Studies on the Growth in Culture of Plant Cells: XXIII ISOLATION OF NUCLEI AND HISTONES FROM CULTURED CELLS OF ACER PSEUDOPLATANUS L.

A technique is described for the isolation of purified nucleifrom suspension culture cells of Acer pseudoplatanus. This involvesa grinding medium containing 70% (v/v) glycerol, 1 mM Mg²⁺,2 mM Ca²⁺, and Tris buffer at pH 7.8, prestorage and disruptionof the cells at –20 °C in a Potter-Elvehjem homogenizer,and purification by filtration and centrifugation in the presenceof Triton X-100. The nuclear yield is c. 25% as assessed bynuclear count or DNA estimation and the nuclei are active inthe RNA synthesizing system of Tautvydas (1971). When the histones of these nuclei are extracted in H₂SO₄ andprecipitated by ethanol, 113 µg histone is obtained perµg nuclear DNA and the histone fraction contains 22% basicamino acids and has a lysine: arginine ratio of 2.6. Acid-ureagel electrophoresis shows the presence of five major histones(H1, H2A, H2B, H3, and H4 in sequence from anode to cathode)having respectively molecular weights of 24 500, 13 500, 13300, 12 800, and 11 000. There is very good correspondence betweencalf thymus histones H3 (reduced form) and H4 and two of theseAcer histones. The other Acer histones differ from the calfthymus histones H1, H2A, H2B in molecular weight but can beprovisionally equated with these by a newly developed differentialstaining reaction. Calf thymus histone H2A appears to be lessrich in lysine than the corresponding Acer histone. Evidence from a pulse-chase experiment with (¹⁴C)lysine and[³H]tryptophan is in favour of the cytoplasmic synthesis ofthe histones.     

Effect of Temperature on Photosynthesis and Photorespiration of Wheat Leaves

Wheat plants were grown in a controlled environment with daytemperatures of 18 ?C and with 500 µ Einsteins m^–28^–1 of photosynthetically active radiation for 16 h. Beforeanthesis and 2 to 3 weeks after, rates of net photosynthesiswere measured for leaves in 2 or 21% O₂ containing 350 vpm CO₂at 13, 18, 23, and 28 ?C and with 500 µEinsteins m^–2s^–1 of photosynthetically active radiation. Also, underthe same conditions of light intensity and temperature, therates of efflux of CO₂ into CO₂-free air were measured and,for mature flag leaves 3 to 4 weeks after anthesis, gross andnet photosynthesis from air containing 320 vpm ¹⁴CO₂ of specificactivity 39?7 nCi µmol^–1. When the O₂ concentration was decreased from 21 to 2% (v/v)the rate of net photosynthesis increased by 32 per cent at thelowest temperature and 54 per cent at the highest temperature.Efflux of CO₂ into CO₂-free air ranged from 38 per cent of netphotosynthesis at 13 ?C to 86 per cent at 28 ?C. Gross photosynthesis,measured by the ¹⁴C assimilated during 40 s, was greater thannet photosynthesis by some 10 per cent at 13 ?C and 17 per centat 28 ?C. These data indicate that photorespiration was relativelygreater at higher temperatures.     

The occurrence and properties of methenyltetrahydrofolate cyclohydrolase in plants

Methenyltetrahydrofolate cyclohydrolase (E.C. 3.5.4.9 [EC] ), whichis responsible for the enzymatic conversion of 5,10-methenyl-H₄FAto 10-formyl-H₄FA, has been found in various plant tissues.The enzyme was partially purified from pea seedlings and someof its properties were investigated. It was unstable, but wasstabilized by the addition of 25% glycerol. The enzyme was purifiedabout 60-fold by fractionation with ammonium sulfate and columnchromatography on DEAE-cellulose in the presence of 25% glycerol.Optimum pH for the reaction was 7.7. Michaelis constants for5,10-methenyl-H₄FA in the forward reaction, and for 10-formyl-H₄FAin the reverse reaction were 4x10^–5M and 2x10^–4M,respectively. The apparent equilibrium constant for the reactionwas calculated as 50. Enzyme activity was greatly inhibitedby the reduced forms of folate derivatives. The probable participationof this enzyme in the regulation of folate coenzyme levels inplant tissues has been suggested. ¹ Studies on the enzymatic synthesis and metabolism of folatecoenzymes in plants, VI. (For Part V, see Reference (5) ). Partof this paper was presented at the 22nd annual meeting of theJapan Vitamin Society held at Hiroshima on October 14, 1970. ² Present address: Sizuoka Eiwa Junior College, Ikeda, Shizuoka. (Received September 9, 1972; )     

Germination of Sporangia of Phytophthora palmivora (Butl.) Butl.

Sporangia of Phytophthora palmivora germinated by either forminggerm tubes or producing zoospores. Two distinct modes of germ-tubedevelopment have been described. Sporangia in distilled waterformed zoospores at 10–34°C with an optimum at 22°Cbut germinated by means of germ tubes at 30 and 34°C only.Zoospore formation was inhibited to varying degrees by cocoapod extract, I.0 per cent (w/v) peptone and yeast extract, 100–500µg1^-1 thiamine, and by very low concentrations of severalamino acids, carbohydrates, and inorganic salts. Germ-tube formation was encouraged at 22°C by 1'0 per cent(w/v) peptone and yeast extract, by cocoa pod extract and exudate,10mM CaCl2, 1–10 mM MgSO₄. 7H₂O, 0.5 per cent (w/v) fructose,galactose, glucose, lactose, maltose, and sucrose, by 100 ppmarginine, aspartic acid, glutarnic acid, glycine, leucine, andtryptophane, and by 100–500 µg 1^-1 thiamine.     

Concurrent Rates of N2 Fixation, Nitrate and Ammonium Uptake by White Clover in Response to Growth at Different Root Temperatures

Nodulated white clover plants (Trifolium repens L. cv. Huia)were grown for 71 d in flowing nutrient solutions containingN as 10 mmol m_–3 NH₄NO₃, under artificial illumination,with shoots at 20/15°C day/night temperatures and root temperaturereduced decrementally from 20 to 5°C. Root temperatureswere then changed to 3, 7, 9, 11, 13, 17 or 25°C, and theacquisition of N by N₂ fixation, NH₄+ and NO₃^– uptakewas measured over 14 d. Shoot specific growth rates (d. wt)doubled with increasing temperature between 7 and 17°C,whilst root specific growth rates showed little response; shoot:root ratios increased with root temperature, and over time at11°C. Net uptake of total N per plant (N₂ fixation + NH₄++ NO₃^–) over 14 d increased three-fold between 3 and 17°C.The proportion contributed by N₂ fixation decreased with increasingtemperature from 51% at 5°C to 18% at 25°C. Uptake ofNH₄+ as a proportion of NH₄+ + NO₃^– uptake over 14 d variedlittle (55–62%) with root temperature between 3 and 25°C,although it increased with time at most temperatures. Mean ratesof total N uptake per unit shoot f. wt over 14 d changed littlebetween 9 and 25°C, but decreased progressively with temperaturebelow 9°C, due to the decline in the rates of NH₄+ and NO₃^–uptake, even though N₂ fixation increased. The results suggestthat N₂ fixation in the presence of sustained low concentrationsof NH₄+ and NO₄^– is less sensitive to low root temperaturethan are either NH₄+ or NO₃^– uptake systems. White clover, Trifolium repens L. cv. Huia, root temperature, nitrogen fixation, ammonium, nitrate     

Evidence for Essential Maintenance Respiration of Leaves of Xanthium Strumarium at High Temperature

Mesophyll resistance to photosynthetic carboxylation (r'_m) wasused as a criterion for leaf integrity. It was measured, at25 °C, in the light, before and after periods of high temperature(3 h at 38 °C) in the dark. During the high temperatureperiods, respiration (R_D) of attached leaves of Xanthium strumariumwas suppressed from 27%-36% by either low [O₂] (1.04% or 0.21%v.v.) or high [CO₂] (840 µl 1^–1) in the ambientair. Neither treatment affected rates of R_D or photo-respirationduring the second period at 25 °C. There was no significant increase of r'_m when R_D was not suppressedduring the high temperature treatment. When R_D was suppressedat high temperatures, r'_m increased from about 3s cm^–1before, to about 26 s cm^–1 after the high temperaturetreatment. The increase depended upon the degree of suppression. It is concluded that increased R_D at high temperature in Xanthiumleaves is partly the result of an increase of energy demandingmaintenance. The subsequent rate of carbon dioxide fixationis reduced when this increase of maintenance-induced respirationis inhibited.     

Acclimation of NO-3 fluxes to low root temperature by Brassica napus in relation to NO-3 supply

Acclimation of NO^–₃ transport fluxes (influx, efflux)in roots of oilseed rape (Brassica napus L. cv. Bien venu) andtheir sensitivity to growth at low root temperature was studiedin relation to external NO^–₃ supply, defined by constantconcentrations ranging from sub- to supra-optimal with respectto plant growth rate. Plants were grown from seed in flowingnutrient solutions containing 250 mmol m^–3 NO^–₃at 17°C for 20d, and solution temperature in half the cultureunits was then lowered decrementally over 3 d to 7°C. Threedays later plants were supplied with NO^–₃ at 1, 10, 100or 1000 mmol m^–3 maintained for 18 d. Dry matter productionwas decreased more by low root zone temperature than low [NO^–₃]_e. Root specific growth rates were inversely related to [NO^–₃]_eand shoot:root ratios increased with time at [NO^–₃]_e between10–1000 mmol m^–3. Net uptake of NO^–₃ at 17°Cwas twice that at 7°C, and at both temperatures it doubledwith increasing [NO^–₃]_e between 1–10 mmol m^–3with further small increases at higher [NO^–₃]_e. Mean unitabsorption rates of NO^–₃ between 0–6 d and 6–14d were linearly related (r² of 0.79–0.99) to log₁₀[NO].Steady-state Q₁₀ (7–17°C) for uptake between 0–6d were 0.91, 1.62, 1.27, and 1.10, respectively, at [NO^–₃]_eof 1, 10, 100, and 1000 mmol m^–3, compared with correspondingvalues of 0.98, 1.38, 1.68, and 1.89 between 6–14 d. Thedata indicated that net uptake rates at 7 and 17°C divergedover time at high [NO^–₃]_e. Short-term uptake rates from1 mol m^–3 NO^–₃ measured at 17°C were higherin plants grown with roots at 7°C than at 17°C; for7°C plants there was a strong inverse linear relationship(r²=0.94) between uptake rate and treatment log₁₀ [NO^–₃]_ewhilst rates in 17°C plants were independent of prior [NO^–₃]_e. Rates of NO^–₃ influx and efflux under different steady-stateconditions of NO^–₃ supply and root temperature were calculatedfrom dilution of ¹⁵N added to culture solutions. Efflux wassubstantial relative to net uptake in all treatments, and wasinversely related to [NO^–₃]_e at 17°C but not at 7°C.Ratios of influx: efflux ranged from 1.6–2.9 at 17°Cand 1.3–1.8 at 7°C, indicating the proportionatelygreater impact of efflux at low root temperature. Ratios ofefflux: net uptake were 0.53–1.56 at 17°C and 1.21–3.58at 7°C. The apparent sensitivities of influx and effluxto steady-state root temperature varied with [NO^–₃]_e.Both fluxes were higher at 17°C than 7°C in the presenceof 100–1000 mmol m^–3 NO^–₃ but the trend wasreversed at 1–10 mmol m^–3 NO. Concentrations oftotal N measured in xylem exudate were at least 2-fold higherat 7°C compared with 17°C, attributable mainly to higherconcentrations of NO^–₃ glutamine and proline. The resultsare discussed in terms of acclimatory and other responses shownby the NO^–₃ transport system under conditions of limitingNO^–₃ supply and low root temperature. Key words: Brassica napus, nitrate supply, efflux, influx, root temperature, xylem exudate     

Influence of Elevated CO2 and Temperature on the Photosynthesis and Respiration of White Clover Dependent on N2 Fixation

Single clonal plants of white clover (Trifolium repens L) grownfrom explants in a Perlite rooting medium, and dependent fornitrogen on N₂ fixation in root nodules, were grown for severalweeks in controlled environments which provided two regimesof CO₂, and temperature 23/18 °C day/night temperaturesat 680 µmol mol^–1 CO₂, (C680), and 20/15 °Cday/night temperatures at 340 µmol mol^–1 CO₂ (C340)After 3–4 weeks of growth, when the plants were acclimatedto the environmental regimes, leaf and whole-plant photosynthesisand respiration were measured using conventional infra-red gasanalysis techniques Elevated CO₂ and temperature increased ratesof photosynthesis of young, fully expanded leaves at the growthirradiance by 17–29%, despite decreased stomatal conductancesand transpiration rates Water use efficiency (mol CO₂ mol H₂O^–1)was also significantly increased Plants acclimated to elevatedCO₂, and temperature exhibited rates of leaf photosynthesisvery similar to those of C340 leaves ‘instantaneously’exposed to the C680 regime However, leaves developed in theC680 regime photosynthesised less rapidly than C340 leaves whenboth were exposed to a normal CO₂, and temperature environmentIn measurements where irradiance was varied, the enhancementof photosynthesis in elevated CO₂ at 23 °C increased graduallyfrom approx 10 % at 100 µmol m^–1 s^–1 to >27 % at 1170 µmol m^–2 s^–1 In parallel, wateruse efficiency increased by 20–40 % at 315 µmolm^–2 s^–1 In parallel, water use efficiency increasedby 20–40 % at 315 µmol m^–2 s^–1 In parallel,water use efficiency increased by 20–40 % at 315 µmolm^–2 s^–1 In parallel, water use efficiency increasedby 20–40 % at 315 µmol m^–2 s^–1 to approx100 % at the highest irradiance Elevated CO₂, and temperatureincreased whole-plant photosynthesis by > 40 %, when expressedin terms of shoot surface area or shoot weight No effects ofelevated CO₂ and temperature on rate of tissue respiration,either during growth or measurement, were established for singleleaves or for whole plants Dependence on N₂, fixation in rootnodules appeared to have no detrimental effect on photosyntheticperformance in elevated CO₂, and temperature Trifolium repens, white clover, photosynthesis, respiration, elevated CO₂, elevated temperature, water use efficiency, N₂ fixation     

Diffusion of Oxyleghaemoglobin

The diffusion of oxyleghaemoglobin, prepared from soybean rootnodules, was measured at 24°C in agar and agarose gels ofvarious strengths, or in 1% agarose containing 0-18% (w/v) bovineserum albumin, to simulate the protein content of the cytoplasmof root nodule cells. Values of D_p, the diffusion coefficient,were unaffected (D_p = 11·8 x 10^-11 m² s^-1; s.e.m. 0·3x 10^-11) until the protein concentration exceeded 6%, abovewhich D_p declined sharply. With 18% bovine serum albumin, theconcentration of total soluble protein calculated to be presentin the cytoplasm of infected cells, where most of the leghaemoglobinis located in vivo, D_p was 5·9 x 10^-11 m² s^-1. Theseresults are discussed in relation to leghaemoglobin-facilitateddelivery of O₂ to the respiring N₂-fixing bacteroids in rootnodule cells.Copyright 1993, 1999 Academic Press Bacteroids, diffusion, Glycine max, N₂ fixation, oxyleghaemoglobin, soybean, root nodules     

Effects of temperature and CO2 concentration on photosynthetic CO2 fixation by Chlorella

The rates of photosynthetic ¹⁴CO₂ fixation by Chlorella vulgarisllh, grown under high CO₂, were determined between 4 to 37°Cwith air containing from 300 to 13,000 ppm ¹⁴CO₂. When the CO₂level was increased, both the rate of photosynthesis and theoptimum temperature for maximum photosynthesis increased. Themaximum photosynthetic rate was reached at 12°C with 300ppm ^l4CO₂. Among the photosynthetic products fromed at 300 ppm ¹⁴CO₂, glycolatedecreased greatly when the temperature was raised from 20 to30°C. At 3,000 ppm ¹⁴CO₂ an insignificant amount of glycolatewas formed at all temperatures, whereas ¹⁴C-incorporation intothe insoluble fraction, sucrose, and the lipid fraction wassignificantly higher than at 300 ppm ¹⁴CO₂. The ¹⁴C in sucrosewas greatly increased and the radioactivity in the insolublefraction decreased when the temperature was raised from 28 to36°C. (Received April 8, 1980; )     

A Comparative Study of the Influence of Salt Type and Concentration on 14CO2 Fixation in Potato Slices at 25{degrees} C and 0{degrees} C

Dark fixation of ¹⁴CO₂ was followed in potato disks under varyingsalt treatments at 0° C and 25° C. It is shown thatthe specific activity of the ¹⁴CO₂ supplied is heavily dilutedby endogenously produced CO₂ and that the apparently greaterfixation of ¹⁴CO₂, at 0° C as compared with that at 25 °C is due to the lower respiration rate at 0° C, with consequentlyless dilution of the ¹⁴CO₂. supplied. At 25° C organic acidformation in response to different salt treatments fulfils thecommon expectation, ¹⁴CO₂ fixation increasing in the presenceof K₂SO₄ and decreasing in CaCl₂ relative to that in KCl. Therole of organic acids in maintaining ionic balance within thecell at 25° C is thereby indicated but at 0° C organicacid adjustments did not follow the normal pattern. At 25°C but not at o° C increasing external concentration of KCIresulted in an increased level of ¹⁴CO₂ fixation.     

Activation of Ribulose Bisphosphate Carboxylase Purified from Wheat Leaves

A stable freeze-dried powder was prepared of partly purifiedribulose bisphosphate carboxylase from wheat leaves. As withpreparations from other leaves it is necessary to incubate theenzyme with Mg^2$ and CO₂ to achieve maximum activity. At 25°C this activity was 0.75 IU mg^–1 protein for a preparationactivated at 50 °C for 10 min; the K_m for CO₂ was 15 µM. The time for reactivation of enzyme that had been inactivatedthrough the absence of CO₂ and Mg^2$ was influenced by the lengthof the inactivating treatment. After a short inactivation periodthe enzyme was reactivated within a few minutes, whereas aftera longer period several hours were needed. Enzyme in the latterstate had some properties in common with enzyme inactivatedby lower temperatures but in the presence of CO₂ and Mg^2$. Theenzyme kinetic characteristics are similarly affected by bothkinds of inactivation; the maximum velocity is decreased butthe affinity for CO₂ is not affected. Reactivation following a long inactivating treatment becomesmore dependent on Mg^2$ concentration as the temperature is increasedfrom 0 to 20 °C.     

Purification and characterization of human lymphoblast N-acetylglucosamine-1-phosphodiester {alpha}-N-acety1g1ucosaminidase

The enzyme N-acetylglucosamine-1-phosphodiester -N-acetylglucosaminidase(EC 3.1.4.45 [EC] ; uncovering enzyme) catalyzes the removal of N-acetylglucosaminefrom the N-acetylglucosamine--phospho-mannose portion of selectedlysosomal enzyme oligosaccharide chains, thereby formimg themannose 6-phosphate signal which is responsible for the targetingof these lysosomal enzymes for transport into lysosomes. Theuncovering enzyme has been purified approximately 7000-foldto electrophoretic homogeneity from Epstein-Barr virus-transformedhuman lymphoblast cells. The purification sequence involvessolubilizing this membrane-bound enzyme with Tergitol NP-10,affinity chromatography on Lentil lectin-Sepharose 4B, ion-exchangechromatography on DEAE-Sephacel, chromatography on zinc(II)-IDA-Sepharose6B, and preparative SDS-PAGE electrophoresis. The purified enzymemigrated as a single band of 114 kDa which was coincident withenzyme activity on analytical SDS-PAGE electrophoresis. Characterizationstudies of the purified enzyme demonstrated that catalytic activitywas maximal at pH 6.95 and that the enzyme retained full activityfollowing incubation for 10 min at 60°C. No requirementwas found for a divalent cation, but Zn²⁺ Hg²⁺ and Cu²⁺ werefound to reduce the enzyme's activity by 30–40%. The highestcatalytic efficiency was observed with N-acetylglucosamine phospho-methylmannosideas a substrate while uridine diphosphate-N-acetylglucosamine,N-acetylglucosamine phosphomannose-uteroferrin, and N-acetylglucosaminephosphate were also cleaved by the enzyme with decreasing efficiency.Acetamino-deoxycastanospermine was a potent inhibitor of thehuman enzyme with a K₁ of 0.35 µM, while N-acetylglucosaminephosphate (K₁ 1.58 mM) and N-acetylglucosamine (K₁ 5.1 mM) inhibitedthe enzyme to a lesser degree. N-acetylglucosamine-1-phosphodiester -N acetylglucosaminidase lymphoid cells targeting mannose-6 phosphate     

The Potential for Photoinhibition of Pinus sylvestris L. Seedlings Exposed to High Light and Low Soil Temperature

The effect of high light and root chilling on gas exchange,chlorophyll fluorescence, and bulk shoot water potential (_shoot)was examined for Pinus sylvestris seedlings. Transferring plantsfrom low light (200 µmol m^–2s^–1, PAR) anda soil temperature of 15 °C to high light (850 µmolm^–2 s^–1) and 1 °C caused >90% decrease innet photosynthesis and leaf conductance measured at 350 mm³dm^-3 CO₂, and a decrease in the ratio of variable to maximumfluorescence (F_v/F_m) from 0.83 to 0.63. The decrease in Fv/Fmwas, however, only marginally greater than when seedlings weretransferred from low to high light but kept at a soil temperatureof 15 °C. Thus, photoinhibition was a minor component ofthe substantial decrease observed for net photosynthesis at1 °C soil temperature. The decrease in net photosynthesisand _shoot at 1 °C was associated with an increase in calculatedintracellular CO₂ concentration, suggesting that non-stomatalfactors related to water stress were involved in inhibitingcarbon assimilation. Measurements at saturating external CO₂concentration, however, indicate that stomatal closure was thedominant factor limiting net photosynthesis at low soil temperature.This interpretation was confirmed with additional experimentsusing Pinus taeda and Picea engelmannii seedlings. Decreasesin gas-exchange variables at 5 °C soil temperature werenot associated with changes in _shoot Thus, hormonal factors,localized decreases in _needles or changes in xylem flux maymediate the response to moderate root chilling.     

Relationship between Germination and PFR Level in Spores of the Fern Lygodium japonicum

The relationship between germination and P_FR level in sporesof the fern Lygodium japonicum was investigated. Percent P_FRestimated from direct spectrophotometric measurement of sporesincreased with the logarithm of total fluence of 660 nm-light.The transformation from P_R to P_FR was saturated by giving ca.200 Jm^–2 of 660 nm-light and half-saturated by ca. 55J^–2 of 660 nm-light. Clear positive correlation was observedbetween % P_FR levels and germination rates in spores irradiatedwith 660 nm and/or 730 nm-light, or with 686 or 700 nm-light.The P_FR percentage in spores was raised to 16–34% by blue(415 nm) light irradiation. This P_FR level was enough to causesome germination when produced by monochromatic light of redto far-red region, but blue light did not cause any germination. After 660 nm-light irradiation, the P_FR level decreased graduallyin darkness (25±1°C) and P_FR completely disappearedin 8 h, but 730 nm-light given even 16 h after 660 nm-lightirradiation inhibited germination. ⁴Present address: Tropical Botanic Garden and Research Institute,Navaranga Road, Trivandrum 695 011, India. (Received March 15, 1983; Accepted June 4, 1983)     

Form of Inorganic Carbon Utilized for Photosynthesis in Chlamydomonas reinhardtii1

The effect of carbonic anhydrase (CA) on time courses of photosynthetic¹⁴C incorporation in the presence of ¹⁴CO₂ or NaH¹⁴CO₃ was studiedwith cells of Chlamydomonas reinhardtii which had been grownunder ordinary air (low-CO₂ cells) or air enriched with 4% CO₂(high-CO₂ cells). Experimental data obtained at 20°C andpH 8.0 suggested that the major form of inorganic carbon utilizedby high-CO₂ cells was CO₂, while that utilized by low-CO₂ cellswas HCO₃^–. The cell suspension showed CA activity which was comparableto that observed in the sonicate of cells. Both activities werehigher in low-CO₂ cells than in high-CO₂ cells. The mechanism by which HCO₃^– is utilized by low-CO₂ cellsof C. reinhardtii is discussed. ³Present address: Department of Biology, Faculty of Science,University of Niigata, Niigata 950-21, Japan. (Received August 4, 1982; Accepted January 19, 1983)     

Purification and some properties of phosphomannomutase from corms of Amorphophallus konjac C. Koch

Phosphomannomutase [Glazer et al.: Biochim. Biophys. Acta 33:522–625 (1959)] was purified 1700-fold in a 39% yieldfrom cell-free extract of konjak (Amorphophallus konjac C. Koch)corms. The molecular weight of the enzyme as determined by gelfiltration was about 62,000. The enzyme required both Mg²+ and-D-glucose-l,6-bisphosphate for activity, although Mg²+ waspartially replaceable by either Co²+ or Ni²+. An apparent equilibriumconstant, K_eq=(mannose-6-phosphate) (mannose-1-phosphate), wasdetermined to be 8.5. Activity was maximal at pH 6.5 to 7.0.Activation energy was 11.1 kcal/mole. The enzyme was the moststable at pH 7.5. The addition of substrate or cofactor markedlyincreased enzyme stability toward heat denaturation. The enzymewas more labile to heat than phosphoglucomutase from konjakcorms. Treatment with various metal ions in Tris buffer inhibited theenzyme. Cu²+ and Zn²+ were the most potent inhibitors amongthe metal ions tested, while Co²+ and Ni²+ were weak. When theenzyme was treated with metal ions in the presence of histidinebuffer, Cu²+ and Zn²+ showed no inhibitory effect on the enzyme,whereas Be²+ inhibited it to an extent similar to that in Trisbuffer. Plots of 1/v versus l/(mannose-l-phosphate) at different fixedconcentrations of glucose-1,6-bisphosphate and 1/v versus 1/(glucose-1,6-bisphosphate)at different fixed concentrations of mannose-1-phosphate wereseries of converging lines. Mannose-1-phosphate at high concentrationswas found to inhibit the enzyme competitively with respect toglucose-l,6-bisphosphate. Apparent K_m and K₁ values for mannose-1-phosphatewere calculated to be 0.2 mM and 1.2 mM, respectively. The K_mvalue for glucose-1,6-bisphosphate was 1.8 µM. ¹This paper constitutes part 5 of a series of studies on konjakmannan biosynthesis. (Received May 24, 1976; )     

Partial Purification and Characterization of Gibberellin 3{beta}-hydroxylase from Immature Seeds of Phaseolus vulgaris L.

Gibberellin 3/ß-hydroxylase,a 2-oxoglutarate-dependentdioxygenase that catalyzes the hydroxylation of GA₂₀ to GA₁,was purified 313-fold from immature seeds of Phaseolus vulgarisL. The mol wt of the enzyme was estimated to be 42,000 by gelfiltration HPLC and SDS-polyacrylamide gel electrophoresis.The enzyme exhibited maximum activity at pH 7.7. The Km valuesfor [2,3-³H]GA20 and [2,3-³H]GA, were 0.29µu and 0.33µm, respectively. The enzyme requires 2-oxoglutarate asa cosubstrate; the K_m value for 2-oxoglutarate was 250µMusing [³H]- GA₂₀ as a substrate. Fe²⁺ and ascorbate significantlyactivated the enzyme at all purification steps, while catalaseand BSA activated the purified enzyme only. The enzyme was inhibitedby divalent cations Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺ and Hg²⁺.3ß-Hydroxylation of [³H]- GA₂₀ was also inhibitedby non-radioactive GA₅, GA₉,GA₁₅, GA₂₀ and GA₄₄. The possiblesite of 3ß-hydroxylation in gibberellin biosynthesisis discussed in terms of the substrate specificity of partiallypurified gibberellin 3ß-hydroxylase. (Received February 29, 1988; Accepted June 3, 1988)     

RNA Synthesis in Phaseolus Chloroplasts: I. RIBONUCLEIC ACID SYNTHESIS IN CHLOROPLAST PREPARATIONS FROM PHASEOLUS VULGARIS L. LEAVES AND SOLUBILIZATION OF THE RNA POLYMERASE

Chloroplast preparations from the young primary leaves of Phaseolusvulgaris L. cv. Canadian Wonder carry out the DNA-dependentincorporation of UTP into RNA at rates between 8 and 14 pmolUTP µg^–1 chlorophyll h^–1. It is estimatedthat 90% of the activity was localized in the chloroplasts.The incorporation proceeded for between 20 and 30 min at 35°C. The maximum rates of RNA synthesis were attained atpH 8.3, in the presence of 15 mM MgCl₂. Chloroplasts were alsoactive, to a lesser extent, with 1.5 mM MnCl₂. The simultaneouspresence of MnCl₂ and MgCl₂ resulted in inhibition of activity.Nuclear material prepared from young P. vulgaris leaves incorporatedUTP at a rate of about 12 pmol UTP µg^–1 DNA h^–1.On a chloroplast (Tritonsoluble) DNA basis chloroplast activitywas over 40-fold that of nuclei. Methods of solubilizing chloroplastRNA polymerase were explored. Yields of over 75% were achieved,but methods suitable for one species were not always successfulwhen applied to another. The highest yields of the P. vulgarisenzyme were obtained using EDTA and KCl. All methods resultedin solubilization of DNA. RNA synthesis by the soluble P. vulgarisenzyme proceeded for more than 40 min at 35 °C.     

Purification, Properties and Phosphorylation of Anaerobically Induced Enolase in Echinochloa phyllopogon and E. crus-pavonis

Enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11 [EC] ) activityis differentially induced by anoxia in the flood-tolerant speciesE. phyllopogon (Stev.) Koss and the flood-intolerant speciesE. crus-pavonis (H.B.K.) Schult. To examine the regulation ofenolase at the protein level, we purified the enzyme from bothspecies to near homogeneity and compared their physico-chemicaland catalytic properties. Enolase purified from E. phyllopogonexhibits optimal activity at pH 7.0, a K_m of 80 µM for2-PGA, a Q₁₀ of 1.97 and an E_a of 12.3 kcal mol^-1. Similarly,enolase from E. crus-pavonis exhibits optimal activity at pH7.0, a K_m of 50 µM for 2-PGA, a Q₁₀ of 2.04 and an E_aof 12.9 kcal mol^-1. The enzyme from both species is thermostable(100% active after 15 min, 50°C) and is a homodimer of 52.5kDa subunits as resolved by SDS-PAGE and immunoblotting. E.phyllopogon enolase was phosphorylated in vitro using either[