Characterization and flocculating properties of a novel bioflocculant produced by <Emphasis Type="Italic">Bacillus circulans</Emphasis>

We studied a novel bioflocculant, PX, that is produced from Bacillus Bacillus circulans X3, and has excellent flocculating activity with regard to its characterization and flocculating properties. The bioflocculant was purified from supernatant by ethanol precipitation, dialysis and gel permeation chromatography (GPC). The major component of PX was an acid polysaccharide including uronic (19.8%), pyruvic (6.5%) and acetic acids (0.7%). It consisted of galactose, mannose, xylitol, rhamnose and galacturonic acid in an approximate molar ration of 5:4.1:3:2:1.2. The molecular weight of PX was about 4.85 × 10⁴ Da as determined by GPC. The infrared spectrum of the bioflocculant indicated the presence of carboxyl, hydroxyl, amino and methoxyl groups. Studies of the flocculating properties revealed that it was stable at 60–100°C and pH 4–10. Moreover, it could flocculate a kaolin suspension over a wide range of pH and temperature in the presence of CaCl₂.     

Immobilization of Xylan-Degrading Enzymes from Scytalidium thermophilum on Eudragit L-100

Summary Xylanase from Scytalidium thermophilum was immobilized on Eudragit L-100, a pH sensitive copolymer of methacrylic acid and methyl methacrylate. The enzyme was non-covalently immobilized and the system expressed 70% xylanase activity. The immobilized preparation had broader optimum temperature of activity between 55 and 65 °C as compared to 65 °C in case of free enzyme and broader optimum pH between 6.0 and 7.0 as compared to 6.5 in case of free enzyme. Immobilization increased the t_1/2 of enzyme at 60 °C from 15 to 30 min with a stabilization factor of 2. The K_m and V_max values for the immobilized and free xylanase were 0.5% xylan and 0.89 μmol/ml/min and 0.35% xylan and 1.01 μmol/ml/min respectively. An Arrhenius plot showed an increased value of activation energy for immobilized xylanase (227 kcal/mol) as compared to free xylanase (210 kcal/mol) confirming the higher temperature stability of the free enzyme. Enzymatic saccharification of xylan was also improved by xylanase immobilization.     

Purification of cytochrome a 1 c 1 from Nitrobacter agilis and characterization of nitrite oxidation system of the bacterium

Cytochrome a ₁ c ₁ was highly purified from Nitrobacter agilis. The cytochrome contained heme a and heme c of equimolar amount, and its reduced form showed absorption peaks at 587, 550, 521, 434 and 416 nm. Molecular weight per heme a of the cytochrome was estimated to be approx. 100,000–130,000 from the amino acid composition. A similar value was obtained by determining the protein content per heme a. The cytochrome molecule was composed of three subunits with molecular weights of 55,000, 29,000 and 19,000, respectively. The 29 kd subunit had heme c.Hemes a and c of cytochrome a ₁ c ₁ were reduced on addition of nitrite, and the reduced cytochrome was hardly autoxidizable. Exogenously added horse heart cytochrome c was reduced by nitrite in the presence of cytochrome a ₁ c ₁; K _m values of cytochrome a ₁ c ₁ for nitrite and N. agilis cytochrome c were 0.5 mM and and 6 M, respectively. V _max was 1.7 mol ferricytochrome c reduced/min·mol of cytochrome a ₁ c ₁ The pH optimum of the reaction was about 8. The nitrite-cytochrome c reduction catalyzed by cytochrome a ₁ c ₁ was 61% and 88% inhibited by 44M azide and cyanide, respectively. In the presence of 4.4 mM nitrate, the reaction was 89% inhibited. The nitrite-cytochrome c reduction catalysed by cytochrome a ₁ c ₁ was 2.5-fold stimulated by 4.5 mM manganous chloride. An activating factor which was present in the crude enzyme preparation stimulated the reaction by 2.8-fold, and presence of both the factor and manganous ion activated the reaction by 7-fold.Cytochrome a ₁ c ₁ showed also cytochrome c-nitrate reductase activity. The pH optimum of the reaction was about 6. The nitrate reductase activity was also stimulated by manganous ions and the activating factor.     

Cytochrome aa 3 from Methylococcus capsulatus (Bath)

A cytochrome aa ₃-type oxidase was isolated with and without a c-type cytochrome (cytochrome c-557) from Methylococcus capsulatus Bath by ion-exchange and hydrophobic chromatography in the presence of Triton X-100. Although cytochrome c-557 was not a constitutive component of the terminal oxidase, the cytochrome c ascorbate-TMPD oxidase activity of the enzyme decreased dramatically when the ratio of cytochrome c-557 to heme a dropped below 1:3. On denaturing gels, the purified enzyme dissociated into three subunits with molecular weights of 46,000, 28,000 and 20,000. The enzyme contains two heme groups (a and a ₃), absorption maximum at 422 nm in the resting state, at 445 and 601 nm in the dithionite reduced form and at 434 and 598 nm in the dithionite reduced plus CO form. Denaturing gels of the cytochrome aa ₃-cytochrome c-557 complex showed the polypeptides associated with cytochrome aa ₃ plus a heme c-staining subunit with a molecular weight of 37,000. The complex contains approximately two heme a, one heme c, absorption maximum at 420 nm in the resting state and at 421, 445, 522, 557 and 601 nm in the dithionite reduced form. The specific activity of the purified enzyme was 130 mol O₂/min · mol heme a compared to 753 mol O₂/min · mol heme a when isolated with cytochrome c-557.Abbreviations MMO methan monooxygenase - sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - TMPD N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride - Na₂EDTA disodium ethylenediamine-tetraacetic acid     

The optimal growth conditions for the biomass production of Isochrysis galbana and the effects that phosphorus, Zn2+, CO2, and light intensity have on the biochemical composition of Isochrysis galbana and the activity of extracellular CA

The effects of phosphorus, Zn²⁺, CO₂, and light intensity on growth, biochemical composition, and the activity of extracellular carbonic anhydrase (CA) in Isochrysis galbana were investigated. A significant change was observed when the concentration of phosphorus in the medium was increased from 5 μmol/L to 1000 μmol/L affecting I. galbana’s cell density, biochemical composition, and the activity of extracellular CA. Phosphorous concentration of 50 μmol/L to 500 μmol/L was optimal for this microalgae. The Zn²⁺ concentration at 10 μmol/L was essential to maintain optimal growth of the cells, but a higher concentration of Zn²⁺ (≥ 1000 μmol/L) inhibited the growth of I. galbana. High CO₂ concentrations (43.75 mL/L) significantly increased the cell densities compared to low CO₂ concentrations (0.35 mL/L). However, the activity of extracellular CA decreased significantly with an increasing concentration of CO₂. The activity of extracellular CA at a CO₂ concentration of 43.75 mL/L was approximately 1/6 of the activity when the CO₂ concentration was at 0.35 mL/L CO₂. Light intensity from 4.0 mW/cm² to 5.6 mW/cm² was beneficial for the growth, biochemical composition and the activity of extracellular CA. The lower and higher light intensity was restrictive for growth and changed its biochemical composition and the activity of extracellular CA. These results indicate that phosphorus, Zn²⁺, CO₂, and light intensity are important factors that impact growth, biochemical composition and the activity of extracellular CA in I. galbana.     

Effect of ubiquinone extraction on the reaction of the mitochondrialbc 1 complex with ferricyanide

Depletion of endogenous ubiquinone by pentane extraction of mitochondrial membranes lowered succinate-ferricyanide reductase activity, whereas quinone reincorporation restored the enzymatic activity as well as antimycin sensitivity. The oxidant-induced cytochromeb extrareduction, normally found upon ferricyanide pulse in intact mitochondria in the presence of antimycin, was lost in ubiquinone-depleted membranes, even if cytochromec was added. Readdition of ubiquinone-2 restored the oxidant-induced extrareduction with an apparent half saturation at 1 mol/molbc ₁ complex saturating at about 5 mol/mol. These findings demonstrate a requirement for the ubiquinone pool of the cytochromeb extrareduction. Since the initial rates of cytochromeb reoxidation upon ferricyanide addition, in the presence of antimycin, did not saturate by any ferricyanide concentration in ubiquinone-depleted mitochondria, a direct chemical reaction between ferricyanide and reduced cytochromeb was postulated. The fact that such direct reaction is much faster in ubiquinone-depleted mitochondria may explain the lower antimycin sensitivity of the succinate ferricyanide reductase activity after removal of endogenous ubiquinone.     

Purification and properties of a F420-nonreactive,membrane-bound hydrogenase from Methanosarcina strain Gö1

The distribution of the F₄₂₀-reactive and F₄₂₀-nonreactive hydrogenases from the methylotrophic Methanosarcina strain Gö1 indicated a membrane association of the F₄₂₀-nonreactive enzyme. The membrane-bound F₄₂₀-nonreactive hydrogenase was purified 42-fold to electrophoretic homogeneity with a yield of 26.7%. The enzyme had a specific activity of 359 mol H₂ oxidized · min^-1 · mg protein^-1. The purification procedure involved dispersion of the membrane fraction with the detergent Chaps followed by anion exchange, hydrophobic and hydroxylapatite chromatography. The aerobically prepared enzyme had to be reactivated anaerobically. Maximal activity was observed at 80°C. The molecular mass as determined by native gel electrophoresis and gel filtration was 77000 and 79000, respectively. SDS gel electrophoresis revealed two polypeptides with molecular masses of 60000 and 40000 indicating a 1:1 stoichiometry. The purified enzyme contained 13.3 mol S^2-, 15.1 mol Fe and 0.8 mol Ni/mol enzyme. Flavins were not detected. The amino acid sequence of the N-termini of the subunits showed a higher degree of homology to cubacterial uptake-hydrogenases than to F₄₂₀-dependent hydrogenases from other methanogenic bacteria. The physiological function of the F₄₂₀-nonreactive hydrogenase from Methanosarcina strain Gö1 is discussed.Abbreviations transmembrane electrochemical gradient of H^- - CoM-SH 2-mercaptoethanesulfonate - F₄₂₀ (N-l-lactyl--l-glutamyl)-l-glutamic acid phospodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - F₄₂₀H₂ reduced F₄₂₀ - HTP-SH 7-mercaptoheptanoylthreonine phosphate - Mb. Methanobacterium - PMSF phenylmethyl-sulfonylfluoride - Cl₃AcOH trichloroacetic acid     

外源赤霉素对紫斑牡丹种子萌发的影响

以紫斑牡丹种子为试验材料,研究不同浓度的赤霉素(GA_3)处理对种子生根以及生根过程中营养物质、酶活性和内源激素水平变化的影响,为探讨紫斑牡丹种子萌发机制提供依据。结果表明:(1)GA_3处理能够促进种子生根,并以300 mg/L GA_3处理对种子生根效果最好,与对照相比可提前14.67 d生根,生根率可达71.00%。(2)与对照相比,GA_3处理可以在0～15 d时促进种子淀粉水解和可溶性糖的积累,并加速可溶性蛋白的消耗,在0～30 d促进过氧化物酶(POD)活性的提高,从而促进种子萌发生根。(3)在种子沙藏生根过程中,种子脱落酸(ABA)含量呈下降趋势,赤霉素(GA)、玉米素核苷(ZR)和吲哚乙酸(IAA)含量均表现出先上升后下降的趋势,与对照相比,GA_3处理可使种子GA、ZR和IAA的含量在沙藏前期明显上升,以解除种子休眠。研究发现,外源GA_3处理可以调控紫斑牡丹种子内源激素含量和POD活性的变化,促进营养物质转化,从而提前解除种子休眠使其萌发。     

Studies on the resolution of cytochrome oxidase

Cytochrome oxidase has been resolved in acetic acid and high salt/detergent media. In 0.5% acetic acid, the smaller subunits of the enzyme are selectively extracted with retention of an insoluble protein fraction containing subunits I–IV, VII. This fraction retains all the heme and copper of the original enzyme in a spectrally unaltered state, and possesses enzymic activity comparable to the unresolved enzyme. The further removal of subunit IV from this fraction results in migration of heme and copper and modification of their spectral characteristics. Resolution of the enzyme in a high salt/detergent medium extracts smaller subunits (V–VII) together with subunit IV and some heme and copper. The heme associated with this enzymically active extract has spectral characteristics that are partially suggestive of hemea ₃. It is suggested that the fraction of subunits I–IV, VII, resolved in dilute acetic acid, may represent the limit of resolution of the cytochrome oxidase complex that remains actively and spectrally indistinguishable from the original enzyme.     

Effect of molecular hydrogen and carbon dioxide on chemo-organotrophic growth of Acetobacterium woodii and Clostridium aceticum

During growth of Acetobacterium woodii on fructose, glucose or lactate in a medium containing less than 0.04% bicarbonate, molecular hydrogen was evolved up to 0.1 mol per mol of substrate. Under an H₂-atmosphere growth of A. woodii with organic substrates was completely inhibited whereas under an H₂/CO₂-atmosphere rapid growth occurred. Under these conditions H₂+CO₂ and the organic substrate were utilized simultaneously indicating that A. woodii was able to grow mixotrophically. Clostridium aceticum differed from A. woodii in that H₂ was only evolved in the stationary phase, that the inhibition by H₂ was observed at pH 8.5 but not at pH 7.5, anf that in the presence of fructose and H₂+CO₂ only fructose was utilized.The hydrogenase activity of fructose-grown cells of C. aceticum amounted to only 12% of that of H₂+CO₂-grown cells. With A. woodii a corresponding decrease of the activity of this enzyme was not observed.     

Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H₂. The enzyme showed a maximal activity of 120±40 mol H₂ oxidized · min^–1 · min^–1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 mol H₂ · min^–1 · mg^–1 with methyl viologen as electron donor, and an apparent K _m for hydrogen oxidation of 5.6±1.7 M. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S^2–, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F₄₂₀ or ferredoxin, nor with FAD, FMN, or NAD(P)⁺. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.Abbreviation CoM-S-S-HTP the heterodisulfide of 7-mercaptoheptanoylthrconine phosphate and coenzyme M (mercaptoethanesulfonic acid)     

Oxidation of ethanol by methanogenic bacteria

Methanogenium organophilum, a non-autotrophic methanogen able to use primary and secondary alcohols as hydrogen donors, was grown on ethanol. Per mol of methane formed, 2 mol of ethanol were oxidized to acetate. In crude extract, an NADP⁺-dependent alcohol dehydrogenase (ADH) with a pH optimum of about 10.0 catalyzed a rapid (5 mol/min·mg protein; 22°C) oxidation of ethanol to acetaldehyde; after prolonged incubation also acetate was detectable. With NAD⁺ only 2% of the activity was observed. F₄₂₀ was not reduced. The crude extract also contained F₄₂₀: NADP⁺ oxidoreductase (0.45 mol/min·mg protein) that was not active at the pH optimum of ADH. With added acetaldehyde no net reduction of various electron acceptors was measured. However, the acetaldehyde was dismutated to ethanol and acetate by the crude extract. The dismutation was stimulated by NADP⁺. These findings suggested that not only the dehydrogenation of alcohol but also of aldehyde to acid was coupled to NADP⁺ reduction. If the reaction was started with acetaldehyde, formed NADPH probably reduced excess aldehyde immediately to ethanol and in this way gave rise to the observed dismutation. Acetate thiokinase activity (0.11 mol/min·mg) but no acetate kinase or phosphotransacetylase activity was observed. It is concluded that during growth on ethanol further oxidation of acetaldehyde does not occur via acetylCoA and acetyl phosphate and hence is not associated with substrate level phosphorylation. The possibility exists that oxidation of both ethanol and acetaldehyde is catalyzed by ADH. Isolation of a Methanobacterium-like strain with ethanol showed that the ability to use primary alcohols also occurs in genera other than Methanogenium.Non-standard abbreviations ADH alcohol dehydrogenase - Ap₅ALi₃ P¹,P⁵-Di(adenosine-5-)pentaphosphate - DTE dithioerythritol (2,3-dihydroxy-1,4-dithiolbutane) - F₄₂₀ N-(N-l-lactyl--l-glutamyl)-l-glutamic acid phosphodiester of 7,8-dimethyl-8-hydroxy-5-deazariboflavin-5-phosphate - Mg. Methanogenium - OD₅₇₈ optical density at 578 nm - PIPES 1,4-piperazine-diethanesulfonic acid - TRICINE N-(2-hydroxy-1,1-bis[hydroxymethyl]methyl)-glycine - Tris 2-amino-2-hydroxy-methylpropane-1,3-diol - U unit (mol substrate/min)     

Simultaneous butyrate oxidation by Syntrophomonas wolfei and catalytic olefin reduction in absence of interspecies hydrogen transfer

Methanogenesis by a Syntrophomonas wolfei/ Methanospirillum hungatei coculture was inhibited in presence of ethylene and the hydrogenation catalyst Pd-BaSO₄. However, butyrate oxidation by S. wolfei continued and ethylene was reduced to ethane. Per mol of butyrate oxidized, 2.4 mol acetate was produced and 0.8 mol ethylene was reduced. Acetylene, propylene and butene were less effective as H₂ acceptors than ethylene, and addition of bromoethanesulfonic acid was necessary to inhibit methanogenesis in the presence of the two longer-chain olefins. Other hydrogenation catalysts were less effective in the order Pd-charcoal < PE-asbestos < Pd-PEI beads < Pt-Al₂O₃, Pd-CaCO₃. Optimal ethylene hydrogenation was achieved with still incubation in presence of 7.2 mg Pd-BaSO₄ and 0.7 g sand per ml medium. The higher catabolic rate of S. wolfei in presence of the methanogen indicated that the biological H₂ removal mechanism was more efficient than the catalytic olefin reduction.Abbreviations BES bromoethane sulfonic acid - VFA volatile fatty acid     

Harvesting of Chlorella vulgaris using a bioflocculant from Paenibacillus sp. AM49

Microbial flocculants for harvesting mass cultured Chlorella vulgaris were screened and that from Paenibacillussp. AM49 was identified as the best. The flocculation efficiency of this bioflocculant increased with the pH within a range of pH 5–11 and was 83%, which was higher than the 72% and 78% produced by aluminum sulfate and polyacrylamide, respectively. The highest flocculation efficiency was with 6.8 mm CaCl₂ as co-flocculant. The bioflocculant from Paenibacillussp. AM49 can be used effectively to harvest C. vulgaris from large-scale cultures.     

Nickel,a component of factor F 430 from Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum, growing on medium supplemented with 2 mol ⁶³NiCl₂/l, was found to take up 1.2 mol ⁶³Ni per g cells (dry weight). More than 70% of the radioisotope was incorporated into a compound, which dissociated from the protein fraction after heat treatment, was soluble in 70% acetone, and could be purified by chromatography on QAE-Sephadex A-25, Sephadex G-25, and DEAE cellulose. The purified ⁶³Ni labelled compound had an absorption spectrum and properties identical to those of factor F ₄₃₀ and is therefore considered to be identical with factor F ₄₃₀.Factor F ₄₃₀, a compound of molecular weight higher than 1000 with an absorbance maximum at 430 nm, has recently been purified from Methanobacterium thermoautotrophicum (Gunsalus and Wolfe, 1978). The structure and function of this compound are not yet known.     

The roles of malate and aspartate in C4 photosynthetic metabolism of Flaveria bidentis (L.)

In C₄ grasses belonging to the NADP-malic enzyme-type subgroup, malate is considered to be the predominant C₄ acid metabolized during C₄ photosynthesis, and the bundle sheath cell chloroplasts contain very little photosystem-II (PSII) activity. The present studies showed that Flaveria bidentis (L.), an NADP-malic enzyme-type C₄ dicotyledon, had substantial PSII activity in bundle sheath cells and that malate and aspartate apparently contributed about equally to the transfer of CO₂ to bundle sheath cells. Preparations of bundle sheath cells and chloroplasts isolated from these cells evolved O₂ at rates between 1.5 and 2 mol · min^–1 · mg^–1 chlorophyll (Chl) in the light in response to adding either 3-phosphoglycerate plus HCO ₃ ^– or aspartate plus 2-oxoglutarate. Rates of more than 2 mol O₂ · min^–1 · mg^–1 Chl were recorded for cells provided with both sets of these substrates. With bundle sheath cell preparations the maximum rates of light-dependent CO₂ fixation and malate decarboxylation to pyruvate recorded were about 1.7 mol · min^–1 · mg^–1 Chl. Compared with NADP-malic enzyme-type grass species, F. bidentis bundle sheath cells contained much higher activities of NADP-malate dehydrogenase and of aspartate and alanine aminotransferases. Time-course and pulse-chase studies following the kinetics of radiolabelling of the C-4 carboxyl of C₄ acids from ¹⁴CO₂ indicated that the photosynthetically active pool of malate was about twice the size of the aspartate pool. However, there was strong evidence for a rapid flux of carbon through both these pools. Possible routes of aspartate metabolism and the relationship between this metabolism and PSII activity in bundle sheath cells are considered.Abbreviations DHAP dihydroxyacetone phosphate - NADP-ME(-type) NADP-malic enzyme (type) - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetic acid - 2-OG 2-oxoglutarate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - Ru5P ribulose 5-phosphate     

Photosynthetic carbon metabolism during leaf ontogeny in Zea mays L.: Enzyme studies

The activities of several enzymes, including ribulose-1,5-diphosphate (RuDP) carboxylase (EC 4.1.1.39) and phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) were measured as a function of leaf age in Z. mays. Mature leaf tissue had a RuDP-carboxylase activity of 296.7 mol CO₂ g^-1 fresh weight h^-1 and a PEP-carboxylase activity of 660.6 mol CO₂ g^-1 fresh weight h^-1. In young corn leaves the activity of the two enzymes was 11 and 29%, respectively, of the mature leaves. In senescent leaf tissue, RuDP carboxylase activity declined more rapidly than that of any of the other enzymes assayed. On a relative basis the activities of NADP malic enzyme (EC 1.1.1.40), aspartate (EC 2.6.1.1) and alanine aminotransferase (EC 2.6.1.2), and NAD malate dehydrogenase (EC 1.1.1.37) exceeded those of both PEP and RuDP carboxylase in young and senescent leaf tissue. Pulse-chase labeling experiments with mature and senescent leaf tissue show that the predominant C₄ acid differs between the two leaf ages. Labeling of alanine in senescent tissue never exceeded 4% of the total ¹⁴C remaining during the chase period, while in mature leaf tissue alanine accounted for 20% of the total after 60 s in ¹²CO₂. The activity of RuDP carboxylase during leaf ontogeny in Z. mays parallels the development of the activity of this enzyme in C₃ plants.Abbreviations RuDP ribulose-1,5-diphosphate - PEP phosphoenol pyruvate - PGA 3-phosphoglycerate     

Purification and Kinetic Properties of 6-Phosphogluconate Dehydrogenase from Rat Small Intestine

6-Phosphogluconate dehydrogenase (6PG) was purified from rat small intestine with 36% yield and a specific activity of 15 U/mg. On SDS/PAGE, one band with a mass of 52 kDa was found. On native PAGE three protein and two activity bands were observed. The pH optimum was 7.35. Using Arrhenius plots, Ea, ΔH, Q₁₀ and Tm for 6PGD were found to be 7.52 kcal/mol, 6.90 kcal/mol, 1.49 and 49.4°C, respectively. The enzyme obeyed “Rapid Equilibrium Random Bi Bi” kinetic model with K_m values of 595 ± 213 μM for 6PG and 53.03±1.99 μM for NADP. 1/V_m versus 1/6PG and 1/NADP plots gave a V_m value of 8.91±1.92 U/mg protein. NADPH is the competitive inhibitor with a K_i of 31.91±1.31 μM. The relatively small K_i for the 6PGD:NADPH complex indicates the importance of NADPH in the regulation of the pentose phosphate pathway through G6PD and 6PGD.     

Phospholipid dependence of membrane-bound phospholipase A2 in ras-transformed NIH 3T3 fibroblasts

Although the activation of phospholipase A₂ (PLA₂) in ras-transformed cells has been well documented, the mechanisms underlying this activation are poorly understood. In this study we tried to elucidate whether the membrane phospholipid composition and physical state influence the activity of membrane-associated PLA₂ in ras-transformed fibroblasts. For this purpose membranes from non-transfected and ras-transfected NIH 3T3 fibroblasts were enriched with different phospholipids by the aid of partially purified lipid transfer protein. The results showed that of all tested phospholipids only phosphatidylcholine (PC) increased PLA₂ activity in the control cells, whereas in their transformed counterparts both PC and phosphatidic acid (PA) induced such effect. Further we investigated whether the activatory effect was due only to the polar head of these phospholipids, or if it was also related to their acyl chain composition. The results demonstrated that the arachidonic acid-containing PC and PA molecules induced a more pronounced increase of membrane-associated PLA₂ activity in ras-transformed cells compared to the corresponding palmitatestearate- or oleate- containing molecular species. However, we did not observe any specific effect of the phospholipid fatty acid composition in non-transformed NIH 3T3 fibroblasts. In ras-transformed cells incubated with increasing concentrations of arachidonic acid, PLA₂ activity was altered in parallel with the changes of the cellular content of this fatty acid. The role of phosphatidic and arachidonic acids as specific activators of PLA₂ in ras-transformed cells is discussed with respect to their possible role in the signal transduction pathways as well as in the processes of malignant transformation of cells.