Mechanism of Electron Flow through the QB Site in Photosystem II. 4. Reaction Mechanism of Plastoquinone Derivatives at the QB Site in Spinach Photosystem II Membrane Fragments

Interactions of externally added plastoquinone (PQ) derivatives(PQ₀-PQ₃) with the photosystem II (PSII) acceptor side wereinvestigated in PSII membrane fragments prepared from spinachby measuring the photoreduction rates of PQ derivatives at variousPQ concentrations, and the following results were obtained. From the kinetic analysis, all the PQ derivatives (PQ₀-PQ₃)except PQ₃ were shown to accept electrons at two sites (theQ_B site and the PQ site) as in the case of Synechococcus vulcanusPSII particles with benzoquinone derivatives [Satoh et al. (1995)Plant Cell Physiol. 36: 597]. Affinities of PQ derivatives at the Q_B site increased as thelength of the isoprene side chain got longer, while those atthe PQ site were not very much different for all the PQ derivativestested in this study. The inhibitory effect of DCMU was noncompetitive, and, therefore,the affinity of PQ₃ for the PQ site was determined while thatfor the Q_B site could not be estimated presumably due to itsfairly high affinity to the site. Based on the results obtained using PQ derivatives, the mechanismof interaction of an authentic PQ, PQ₉, at the Q_B site is discussed. (Received May 2, 1996; Accepted July 24, 1996)     

Mechanism of Electron Flow through the QB Site in Photosystem II. 1. Kinetics of the Reduction of Electron Acceptors at the QB and Plastoquinone Sites in Photosystem II Particles from the Cyanobacterium Synechococcus vulcanus

Various derivatives of benzoquinone (BQ) were found to be reducedat two sites [the Q_B and plastoquinone (PQ) sites] in photosystemII particles from Synechococcus vulcanus, and the relationshipbetween the structures of BQs and the kinetics of such reductionat each site were studied. Affinities of BQs for both the Q_B and the PQ site and the maximumturnover rates at the two sites were estimated by computer simulationof the dependence on the concentration of the BQ of the rateof oxygen evolution. Affinities of BQs for the Q_B sites determinedby this method agreed well with those determined from competitionbetween BQs and DCMU for the Q_B site. All methyl-substituted BQs had low affinities for the Q_B site,and tetramethyl-p-benzoquinone had almost no affinity. An increasein the number of chlorine atoms in the quinone ring increasedthe affinity, and the position of such substitutions had a greateffect on the affinity when two positions on the ring were occupiedby two chlorine atoms or methyl groups. The affinities of BQs for the PQ site were almost the same forall BQs tested in this experiment but the maximum turnover ratesat this site varied greatly from one derivative to another. The results are consistent with the hypothesis that the bindingof the PQ molecule to the Q_B site is attributable not to itsquinone ring but to its isoprenoid chain and, moreover, thatthe electron transfer through the Q_B site occurs not by replacementof the PQ molecule but by donation of electrons to another PQmolecule. (Received September 20, 1994; Accepted February 27, 1995)     

Binding Affinities of Oxidized and Reduced Forms of Tetrahalogenated Benzoquinones to the QB Site in Oxygen-Evolving Photosystem II Particles from Synechococcus elongatus

Binding affinities of the Q_B site for four tetrahalogenatedbenzoquinones (THBQs) were investigated by measuring their abilityto serve as electron acceptors or act as inhibitors of oxygenevolution in Synechococcus photosystem II particles. Iodanil,bromanil and chloranil but not fluoranil induced a rapid oxidationof Q_A^– and doubled the area over the fluorescence inductioncurve, indicating dark oxidation of Q₄₀₀. Analyses of thesetwo THBQ-induced reactions and inhibition of the acceleratedQ_A^– oxidation by DCMU yielded binding constants of thequinones comparable to those determined from measurements ofoxygen evolution. Generally, THBQs bound tightly to the Q_B site.However, the binding affinity varied in a wide range with THBQs.The Q_B site bound iodanil with an extremely high affinity butfluoranil relatively weakly. The hydroquinone forms of the THBQsalso bound to the Q_B site and inhibited Q_A^– oxidationby Q_B. The concentrations of the hydroquinones required for50% inhibition of Q_A^– oxidation suggest that the Q_B sitebinds the hydroquinones more weakly than the corresponding quinonesexcept for fluoranil, which binds to the Q_B site more tightlyin its reduced form than in oxidized one. The abilities of THBQsto function as electron acceptors or inhibitors of oxygen evolution,and as oxidants of Q₄₀₀ in the dark, are discussed in relationto the binding affinities of the quinones to the Q_B site. ⁴Present address: Department of Biology, Faculty of Science,Toho University. Miyama 2-2-1, Funabashi, 274 Japan     

Kinetic Properties of Four Enzymes Involved in Proline Metabolism in Cold-acclimated Wheat

Two varieties of wheat (Triticum aestivum L.) a winter (Kharkov)and a spring (Glenlea), were acclimated under controlled conditionsat 5 °C and 25 °C (12 h photoperiod). Kinetic properties(K_m1 V_max/Km ratio and Q₁₀ as a function of reduction of substrateconcentration) were investigated for enzymatic systems involvedin two pathways of proline metabolism: the glutamic acid andthe ornithine pathways. Four enzymes were studied, namely prolinedehydrogenase (PDH, EC 1.5.1.2 [EC] ), glutamate dehydrogenase (GDH,EC 1.4.1.2 [EC] -4), glutamine synthetase (GS, EC 6.3.1.2 [EC] ) and ornithinetransaminase (OT, EC 2.6.1.13 [EC] ). Kinetic properties of thesefour enzymes proved to be modulated by cold acclimation, especiallyin Kharkov, the winter cultivar, which accumulates proline.Firstly, the synthesis of precursors of proline may be augmentedand the degradation of proline lessened by either decreasingthe K_m values of OT or increasing the K_m values of PDH. Secondly,the catalytic efficiency (V_max ratio) of GDH, GS, and OT isincreased. Thirdly, the lower values of Q₁₀ indicate a highcapacity of reaction of GS and OT.     

Activation of 20S Proteasomes from Spinach Leaves by Fatty Acids

In order to clarify the mechanism of activation of plant 20Sproteasomes by fatty acids, we examined the effects of oleic,linoleic and linolenic acids on the three peptidase activitiesof purified 20S proteasomes from spinach leaves and comparedthem with the effects of SDS, a previously characterized activatorof 20S proteasomes. The three fatty acids all activated thehydrolysis of succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide(Suc-LLVYMCA) and benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide(Cbz-LLE-2NA) at low concentrations (one-third to one-sixthof that required for activation by SDS). The range of concentrationsof linolenic acid for the activation of Suc-LLVY-MCA hydrolysiswas very narrow. All the fatty acids inhibited the hydrolysisof tert-butoxycarbonyl-Leu-Arg-Arg-4-methylcoumaryl-7-amide(Boc-LRR-MCA)at extremely low concentrations (one-fifth to one-fifteenthof that required for the activation of the hydrolysis of Suc-LLVY-MCAand Cbz-LLE-2NA). In the case of hydrolysis of Suc-LLVY-MCA,SDS and the three fatty acids increased the V_max value and decreasedthe apparent K_m value to similar relative extents. In the caseof hydrolysis of Boc-LLE-MCA, SDS and the three fatty acidsalso decreased the K_m and increased the V_max. However, SDS markedlyincreased V_max. The curves representing the SDS-dependent activationwere shifted to a lower range by the addition of linoleic acid,but the maximum activity at the optimum concentration of SDSwas essentially unchanged. These results suggest that the activationby SDS and that by the fatty acids has an additive effect. Theresults imply that fatty acids, such as linolenic acid, mightact as physiological regulators in plant cells. (Received April 10, 1995; Accepted December 22, 1995)     

Suppression of the High Affinity Phosphate Uptake System: A Mechanism of Arsenate Tolerance in Holcus lanatus L.

In Holcus lanatus L. phosphate and arsenate are taken up bythe same transport system. Short-term uptake kinetics of thehigh affinity arsenate transport system were determined in excisedroots of arsenate-tolerant and non-tolerant genotypes. In tolerantplants the V_max of ion uptake in plants grown in phosphate-freemedia was decreased compared to non-tolerant plants, and theaffinity of the uptake system was lower than in the non-tolerantplants. Both the reduction in V_max and the increase in K_m ledto reduced arsenate influx into tolerant roots. When the twogenotypes were grown in nutrient solution containing high levelsof phosphate, there was little change in the uptake kineticsin tolerant plants. In non-tolerant plants, however, there wasa marked decrease in the V_max to the level of the tolerant plantsbut with little change in the K_m. This suggests that the lowrate of arsenate uptake over a wide range of differing rootphosphate status is due to loss of induction of the synthesisof the arsenate (phosphate) carrier. Key words: Arsenate, Holcus lanatus L., phosphate uptake, tolerance mechanisms, uptake mechanisms     

Variations in the Contents and Kinetic Properties of Ribulose-1,5-bisphosphate Carboxylases among Rice Species

The K_m(CO₂) ancl V_max of ribulose 1,5-bisphosphate (RuBP) carboxylaseand its protein ratio to total soluble protein from Oryza speciesincluding cultivars (25 varieties) and wild types (11 species,21 strains) were surveyed. Their variabilities among cultivarsof O. sativa were very small. The averages of the K_m(CO₂) andV_max values and the ratio of carboxylase to soluble protein,and their standard errors were 10.2?1.0µM, 1.72?0.13units.mg^–1(pH 8.0 and 25?C) and 52?2%, respectively. However, some differencesseemed to exist based on genome constitution in the Oryza genus.RuBP carboxylases from the species with the A^gA^g genome, O.graberrima and O. breviligulate, exhibited low K_m(CO₂) values(8.0?0.8 µM). High V_max was associated with the CC genome,O. eichingeri and O. officinalis (2.08?0.15 units.mg^–1).A higher ratio of RuBP carboxylase protein to soluble proteinwas found for the AA genome, O. sativa and O. perennis. (Received September 24, 1986; Accepted April 15, 1987)     

Control of the maximal chlorophyll fluorescence yield by the Q<Subscript>B</Subscript> binding site

Differences in maximal yields of chlorophyll variable fluorescence (F_m) induced by single turnover (ST) and multiple turnover (MT) excitation are as great as 40%. Using mutants of Chlamydomonas reinhardtii we investigated potential mechanisms controlling F_m above and beyond the Q_A redox level. F_m was low when the Q_B binding site was occupied by PQ and high when the Q_B binding site was empty or occupied by a PSII herbicide. Furthermore, in mutants with impaired rates of plastoquinol reoxidation, F_m was reached rapidly during MT excitation. In PSII particles with no mobile PQ pool, F_m was virtually identical to that obtained in the presence of PSII herbicides. We have developed a model to account for the variations in maximal fluorescence yields based on the occupancy of the Q_B binding site. The model predicts that the variations in maximal fluorescence yields are caused by the capacity of secondary electron acceptors to reoxidize Q_A^–.     

Alteration in the acceptor side of photosystem II of chloroplast by high light

The effect of high light on the acceptor side of photosystem II of chloroplasts and core particles of spinach was studied. BothV _max and apparentK _m for DCIP were altered in photoinhibited photosystem II core particles. The double reciprocal plot analysis as a function of actinic light showed increased slope in chloroplasts photoinhibited in the presence of DCMU. Exposure of chloroplasts to high light in the presence of DCMU did not protect the chloroplast against high light induced decrease in F_m, level. Further the high light stress induced decrease inF _m level was not restored by the addition of DCMU. These results suggest that the high light stress induced damage to chloroplast involves alteration in the binding site forQ _B on the DI protein on the acceptor side of photosystem II     

Spare quinones in the QB cavity of crystallized photosystem II from Thermosynechococcus elongatus

The recent crystallographic structure at 3.0 Å resolution of PSII from Thermosynechococcus elongatus has revealed a cavity in the protein which connects the membrane phase to the binding pocket of the secondary plastoquinone Q_B. The cavity may serve as a quinone diffusion pathway. By fluorescence methods, electron transfer at the donor and acceptor sides was investigated in the same membrane-free PSII core particle preparation from T. elongatus prior to and after crystallization; PSII membrane fragments from spinach were studied as a reference. The data suggest selective enrichment of those PSII centers in the crystal that are intact with respect to O₂ evolution at the manganese-calcium complex of water oxidation and with respect to the integrity of the quinone binding site. One and more functional quinone molecules (per PSII monomer) besides of Q_A and Q_B were found in the crystallized PSII. We propose that the extra quinones are located in the Q_B cavity and serve as a PSII intrinsic pool of electron acceptors.     

Functional Structure of the Oxygen-Evolving Unit of Photosystem II as Determined by Radiation Inactivation

The size of the complex that is essential for the electron-transferactivity from the oxygen-evolving center to the secondary electronacceptor, Q_B, is about 250 kDa, as determined by target-sizeanalysis after the radiation inactivation of functions of photosystemII (PS II). Inter-Chl tranfer of excitation energy was insensitiveto the radiation inactivation indicating that the masses ofCP47, CP43, and light-harvesting Chi a/b proteins are not includedin the functional size of the oxygen-evolving PS II complex.The transfer of electrons from the secondary electron donor,Z, to Q_B was catalyzed by a unit of only 65 kDa. The sizes ofthe complexes involved in these light-induced functions of PSII were dependent on the intensity of actinic light. Under saturatingintensities of light, the functional size of the complex fortransfer of electrons from Z to Q_B was 38 kDa, with a correspondingdecrease in the size of the oxygen-evolving PS II from 250 kDato 125 kDa [Takahashi, Mano and Asada (1985) Plant Cell Physiol.26: 383]. The protein of about 30 kDa functions in the photoreductionof the pheophytin molecule, as well as in the electron transferfrom Z to Q_A. Under low-intensity light, complexes having thesame sizes as those of the basal functional complexes undersaturating-intensity light are further required, probably tostabilize separated charges in the PS II reaction center andthe oxygen-evolving center. (Received June 20, 1990; Accepted September 18, 1990)     

Presence in Photosystem II Core Complexes of a 34-Kilodalton Polypeptide Required for Water Photolysis

Photosystem II (PSII) reaction center core complexes have been isolated and characterized from wild type (WT) Scenedesmus obliquus and from its LF-1 mutant. LF-1 thylakoids are blocked on the oxidizing side of PSII and have a reduced Mn content. Visible absorption and low temperature fluorescence spectra of both core complexes are identical and resemble those reported for spinach (Satoh, Butler 1978 Plant Physiol 61: 373-379). Lithium dodecyl sulfate-polycrylamide gel electrophoresis reveals that a protein alteration, originally observed in thylakoid membranes (Metz, Wong, Bishop 1980 FEBS Lett 114: 61-66), is retained in the PSII core particles. That is, a 34-kilodalton (kD) polypeptide, present in the WT core complex, is missing in the mutant, and the core complex of the mutant contains a 36-kD protein not present in the WT. The 34-kD intrinsic protein is also observed in O₂-evolving PSII preparations and PSII core complexes from spinach. It is distinct from the 33-kD extrinsic protein first reported by T. Kuwabara and N. Murata (1979 Biochim Biophys Acta 581: 228-236). We suggest that the 34-kD protein is a site of Mn binding in the PSII membrane.     

Biochemical Properties of the Plasmalemma ATPase of Jerusalem Artichoke (Helianthus tuberosus L.) Tubers in Relation to Dormancy

Enzymological properties of the ATPase of plasmalemma-enrichedfractions obtained from the parenchyma of Jerusalem artichoke(Helianthus tuberosus L.) tubers were studied in relation tothe morphogenetic properties of the tubers. Preparations fromdormant tubers showed higher V_max and K_m values than those fromnon-dormant tubers. These differences persisted after solubilizationof the enzyme with octylglucoside, but were slightly modifiedafter addition of K$ to the reaction medium. The implicationsof these results are discussed in relation to the morphogeneticproperties of the tubers (Received August 24, 1987; Accepted March 19, 1988)     

Efficiency of K+ Utilization by Barley Varieties: Activation of Pyruvate Kinase

Barley (Hordeum vulgare L.) varieties differed in their raponseto [K⁺]₀, in terms of their utilization efficiencies (UE = freshweight. concentration of [K⁺]₁^–1). At low [K⁺]₀, Compana,an efficient-non-responder demonstrated superior utilizationof absorbed K⁺. On the other hand, at high [K⁺]₀, Fergus (anefficient responder) and BT 334 (an inefficient responder) hadhigher UE values for K⁺ than Compana which performed poorlyat this [K⁺]₀. Kinetic parameters for K⁺ activation of the enzyme pyruvatekinase from 12 barley varieties, representing a range of UEvalues, were determined. Varieties showed substantial differencesin their V_max values (P<0·01). Compana, an efficientvariety, had the highest V_max (31 µmol g^–1 freshwt. h^–1) which was about 50% higher than that of Mingo,an inefficient variety. By contrast, K_m values for the enzymeswere not significantly different among varieties The mean valuesfor all varieties (3·9±0·15 mol m^–3K⁺) is far below the estimated cytoplasmic [K⁺] (100-200 molm^–3). It is, therefore, unlikely that differences in theutilization of K⁺ by these varieties can be explained on thebasis of differential requirements for (K⁺) activation of theseenzymes. Alternative possibilities for differences in the utilizationof K⁺ are discussed. Key words: K⁺ utilization efficiency, Pyruvate kinase, Barley varieties     

Characteristics of the Cl- Action Site in the O2 Evolving Reaction in PS II Particles: Electrostatic Interaction with Ions

The role of Cl^– in the reactivation of O₂ evolution inphotosystem II (PS II) particles derived from spinach chloroplastswas studied in the presence of various salts. Multivalent ion(especially anion) salts were found to strongly suppress thereactivation of O₂ evolution by Cl^– in the Cl^–-depletedPS II particles in a competitive manner. The effectiveness ofanions in the suppression of Cl^–-supported O₂ evolutionwas in the order of trivalent>divalent>monovalent ones.Multivalent anions similarly suppressed O₂ evolution in theuntreated PS II particles under low and moderate Cl^– concentrations.pH dependence of the Cl^–-affinity (K_m) value for Cl^–)was also studied. Within the pH range 5.5 to 8 the K_m valuebecame higher as the pH of the medium increased. These resultssuggest that the membrane surface in the vicinity of the Cl^–action site is net positively charged and attracts Cl^–electrostatically, and that the site is almost freely accessibleto various anions. The origin and role of the local net positivedomain and the role of peripheral proteins are discussed. (Received May 27, 1985; Accepted October 8, 1985)     

A carrier-mediated mechanism for pyridoxine uptake by human intestinal epithelial Caco-2 cells: regulation by a PKA-mediated pathway

Vitamin B₆ is essential for cellular functions and growth due to its involvement in important metabolic reactions. Humans and other mammals cannot synthesize vitamin B₆ and thus must obtain this micronutrient from exogenous sources via intestinal absorption. The intestine, therefore, plays a central role in maintaining and regulating normal vitamin B₆ homeostasis. Due to the water-soluble nature of vitamin B₆ and the demonstration that transport of other water-soluble vitamins in intestinal epithelial cells involves specialized carrier-mediated mechanisms, we hypothesized that transport of vitamin B₆ in these cells is also carrier mediated in nature. To test this hypothesis, we examined pyridoxine transport in a model system for human enterocytes, the human-derived intestinal epithelial Caco-2 cells. The results showed pyridoxine uptake to be 1) linear with time for up to 10 min of incubation and to occur with minimal metabolic alteration in the transported substrate, 2) temperature and energy dependent but Na⁺ independent, 3) pH dependent with higher uptake at acidic compared with alkaline pHs, 4) saturable as a function of concentration (at buffer pH 5.5 but not 7.4) with an apparent Michaelis-Menten constant (K_m) of 11.99 ± 1.41 µM and a maximal velocity (V_max) of 67.63 ± 3.87 pmol · mg protein^-1 · 3 min^-1, 5) inhibited by pyridoxine structural analogs (at buffer pH 5.5 but not 7.4) but not by unrelated compounds, and 6) inhibited in a competitive manner by amiloride with an apparent inhibitor constant (K_i) of 0.39 mM. We also examined the possible regulation of pyridoxine uptake by specific intracellular regulatory pathways. The results showed that whereas modulators of PKC, Ca⁺²/calmodulin (CaM), and nitric oxide (NO)-mediated pathways had no effect on pyridoxine uptake, modulators of PKA-mediated pathway were found to cause significant reduction in pyridoxine uptake. This reduction was mediated via a significant inhibition in the V_max, but not the apparent K_m, of the pyridoxine uptake process. These results demonstrate, for the first time, the involvement of a specialized carrier-mediated mechanism for pyridoxine uptake by intestinal epithelial cells. This system is pH dependent and amiloride sensitive and appears to be under the regulation of an intracellular PKA-mediated pathway. vitamin B₆; intestinal transport; transport regulation; Caco-2 cell     

Stigmatellin Probes the Electrostatic Potential in the QB Site of the Photosynthetic Reaction Center

The electrostatic potential in the secondary quinone (Q_B) binding site of the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides determines the rate and free energy change (driving force) of electron transfer to Q_B. It is controlled by the ionization states of residues in a strongly interacting cluster around the Q_B site. Reduction of the Q_B induces change of the ionization states of residues and binding of protons from the bulk. Stigmatellin, an inhibitor of the mitochondrial and photosynthetic respiratory chain, has been proven to be a unique voltage probe of the Q_B binding pocket. It binds to the Q_B site with high affinity, and the pK value of its phenolic group monitors the local electrostatic potential with high sensitivity. Investigations with different types of detergent as a model system of isolated RC revealed that the pK of stigmatellin was controlled overwhelmingly by electrostatic and slightly by hydrophobic interactions. Measurements showed a high pK value (>11) of stigmatellin in the Q_B pocket of the dark-state wild-type RC, indicating substantial negative potential. When the local electrostatics of the Q_B site was modulated by a single mutation, L213Asp→Ala, or double mutations, L213Asp-L212Glu→Ala-Ala (AA), the pK of stigmatellin dropped to 7.5 and 7.4, respectively, which corresponds to a >210 mV increase in the electrostatic potential relative to the wild-type RC. This significant pK drop (ΔpK > 3.5) decreased dramatically to (ΔpK > 0.75) in the RC of the compensatory mutant (AA+M44Asn→AA+M44Asp). Our results indicate that the L213Asp is the most important actor in the control of the electrostatic potential in the Q_B site of the dark-state wild-type RC, in good accordance with conclusions of former studies using theoretical calculations or light-induced charge recombination assay.     

Nutrient-limited growth rates: quantitative benefits of stress responses and some aspects of regulation

Young sunflower plants (Helianthus annuus L.) under stress oflow nitrate or phosphate availability exhibited increases inroot: shoot ratio and in kinetic parameters for uptake. Theyshowed no significant changes in photosynthetic utilizationof either nutrient. Increases in root: shoot ratio were achievedby early and persistent suppression of shoot growth, but notroot growth. Affinity for phosphate uptake, 1/K_m(P), increasedwith phosphate stress, as did affinity for nitrate uptake, 1/K_m(N),with nitrate stress. Maximal uptake rate, V_max, for phosphateuptake increased with phosphorus stress; V_max for nitrate didnot increase with nitrogen stress. Phosphate V_max was relatedstrongly to root nutrient status. Decreases in V_max with plantage were not well explained by changes in age structure of roots.Estimated benefits of acclimatory changes in root: shoot ratioand uptake kinetics ranged up to 2-fold increases in relativegrowth rate, RGR. The relation of RGR to uptake physiology followedpredictions of functional balance moderately well, with somesystematic deviations. Analyses of RGR using growth models implyno significant growth benefit from regulating Vmax, specifically,not from down-regulating it at high nutrient availability. Quantitativebenefits of increases in root: shoot ratio and uptake parametersare predicted to be quite small under common conditions whereinnutrient concentrations are significantly depleted by uptake.The root: shoot response is estimated to confer the smallestbenefit under non-depleting conditions and the largest benefitunder depleting conditions. Even then, the absolute benefitis predicted to be small, possibly excepting the case of heterogeneoussoils. Depleting and non-depleting conditions are addressedwith very different experimental techniques. We note that atheoretical framework is lacking that spans both these cases,other than purely numerical formulations that are not readilyinterpreted. Key words: Nutrient stress, nutrient uptake, nutrient use efficiency, relative growth rate, Helianthus annuus     

Affinity of RuBP Carboxylases for Carbon Dioxide and Inhibition of the Enzymes by Oxygen

Ribulose bisphosphate carboxylase (E.C.4.1.1.39) was purifiedfrom leaves of Triticum aestivum, Hordeum vulgare, Spinaceaoleracea, Petroselinum crispum, salad mustard-most likely Brassicanapus, Helianthus annuus, Solanum tuberosum, Beta vulgaris,Lolium perenne, Equisetum arvense, Zea mays, Ginkgo biloba,Pteris aquilina, Salix babylonica, Chamaecyparis lawsonianaand Atrichum undulatum by density gradient centrifugation andgel filtration or by ammonium sulphate fractionation, densitygradient centrifugation, ion-exchange chromatography and gelfiltration. Purified enzymes were freeze-dried and then storedat 0 °C to 4 °C. Portions of each enzyme preparationwere reactivated at 25 °C for 5 h in the presence of 10mM HCO₂ and 20 mM MgCl₂-RuBP carboxylase activities were measuredat four different concentrations of CO₂ at 25 °C and pH8.2 in solutions equilibrated with pure nitrogen or air (21%O₂, 79% N₂). K_m(CO₂), V_max and K₁(O₂) values were computed fromthe results. Significant differences were found in the K_m(CO₂)values for enzymes isolated from different species. Sensitivityof the enzymes to oxygen was less variable.     

苏云金杆菌预处理小菜蛾对有机磷和氨基甲酸酯杀虫剂的增效作用

研究了小菜蛾Plutella xylostella幼虫经苏云金杆菌Bacillus thuringiensis预处理后,对有机磷和氨基甲酸酯杀虫剂敏感性的变化以及预处理对小菜蛾幼虫体内乙酰胆碱酯酶、羧酸酯酶、谷胱甘肽S-转移酶和谷胱甘肽的含量的影响。结果表明：苏云金杆菌预处理抗性小菜蛾幼虫后,其对甲胺磷、水胺硫磷和克百威的敏感性分别为未处理组的6.74、8.83和8.50倍;处理敏感小菜蛾幼虫后则分别为未处理组的2.96、1.69和3.88倍。苏云金杆菌预处理抗性小菜蛾,未处理组乙酰胆碱酯酶的K_m和V_max值分别为预处理组的1.86和1.56倍,所使用的6种杀虫剂对乙酰胆碱酯酶的K_I值,处理组为未处理组的1.80～2.66倍,苏云金杆菌预处理抗性小菜蛾对羧酸酯酶的K_m、K_I影响不大,但能显著地抑制羧酸酯酶和谷胱甘肽S-转移酶的活性并导致谷胱甘肽含量下降(对照分别为处理的2.02、1.76和1.66倍)。苏云金杆菌预处理敏感小菜蛾,对乙酰胆碱酯酶的K_m、V_max、K_I值和羧酸酯酶的K_m、K_I值以及谷胱甘肽含量影响不大,但能显著地抑制羧酸酯酶和谷胱甘肽S-转移酶的活性(对照分别为处理的1.54和1.64倍)。