Purification and characterization of fatty acid synthetase from Cryptococcus neoformans

Fatty acid synthetase has been purified from Cryptococcus neoformans 450 fold to a specific activity of 3.6 units per mg protein with an overall yield of 23%. The purified enzyme contained two non-identical subunits, Mr approximately 2.1×10⁵ and 1.8×10⁵. Under optimum conditions, 100 mM KCl and pH 7.5, apparent Km values for the substrates were: Acetyl CoA, 19 M; Malonyl CoA, 5 M; and NADPH, 6 M. Product inhibition patterns were determined to be: CoA, competitive versus acetyl CoA and malonyl CoA, uncompetitive versus NADPH; NADP, competitive versus NADPH, uncompetitive versus acetyl CoA and malonyl CoA; Palmitoyl CoA, competitive versus malonyl CoA, noncompetitive versus acetyl CoA and NADPH; Bicarbonate, uncompetitive versus malonyl CoA. These product inhibition patterns are consistent with the multisite ping-pong mechanism previously proposed for the avian fatty acid synthetase complex. The cryptococcal fatty acid synthetase was inhibited by the polyanionic polymers, heparin and dextran sulfate, an effect never before demonstrated for a fatty acid synthetase. This inhibition exhibited a marked dependence on the length of the polymer chain, with dextran sulfate fractions with Mr of 6×10⁵ and above having K _i values below 100 nanomolar. A model is presented that involves initial binding of the anionic polymer to the enzyme complex at a region of high positive charge density, followed by interaction of the end of the tethered polymer with the catalytic site. This study represents the first purification of fatty acid synthetase from a basidiomycete.     

An NADP-linked acetoacetyl CoA reductase from Zoogloea ramigera

Zoogloea ramigera I-16-M was found to contain two stereospecific acetoacetyl CoA reductases; one was NADP⁺-linked and d(-)--hydroxybutyryl CoA specific and the other was NAD⁺-linked and l(+)-isomer specific. The NADP⁺-linked enzyme, purified approximately 150-fold, had a pH optimum for the reduction of acetoacetyl CoA at 8.1, but no definite pH optimum for the oxidation of -hydroxybutyryl CoA. The apparent Michaelis constants for acetoacetyl CoA and NADPH were 8.3 and 21 M, respectively. The enzyme was markedly inhibited by acetoacetyl CoA at concentrations higher than 10 M.The incorporation of [1-¹⁴C]acetyl CoA into poly--hydroxybutyrate (PHB) by bacterial crude extract (containing -ketothiolase, acetoacetyl CoA reductases, enoyl CoA hydratases and PHB synthases) or by a system reconstituted from purified preparations of -ketothiolase, acetoacetyl CoA reductase and PHB synthase, was observed only in the presence of NADPH, but not NADH. Among various enzymes involved in PHB metabolism, only the specific activity of glucose 6-phosphate dehydrogenase was elevated 5-fold within 2 h after the addition of glucose to the cells grown in the basal medium.These findings suggest that, in Z. ramigera I-16-M, acetoacetyl CoA is directly reduced to d(-)--hydroxybutyryl CoA by the NADP⁺-dependent reductase, and PHB synthesis is at least partially controled by NADPH availability through glucose 6-phosphate dehydrogenase.Non-Standard Abbreviation PHB poly--hydroxybutyrate     

Effect of acute and chronic epinephrine administration on clearance and metabolism of [3H]-epinephrine in trout (Oncorhynchus mykiss)

Several studies have measured the rate of catecholamine clearance, metabolism, and tissue accumulation in fish. However, no information is available on the effect of repeated stress or high circulating catecholamine levels on catecholamine clearance and metabolism. We measured the clearance and metabolism of [³H]-epinephrine (approximately 0.1 g·kg^-1) in SW-acclimated rainbow trout subjected to acute (five injections in 1 day) and chronic (4 days; five injections per day) administration of 4.0 g·kg^-1 epinephrine or saline. In addition, a saturation experiment, where 4.0 g·kg^-1 of unlabelled epinephrine was injected concurrently with [³H]-epinephrine, investigated whether catecholamine clearance and metabolism are affected by high circulatin levels. Neither the rate constants for catecholamine clearance, nor the post-injection proportions of unmetabolised [³H]-epinephrine, deaminated [³H]-epinephrine and O-methylated [³H]-epinephrine were affected by the acute or chronic injection protocols. The concurrent injection of [³H]-epinephrine and 4.0 g·kg^-1 of unlabelled epinephrine resulted in an elevated postinjection ³H:¹⁴C ratio, but increased proportions of O-methylated [³H]-epinephrine and reduced proportions of unmetabolised [³H]-epinephrine. We conclude that in fish (1) catecholamine clearance and metabolism are unlikely to be compromised by repeated exposure to acute stressors; (2) catecholamine extraction and/or metabolism is enhanced when circulating levels are high; and (3) there is a marked capacity to rapidly (minutes) clear and inactivate catecholamines that are released in response to stressful stimuli.Abbreviations CA catecholamines - dpm disintegrations per minute - MAO monoamine oxidase - COMT catecholamine-O-methyltransferase - MOPEG 3-methoxy-4-hydroxyphenylglycol - VMA 3-methoxy-4-hydroxymendelic acid - SW seawater - HPLC high-performance liquid chromatography     

Fatty-acid synthesis in plastids from maturing safflower and linseed cotyledons

Plastids isolated from maturing, nongreen safflower (Carthamus tinctorius L.) cotyledons yielded unesterified fatty acids as the predominant product of fatty-acid synthesis from [1-¹⁴C]acetate. Exogenous reduced pyridine nucleotides were not required for this synthesis, but [1-¹⁴C]acetate incorporation was absolutely dependent on addition of ATP. Linseed (Linum usitatissimum L.) cotyledons are green during development and plastids isolated from them resembled leaf chloroplasts with developed grana. In contrast to the safflower plastids, those from linseed were able to carry out fatty-acid synthesis at low irradiances without the addition of either pyridine nucleotides or ATP. Intact linseed cotyledons were capable of net photosynthesis at rates up to 95 mol·mg^-1 chlorophyll·h^-1. However, the low-light environment inside the linseed capsule (approx. 15% of external) means that photosynthesis will not contribute appreciably to the carbon economy of the developing seed and its main role may be to supply cofactors for fatty-acid synthesis.Abbreviations ACP acyl carrier protein - DHAP dihydroxyacetone phosphate - PC phosphatidylcholine - PEP phosphoenolpyruvate - UFA unesterified fatty acids     

Compartmentation and flux characteristics of ammonium in spruce

Using ¹³NH ₄ ⁺ as a tracer, compartmental analyses for NH ₄ ⁺ were performed in non-mycorrhizal roots of intact Picea glauca (Moench) Voss. seedlings at four different concentration regimes of external NH ₄ ⁺ ([NH ₄ ⁺ ]_o), i.e. 0, 10, 100, and 1500 M. Three kinetically distinct compartments were identified, with half-lives of exchange of approximately 2 s, 30 s, and 14 min, assumed to represent surface adsorption, Donnan free space, and cytoplasm, respectively. No significant differences were found in half-lives of exchange with changes in [NH ₄ ⁺ ]_o. Influx was calculated to be 0.96 mol·g^–1·h^–1 in N-deprived plants (measured at 10 M [NH ₄ ⁺ ]_o), while under steady-state conditions it was 0.21 mol·g^–1h^–1 at 10 M [NH ₄ ⁺ ]_o, 1.96 mol·g^–1h·^–1 at 100 M [NH ₄ ⁺ ]_o, and 6.45 mol·g^–1·h^–1 at 1.5 mM [NH ₄ ⁺ ]_o. Efflux measured over the same range constituted approximately 9% of influx in N-deprived plants, 10% at 10 M, 28% at 100 M, and 35% at 1.5 mM [NH ₄ ⁺ ]_o. Cytoplasmic [NH ₄ ⁺ ] was estimated at 6 m M in N-deprived plants, 2 mM at 10 M [NH ₄ ⁺ ]_o, 14 mM at 100 M, and 33 mM at 1.5 mM. Free-space [NH ₄ ⁺ ] was 84 M, 50 M, 700 M, and 8 mM, respectively. In comparison with previously published data on fluxes and compartmentation of NO ₃ ^– in white-spruce seedlings, results of this study identify a pronounced physiological preference of this species for NH ₄ ⁺ over NO ₃ ^– as an inorganic N source in terms of uptake and intracellular accumulation. The significant ecological importance of this N-source preference is discussed.The research was supported by a Natural Sciences and Engineering Research Council, Canada, grant to Dr. A.D.M. Glass and a University of British Columbia Graduate Fellowship to Herbert J. Kronzucker. Our thanks go to Dr. M. Adam and Mr. P. Culbert at the particle accelerator facility TRIUMF on the University of British Columbia campus for providing ¹³N, to Drs. R.D. Guy and S. Silim for providing plant material, and to Dr. M.Y. Wang, Mr. J. Bailey, Mr. J. Mehroke and Mr. P. Poon for essential assistance in experiments.     

Comparative effects of CGA-92194, cyometrinil,and flurazole on selected metabolic processes of isolated soybean leaf cells

The potential adverse phytotoxic effects of the herbicide safeners CGA-92194 {-[1,3-dioxolan-2-yl-methoxy)imino]benzeneacetonitrile}, cyometrinil [-(cyanomethoxy)imino-benzeneacetonitrile] and flurazole [phenylmethyl 2-chloro-4-(trifluoromethyl)-5-thiazole-carboxylate] on selected metabolic processes of enzymatically isolated leaf cells of soybean [Glycine max (L.) Merr.] were compared in time- and concentration-course studies. CO₂ fixation, protein synthesis, RNA synthesis, DNA synthesis, and lipid synthesis were assayed by the incorporation of NaH¹⁴CO₃, [¹⁴C]-leucine, [¹⁴C]-uracil, [³H]thymidine, and [¹⁴C]-acetate, respectively, into the isolated cells. CGA-92194 and cyometrinil behaved similarly, and at low concentrations (0.1, 1, and 10 M) they stimulated rather than inhibited the five metabolic processes assayed, following incubation periods of up to 2 h. At the highest concentration of 100 M, both safeners inhibited all metabolic processes of the soybean leaf cells but neither compound exhibited rapid and distinct inhibitions as might be expected in the case of inhibition of a primary target site by a potent inhibitor. At low concentrations and early incubation periods (30 and 60 min), flurazole effects on all metabolic processes were also stimulatory rather than inhibitory. However, the stimulation of CO₂ fixation by 0.1 and 1.0 M was highly significant. At 100 M flurazole was extremely potent on all metabolic processes of soybean leaf cells examined. At the 2-h incubation period, flurazole also inhibited all metabolic processes at concentrations lower than 100 M. The sensitivity of the five metabolic processes to flurazole decreased in the following order: photosynthesis = lipid synthesis >DNA synthesis>protein synthesis>RNA synthesis.Contribution No. 534, Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg VA 24061 USA.     

Δ5 Desaturase activity in rat kidney microsomes

Rat kidney microsomal fraction is able to catalyze the enzymatic desaturation of eicosatrienoic acid (20:3n-6) to arachidonic acid (20:4n-6) by the 5 desaturase pathway, in the presence of reduced nicotinamide adenine dinucleotide (NADH), adenosinetriphosphate (ATP) and coenzyme A (CoA). The substrate of the reaction [1-¹⁴C]eicosa-8,11,14trienoic acid (20:3n-6), was separated from the product [1-¹⁴C]eicosa-5,8,11,14-tetraenoic acid (20:4n-6) by reverse phase high-pressure liquid chromatography (RP-HPLC). These fatty acids were individually collected by monitoring the eluent at 205 nm and their radioactivity was measured by liquid scintillation counting. The 5 desaturase activity in kidney microsomes increased linearly with the substrate concentration up to 20 M. Enzymatic activity was sensitive to pH with the maximum at 7.0 and was proportional with incubation time up to 10 min. The apparent Km and Vmax of 5 desaturase were 56 M and 60 pmoles·min^–1·mg^–1 microsomal protein, respectively. Neither the cytosolic renal fraction nor the cytosolic liver fraction enhanced the 5 desaturase activity. Contrary to a report but in accordance to others, the present results suggest that rat kidneys can synthesize arachidonic acid at least to satisfy partially their needs for eicosanoid production.     

Heterogeneous Long Chain Acyl-CoA Synthetases Control Distribution of Individual Fatty Acids in Newly-Formed Glycerolipids of Neuronal Cells Undergoing Neurite Outgrowth

Using PC12 cells undergoing neurite outgrowth, we studied the activation of various fatty acids, of different chain lengths and degrees of saturation, by long chain acyl-CoA synthetases (LCASs). Cells treated with nerve growth factor (NGF) were labeled with [³H]glycerol, [³H]oleic acid (OA) or [³H]arachidonic acid (AA) in the presence of other unlabeled fatty acids of endogenous or exogenous origin. Triacsin C (4.8 M), an inhibitor of acyl-CoA synthetase, decreased the incorporation of exogenous [³H]OA into glycerolipids by 30–90%, and increased by about 60% the accumulation of free [³H]OA in the cells. However it did not affect the incorporation of endogenous fatty acids nor of exogenous [³H]AA into phospholipids, suggesting that LCASs which activate exogenous AA and at least some endogenous fatty acids are relatively insensitive to this drug. Activities of the LCAS that is specific for AA (ACS), or of the non-specific LCAS which activates OA and other fatty acids (OCS), were much higher in microsomal and cytoplasmic fractions than in mitochondria or nuclei. The Vmax and Km values of ACS and OCS in microsomes were 12 and 0.7 nmol/min/mg protein and 70 and 37 M, respectively; and in cytoplasm, 6 and 0.6 nmol/ min/mg protein and 38 and 60 M, respectively. Triacsin C (2–33 M) did not affect ACS activity in microsomal or cytoplasmal fractions, but inhibited OCS activities dose-dependently and competitively: IC₅₀ and apparent Ki values were 13.5 M and 14 M in microsomes, and 3.8 M and 4 M in cytoplasm. NGF stimulated the activities of the LCASs, and, consistently, the incorporation of the various fatty acids into glycerolipids. These data indicate that LCASs are heterogeneous with respect to their intracellular locations, substrate specificities, kinetic characteristics and sensitivities to triacsin C; and that this heterogeneity affects the extents to which individual fatty acids are utilized to form glycerolipids.     

Some regulatory aspects of [14C]methylamine influx intoPisum sativum L. cv. Feltham First seedlings

[¹⁴C]Methylamine influx intoPisum sativum L. cv. Feltham First seedlings showed Michaelis-Menten-type kinetics with apparentV _max=49.2 mol·g^-1 FW·h^-1 and apparentK _m=0.51 mM. The competitive interactions between ammonium and methylamine were most obvious when biphasic kinetics were assumed with saturation of the first phase at 0.05 mM. The inhibitor constant for ammonium (K _i)=0.027 mM. When [¹⁴C]methylamine was used in trace amounts with ammonium added as substrate, the influx of tracer showed Michaelis-Menten-type kinetics with apparentV _max=3.46 mol·g^-1 FW·h^-1 and apparentK _m=0.15 mM. The initial rate of net ammonium uptake corresponded with that found when [¹⁴C]methylamine was used to trace ammonium influx. The latter was also stimulated by high pH_o and inhibited by nitrate. Ammonium pretreatment±methionine sulphoximine or glutamine pretreatment of the seedlings inhibited subsequent [¹⁴C]methylamine influx, while methylamine or asparagine pretreatment stimulated [¹⁴C]methylamine influx. There was also a stimulatory effect of prior inoculation withRhizobium. The results are discussed in terms of current models for the regulation of ammonium uptake in plants.     

Serum release of hepatic calcium-binding protein regucalcin by liver injury with galactosamine administration in rats

The incorporation and 9 desaturation of exogenous [¹⁴C]stearic acid were studied in HTC 7288c cells in suspension. We examined the uptake of the acid over a wide range of concentrations (0–160 M) after incubating the cells for 6 h in a chemically-defined medium. Under this experimental condition, the uptake of the labeled acid was more extensive than that obtained from static cultures or from monolayer of isolated hepatocytes of rats. At an external concentration of 160 Mca. 52 nmoles of acid per mg of cellular protein was taken up. The production of oleic acid from [¹⁴C]stearate (9 desaturation) correlated well with the uptake curve between 0–80 M concentration. For higher stearate concentrations, the biosynthesis of oleic acid declined substantially and a plateau of 22 nmoles/mg cellular protein was reached. The incorporation and desaturation of an initial exogeneous concentration of [¹⁴C]stearic acid (80 M) was also studied from 0–6 h. The results obtained demonstrated that the uptake of the substrate into cellular lipids was fast and non saturable. Quantitative gas-liquid chromatography of total cellular lipids under the different experimental conditions demonstrated a negative correlation between the decrease in the palmitic and palmitoleic acids and the increase in the intracellular levels of stearic and oleic acids. These analytical modifications took place with no changes in the saturated/monoenoic fatty acid ratio. This work also demonstrated a significant contribution of the stearoyl-CoA desaturase system to the high levels of oleic acid present in this kind of hepatoma cells.Abbreviations FAME Fatty Acid Methyl esters - GLC gas-liquid chromatography - HEPES N-2-hydroxyethyl-piperazine-N-2-ethanosulfonic acid - HTC Hepatoma Tissue Culture - IMEM-Zo Improved Minimal Essential Medium-zinc optional     

Silicon amelioration of aluminium toxicity in teosinte (Zea mays L. ssp. mexicana)

The influence of different Al concentrations, (0, 60 and 120 M Al) on growth and internal concentrations of Al, Si and selected organic acids was analysed in plants of teosinte (Zea mays L. ssp. mexicana), a wild form of maize from acid soils from Mexico. The plants were grown in nutrient solutions (pH 4.0) with or without 4 M silicon. Analysis with the GEOCHEM speciation program did not reveal differences between free activities of Al³⁺ in solutions with and without 4 M Si, but solutions with Si yielded lower concentrations of monomeric Al species, [Al]_mono, when analysed by a modified aluminon method. Plants grown on solutions with similar [Al]_mono, but differing in silicon, showed highly significant differences in growth and tissue concentrations of Al and organic acids. Silicon prevented growth inhibition at [Al]_mono concentrations as high as 35 M, while plants grown without Si suffered severe growth reductions with 33 M [Al]_mono. In solutions with similar [Al]_mono concentrations plants with Si had lower tissue Al concentrations and higher concentrations of malic acid than plants without Si. In view of both the significant influence of Si on the response of plants to Al toxicity and the fact that some soluble Si is always present in soil solutions, the addition of low Si concentrations to nutrient solutions used for Al-tolerance screening is recommended.     

Biosynthesis of saturated very long chain fatty acids by purified membrane fractions from leek epidermal cells

Elongation of fatty acids by microsomal fractions obtained from leek epidermal cells was measured by the incorporation of [1-¹⁴C]stearate, [1-¹⁴C]stearyl CoA, and [1-¹⁴C]stearyl ACP in the presence of malonyl CoA and NADPH. Stearoyl CoA appears to be the primer of the elongase (s) rather than stearoyl ACP. There are at least two elongases, the first elongating C₁₈ to C₂₀, the second synthesizing C₂₂ to C₃₀ fatty acids from C₂₀. The main site of the elongase (s) is a subcellular fraction enriched in endoplasmic reticulum. The plasma membrane-enriched fraction, which contains large amounts of saturated very long chain fatty acids, synthesizes only minor amounts of them.     

Intramyelinic conversion of cerebrosides into acylgalactosylceramides

Acylgalactosylceramide (AGC) synthesis was measured in vivo, and in a cell free system. 24 hours post-injection of [³H]palmitic acid into rat brain, more than 60% of the AGC radioactivity was associated with an ester linkage. Isolated rat myelin was incubated in the presence of [¹⁴C]palmitic acid, 2mM ATP, 50 M CoA and 10 mM MgCl₂ and acylation of myelin cerebrosides occurred at a linear rate for at least 60 min. Incubation of isolated myelin under standard conditions with [³H] cerebrosides and [¹⁴C]palmitic acid produced double labeled AGC. Labeling of AGC was maximum at pH 7.5 and 37°C and appeared to be enzyme mediated inasmuch as it was reduced by myelin incubation with trypsin and drastically reduced by preheating the myelin for 5 min at 80°C. Omission of ATP, CoA, MgCl₂ or all three did not reduce fatty acid incorporation into AGC when compared to the values in the complete system. Addition of Triton X100 or Sodium Dodecyl Sulfate had little or no effect on the acylation of cerebrosides. Pulse chase experiments indicated that the reaction involved the net addition of fatty acid to the cerebrosides, rather than a rapid fatty acid exchange.     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with “antisense” rbcS

Tobacco (Nicotiana tabacum L.) plants transformed with antisense rbcS to decrease the expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) have been used to investigate the contribution of Rubisco to the control of photosynthesis in plants growing at different irradiances. Tobacco plants were grown in controlled-climate chambers under ambient CO₂ at 20°C at 100, 300 and 750 mol·m^–2·s^–1 irradiance, and at 28°C at 100, 300 and 1000 mol·m^–2·s^–1 irradiance. (i) Measurement of photosynthesis under ambient conditions showed that the flux control coefficient of Rubisco (C _infRubisco ^supA ) was very low (0.01–0.03) at low growth irradiance, and still fairly low (0.24–0.27) at higher irradiance. (ii) Short-term changes in the irradiance used to measure photosynthesis showed that C _infRubisco ^supA increases as incident irradiance rises, (iii) When low-light (100 mol·m^–2·s^–1)-grown plants are exposed to high (750–1000 mol·m^–2·s^–1) irradiance, Rubisco is almost totally limiting for photosynthesis in wild types. However, when high-light-grown leaves (750–1000 mol·m^–2·s^–1) are suddenly exposed to high and saturating irradiance (1500–2000 mol·m^–2·s^–1), C _infRubisco ^supA remained relatively low (0.23–0.33), showing that in saturating light Rubisco only exerts partial control over the light-saturated rate of photosynthesis in sun leaves; apparently additional factors are co-limiting photosynthetic performance, (iv) Growth of plants at high irradiance led to a small decrease in the percentage of total protein found in the insoluble (thylakoid fraction), and a decrease of chlorophyll, relative to protein or structural leaf dry weight. As a consequence of this change, high-irradiance-grown leaves illuminated at growth irradiance avoided an inbalance between the light reactions and Rubisco; this was shown by the low value of C _infRubisco ^supA (see above) and by measurements showing that non-photochemical quenching was low, photochemical quenching high, and NADP-malate dehydrogenase activation was low at the growth irradiance. In contrast, when a leaf adapted to low irradiance was illuminated at a higher irradiance, Rubisco exerted more control, non-photochemical quenching was higher, photochemical quenching was lower, and NADP-malate dehydrogenase activation was higher than in a leaf which had grown at that irradiance. We conclude that changes in leaf composition allow the leaf to avoid a one-sided limitation by Rubisco and, hence, overexcitation and overreduction of the thylakoids in high-irradiance growth conditions, (v) Antisense plants with less Rubisco contained a higher content of insoluble (thylakoid) protein and chlorophyll, compared to total protein or structural leaf dry weight. They also showed a higher rate of photosynthesis than the wild type, when measured at an irradiance below that at which the plant had grown. We propose that N-allocation in low light is not optimal in tobacco and that genetic manipulation to decrease Rubisco may, in some circumstances, increase photosynthetic performance in low light.Abbreviations A rate of photosynthesis - C _infRubisco ^supA flux control coefficient of Rubisco for photosynthesis - c_i internal CO₂ concentration - qE energy-dependent quenching of chlorophyll fluorescense - qQ photochemical quenching of chlorophyll fluorescence - NADP-MDH NADP-dependent malate dehydrogenase - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - RuBP ribulose-1,5-bisphosphate This work was supported by the Deutsche Forschungsgemeinschaft (SFB 137).     

Control of gluconeogenesis by phosphoenolpyruvate carboxykinase in cotyledons ofCucurbita pepo L.

3-Mercaptopicolinic acid, a non-competitive inhibitor of phosphoenolpyruvate carboxykinase (EC 4.1.1.19) was used to study the control of gluconeogenesis by this enzyme in germinating marrow (Cucurbita pepo) cotyledons. In vitro, phosphoenolpyruvate carboxykinase was inhibited by 3-mercaptopicolinic acid, with aKi of 5.9 M. At 25°C the inhibitor caused an increase in the label incorporated from [2-¹⁴C]acetate into CO₂, and a decrease in the label incorporated into the insoluble and neutral fractions. Phosphoenolpyruvate carboxykinase had a flux control coefficient for gluconeogenesis (C _PEPCK ^J ) of between 0.7 and 1.0. 3-Mercaptopicolinic acid was a less effective inhibitor of phosphoenolpyruvate carboxykinase at lower temperatures (Ki = 8.6 M at 17°C, 13.3 M at 10°C) and had similar effects on the metabolism of [2-¹⁴C]acetate by marrow cotyledons when the temperature was reduced to 17°C and 10°C. The control coefficient for this enzyme did not change with temperature, indicating that phosphoenolpyruvate carboxykinase exerts a high degree of control over gluconeogenesis at all temperatures examined.Abbreviations PEP Phosphoenolpyruvate - PEPCK PEP carboxykinase The authors thank Dr. Ian Woodrow (University of Melbourne, Australia) for helpful discussions. This work was supported by a grant from the Science and Engineering Research Council, U.K. (GR/F 50978).     

Translocation and metabolism of glycine betaine by barley plants in relation to water stress

The glycine betaine which accumulated in shoots of young barley plants (Hordeum vulgare L.) during an episode of water stress did not undergo net destruction upon relief of stress, but its distribution among plant organs changed. During stress, betaine accumulated primarily in mature leaves, whereas it was found mainly in young leaves after rewatering. Well-watered, stressed, and stressed-rewatered plants were supplied with [methyl-¹⁴C]betaine (8.5 nmol) via an abraded spot on the second leaf blade, and incubated for 3 d. In all three treatments the added ¹⁴C migrated more or less extensively from the second leaf blade, but was recovered quantitatively from various plant organs in the form of betaine; no labeled degradation products were found in any organ. When 0.5 mol of [methyl-¹⁴C]betaine was applied via an abraded spot to the second leaf blades of well-watered, mildly-stressed, and stressed-rewatered plants, ¹⁴C was translocated out of the blades at velocities of about 0.2–0.3 cm/min which were similar to velocities found for applied [¹⁴C]sucrose. Heat-girdling of the sheath prevented export of [¹⁴C]betaine from the blade. When 0.5 mol [³H]sucrose and 0.5 mol [¹⁴C]betaine were suppled simultaneously to second leaf blades, the ³H/¹⁴C ratio in the sheath tissue was the same as that of the supplied mixture. After supplying tracer [¹⁴C]betaine aldehyde (the immediate precursor of betaine) to the second leaf blade, the ¹⁴C which was translocated into the sheath was in the form of betaine. Thus, betaine synthesized by mature leaves during stress behaves as an inert end product and upon rewatering is translocated to the expanding leaves, most probably via the phloem. Accordingly, it is suggested that the level of betaine in a barley plant might serve as a useful cumulative index of the water stress experienced during growth.     

In-vivo control of valine and leucine synthesis in the duckweed Spirodela polyrhiza

Duckweed colonies were grown on 1 l of nutrient solution supplied with 10 M l-[¹⁴C]leucine or with 25 M l-[¹⁴C]valine. Under these conditions the exogenously supplied amino acid did not inhibit growth, but caused in the plants a moderately increased pool of that amino acid, which remained essentially constant during the culture period. The effect of the increased pool of valine or leucine on the biosynthesis of these amino acids was determined from isotope dilution in the protein-bound valine and-or leucine. An increase in the leucine pool from 1.1 to 5.0 nmol mg^–1 dry weight resulted in a 21% reduction of metabolite flow through the common part of the valine-leucine biosynthetic pathway; leucine synthesis was reduced by 35%, but valine synthesis by only 5% and isoleucine synthesis was apparently unaffected. An increase in the valine pool from 3.2 to 6.6 nmol mg^–1 dry weight reduced the metabolite flow through the valine-leucine pathway by 48%, valine synthesis by 70%, and leucine synthesis from pyruvate by 29%, which was compensated by leucine synthesis from exogenous valine, whereas the synthesis of isoleucine was not changed. It is concluded that the biosynthesis of valine and leucine is mainly controlled by feedback inhibition of acetohydroxyacid synthetase. In vivo, the feedback inhibition can be exerted in such a way that synthesis of acetolactate (the precursor of valine and leucine) is appreciably reduced, whereas synthesis of acetohydroxybutyrate (the isoleucine precursor) is not inhibited.     

Light response of D1 turnover and photosystem II efficiency in the seagrassZostera capricorni

Biochemical and biophysical parameters, including D1-protein turnover, chlorophyll fluorescence, oxygen evolution activity and zeaxanthin formation were measured in the marine seagrassZostera capricorni (Aschers) in response to limiting (100 mol·m^–2·^–1), saturating (350 mol·m^–2·s^–1) or photoinhibitory (1100 mol·m^–2·s^–1) irradiances. Synthesis of D1 was maximal at 350 mol·m^–2·s^–1 which was also the irradiance at which the rate of photosynthetic O₂ evolution was maximal. Degradation of D1 was saturated at 350 mol·m^–2·s^–1. The rate of D1 synthesis at 1100 mol·m^–2·s^–1 was very similar to that at 350 mol·m^–2·s^–1 for the first 90 min but then declined. At limiting or saturating irradiance little change was observed in the ratio of variable to maximal fluorescence (Fv/Fm) measured after dark adaptation of the leaves, while significant photoinhibition occurred at 1100 mol·m^–2·s^–1. The proportion of zeaxanthin in the total xanthophyll pool increased with increasing irradiance, indicative of the presence of a photoprotective xanthophyll cycle in this seagrass. These results are consistent with a high level of regulatory D1 turnover inZostera under non-photoinhibitory irradiance conditions, as has been found previously for terrestrial plants.We would like to thank Professor Peter Böger (Department of Plant Biochemistry, University of Konstanz, Germany) for the kind gift of D1 antibodies. This work was partly supported by a University of Queensland Enabling Grant to CC.     

Effect of 2-hydroxybenzoate on the rate of naphthalene mineralization in soil

The effect of 2-hydroxybenzoate (2-OHB, salicylate) on the mineralization rate of [¹⁴C]naphthalene, the population density of naphthalene-degrading bacteria, and the concentration of genes encoding for naphthalene dioxygenase in a soil bacterial community was investigated. Six different concentrations of 2-OHB (10, 20, 50, 100, 150 and 200 g g^–1 soil) were tested in 100-g portions of soil. The addition of 10, 20 or 50 g 2-OHB g^–1 soil produced a general increase in total soil bacterial population density, whereas the addition of 100 g or 200 g 2-OHB g^–1 soil specifically increased the proportion of naphthalene degraders relative to the total population. The addition of 50 g 2-OHB g^–1 soil produced a fourfold increase (the maximum observed) in the rate of naphthalene mineralization relative to the rate in unamended soil. The concentration of 2-OHB ( 100 g/g) added to soil correlated with the population density of naphthalene degraders (r=0.961). Addition of up to 200 g 2-OHB g^–1 correlated with the abundance of DNA sequences homologous to known naphthalene dioxygenase genes (nahAB) (r=0.958). However, mineralization of [¹⁴C]naphthalene was stimulated significantly only by the addition of 50 g 2-OHB g^–1 soil. Results of the mineralization experiments were supported by the detection of nahAB mRNA extracted directly from soil. The specificity of the effect of 2-OHB on naphthalene biodegradation was confirmed in a control experiment using equivalent concentrations of 4-OHB which repressed naphthalene mineralization by about 50%. Addition of ammonium nitrate to the soil also increased the rate of naphthalene mineralization. Ammonium nitrate added together with 2-OHB reduced the mineralization enhancement effect of either compound alone. The study confirmed that specific induction of biodegradative genes can enhance chemical pollutant removal in situ. Correspondence to: O. A. Ogunseitan