N-terminal peptide from the LS of spinach ribulose-P₂ carboxylase contained the site of methylation, which was identified as lysine-14. Thus, the methyltransferase activity can be attributed to S-adenosylmethionine:ribulose-P₂ carboxylase LS (lysine) `N-methyltransferase, a previously undescribed chloroplast enzyme. The partially purified enzyme was specific for ribulose-P₂ carboxylase and exhibited apparent K_m values of 10 micromolar for S-adenosyl-l-methionine and 18 micromolar for ribulose-P₂ carboxylase, a V_max of 700 picomoles CH₃ groups transferred per minute per milligram protein, and a broad pH optimum from 8.5 to 10.0. S-Adenosylmethionine:ribulose-P₂ carboxylase LS (lysine)^εN-methyltransferase was capable of incorporating 24 ³H-methyl groups per spinach ribulose-P₂ carboxylase holoenzyme, forming 1 mole of trimethyllysine per mole of ribulose-P₂ carboxylase LS, but was inactive on ribulose-P₂ carboxylases that contain a trimethyllysyl residue at position 14 in the LS. The enzyme did not distinguish between activated (Mg²⁺ and CO₂) and unactivated forms of ribulose-P₂ carboxylase as substrates. However, complexes of activated ribulose-P₂ carboxylase with the reaction-intermediate analogue 2′-carboxy-d-arabinitol-1,5-bisphosphate, or unactivated spinach ribulose-P₂ carboxylase with ribulose-1,5-bisphosphate, were poor substrates for tobacco LS ^εN-methyltransferase.     

Correlation of redox levels of component electron carriers with total electron flux in an electron-transport system. P-700 and the photoreduction of NADP+ in chloroplast fragments

A mathematical analysis is described which measures the effects of actinic light intensity and concentration of an artificial electron donor on the steady-state light-induced redox level of a reaction-center pigment (e.g. P-700) and on the overall light-induced electron flux (e.g. reduction of NADP⁺). The analysis led to a formulation (somewhat similar to the Michaelis-Menten equation for enzyme kinetics) in which a parameter, $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, is defined as the actinic light intensity that, at a given concentration of electron donor, renders the reaction-center pigment half oxidized and half reduced. To determine the role of a presumed reaction-center pigment, $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ is compared with another parameter, equivalent to $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, that is obtained independently of the reaction-center pigment by measuring the effect of actinic light intensity and concentration of electron donor on the overall electron flow.The theory was tested and validated in a model system with spinach Photosystem I chloroplast fragments by measurements of photooxidation of P-700 and light-induced reduction of NADP⁺ by reduced 2,6-dichlorophenolindophenol. A possible extension of this mathematical analysis to more general electron-transport systems is discussed.     

Relationship of Ribulose-1,5-bisphosphate Carboxylase-Oxygenase Specific Activity to Subunit Composition

Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBPCase, EC 4.1.1.39) was isolated from Nicotiana sylvestris and from two cultivars and three nuclear substitution lines of Nicotiana tabacum. Isoelectric focusing patterns, supported by amino acid analyses and tryptic peptide mapping, were used to divide these enzymes into two categories: (a) RuBPCase with variable large subunits and identical small subunits; and (b) RuBPCase with identical large but different small subunits. Specific activities for both the carboxylation and oxygenation reactions were determined for all six RuBPCase enzymes under standard conditions of activation and assay. High, intermediate, and low levels of carboxylase (880, 530, and 340 nanomoles HCO₃⁻ per milligram per minute) and oxygenase (66, 45, and 35 nanomoles O₂ per milligram per minute) activity were noted. The carboxylase to oxygenase ratios ranged from 9 to 14.