Studies on fructosyl transferase from Agave americana

The possible role of fructosyl transferase in the biosynthesis of fructosans in Agave americana was investigated. This enzyme was extracted from A. americana stem and purified 17.5-fold by salt fractionation and DEAE-cellulose chromatography. The optimum conditions for the enzyme were pH 6. 1, temperature 37°, substrate concentration 20% and K_m 3.6 × 10^?1 M; Ag⁺, Pb ²⁺, Hg²⁺, Al³⁺, Sn²⁺, CN^? acted as inhibitors and Ca²⁺, Mg²⁺, Co²⁺ and Li⁺ actemd as activators. Only sugars of the type F ～ R (R-aidose), e.g. sucrose and raffinose acted as substrates for the enzyme. The donor acceptor specificity of the enzyme was studied extensively. Sugars sucrose. None of the intermediates of fructosan biosynthesis from sucrpse acted as fructose donors. The possible acceptors from sucrose and raffinose. The enzyme was capable of building up oligosaccharides up to FIOG from sucrose. None of the intermediates of fructosan biosynthesis from sucrose acted as fructose donors. The possible mechanism of fructosan biosynthesis from sucrose is discussed.     

Enhancement of Terpenoid Biosynthesis from Mevalonate in a Fraction of the Latex from Euphorbia lathyris

A latex pellet fraction from Euphorbia lathyris incorporates mevalonate into triterpenols and their fatty acid esters. Conditions for improved incorporation were determined. CaCl₂ or CaCl₂ plus MnCl₂ stimulated biosynthesis, and the metal ion chelator, ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) enhanced stimulation. Ethylenediaminetetraacetic acid was almost as effective as EGTA, but phthalic acid and citric acid were relatively poor stimulators. The concentration of the Ca²⁺-EGTA complex was directly measured, and the incorporation data are best fitted by a curve that shows that the receptor for the complex is saturable. In the presence of the metal-chelate complex, the addition of fructose, 1,6-bisphosphate plus aldolase (triose-P) or malate provided additional stimulation. Incorporation was maximum at 40 micromolar R-mevalonate, and inhibition occurred at higher concentrations. The apparent K_m for R-mevalonate was 15 micromolar. Under improved reaction conditions, the rate of triterpenoid biosynthesis from mevalonate is 25 times faster than was previously observed (GJ Piazza, EJ Saggese, KM Spletzer [1987] Plant Physiol 83: 177-180).     

Properties of phenylalanine ammonia-lyase extracted from Cucumis sativus hypocotyls

Some of the in vitro properties of PAL from gherkin hypocotyls were investigated. No metal ion requirement for this enzyme could be demonstrated but there were indications that PAL was a sulphydryl enzyme. Kinetic analysis suggested that PAL exhibited negative homotropic cooperativity. Two K_m values were determined, these were K_m^H, 2·9 × 10^?4 M and K_m^L, 4·3 × 10^?5 M. Strong inhibition of the enzyme was exerted by d-phenylalanine, trans-cinnamic acid, o-coumaric acid, gallic acid, quercetin and kaempferol. Kinetic studies on the inhibition patterns of these compounds established that d-phenylalanine linearized the curvilinear kinetics, trans-cinnamic acid and o-coumaric acid acted as competitive inhibitors whilst gallic acid, quercetin and kaempferol acted as mixed inhibitors. Using a number of desensitization techniques PAL was partially desensitized to inhibition by the mixed inhibitors. These results led to the conclusion that PAL may have a regulatory role in phenol, coumarin and flavonoid biosynthesis.     

Effects of environmental conditions on isoprene emission from live oak

Live-oak plants (Quercus virginiana Mill.) were subjected to various levels of CO₂, water stress or photosynthetic photon flux density to test the hypothesis that isoprene biosynthesis occurred only under conditions of restricted CO₂ availability. Isoprene emission increases as the ambient CO₂ concentration decreased, independent of the amount of time that plants had photosynthesized at ambient CO₂ levels. When plants were water-stressed over a 4-d period photosynthesis and leaf conductance decreased 98 and 94%, respectively, while isoprene emissions remained constant. Significant isoprene emissions occurred when plants were saturated with CO₂, i.e., below the light compensation level for net photosynthesis (100 mol m^-2 s^-1). Isoprene emission rates increased with photosynthetic photon flux density and at 25 and 50 mol m^-2 s^-1 were 7 and 18 times greater than emissions in the dark. These data indicate that isoprene is a normal plant metabolite and not — as has been suggested — formed exclusively in response to restricted CO₂ or various stresses.Abbreviation PPFD photosynthetic photon flux density     

Plastid-associated Porphobilinogen Synthase from Toxoplasma gondii: KINETIC AND STRUCTURAL PROPERTIES VALIDATE THERAPEUTIC POTENTIAL*

Apicomplexan parasites (including Plasmodium spp. and Toxoplasma gondii) employ a four-carbon pathway for de novo heme biosynthesis, but this pathway is distinct from the animal/fungal C4 pathway in that it is distributed between three compartments: the mitochondrion, cytosol, and apicoplast, a plastid acquired by secondary endosymbiosis of an alga. Parasite porphobilinogen synthase (PBGS) resides within the apicoplast, and phylogenetic analysis indicates a plant origin. The PBGS family exhibits a complex use of metal ions (Zn²⁺ and Mg²⁺) and oligomeric states (dimers, hexamers, and octamers). Recombinant T. gondii PBGS (TgPBGS) was purified as a stable ∼320-kDa octamer, and low levels of dimers but no hexamers were also observed. The enzyme displays a broad activity peak (pH 7–8.5), with a K_m for aminolevulinic acid of ∼150 μm and specific activity of ∼24 μmol of porphobilinogen/mg of protein/h. Like the plant enzyme, TgPBGS responds to Mg²⁺ but not Zn²⁺ and shows two Mg²⁺ affinities, interpreted as tight binding at both the active and allosteric sites. Unlike other Mg²⁺-binding PBGS, however, metal ions are not required for TgPBGS octamer stability. A mutant enzyme lacking the C-terminal 13 amino acids distinguishing parasite PBGS from plant and animal enzymes purified as a dimer, suggesting that the C terminus is required for octamer stability. Parasite heme biosynthesis is inhibited (and parasites are killed) by succinylacetone, an active site-directed suicide substrate. The distinct phylogenetic, enzymatic, and structural features of apicomplexan PBGS offer scope for developing selective inhibitors of the parasite enzyme based on its quaternary structure characteristics.     

The cytosolic isoenzyme of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase in Spinacia oleracea and other higher plants: extreme substrate ambiguity and other properties

The cytosolic isoenzyme of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS-Co: EC 4.1.2.15) in Spinacia oleracea, Solanum tubersosum and many other higher plants was found to use a diversity of substrates. Diose (glycolaldehyde), triose (D-glyceraldehyde, L-glyceraldehyde and DL-glyceraldehyde 3-phosphate), tetrose (D-erythrose, L-erythrose, D-erythrose 4-phosphate, D-threose and L-threose), and pentose (D-ribose 5-phosphate and D-arabinose 5-phosphate) were utilized in combination with phosphoenolpyruvate (PEP) to make the corresponding 2-keto-3-deoxy sugar acids. Glyoxylate was also utilized by DS-Co. Glycoladehyde exhibited the highest reaction velocity when substrates were tested at 3 mM concentrations. Pentoses were poor substrates except when phsophorylated, an effect which is probably due to an increased fraction of the molecules being in the open-chain form. Little stereoselective discrimination exists since comparable velocities were demonstrated with the D and L isomers of glyceraldehyde, erythrose or threose. The enzyme is not a reversible aldolase since pyruvate failed to substitute for PEP. The use of D-erythrose 4-phsophate or glycolaldehyde resulted in K_m values of 1.95 mM and 8.60 mM, respectively. However, glycolaldehyde exhibited the largest V_maxK_m ratio, suggesting a greater catalytic efficiency for this substrate. Glycolaldehyde is an ideal substrate for inexpensive assays of DS-Co that are absolutely selective in the presence of two other plant enzymes which also utilize erythrose 4-phosphate and PEP. The spinach DS-Co enzymes required divalent metals for activity. The presence of 20 mM Mg²⁺, 1 mM Co²⁺ and 1 mM Mn²⁺ yielded relative activities of 100, 70 and 15, respectively. The pH optimum was 9.5 and temperature optimum for activity was 49°C. The molecular masses of DS-Co from spinach, tobacco and pea were all in the range of 400 kDa. The possible roles of DS-Co in biosynthesis of α-ketoglutarate and aromatic amino acids, in biosynthesis of components of cell wall and phytotoxin, and in acting as a sink for 2-and 3-carbon sugars are discussed.     

Purification and Properties of Pantothenate Kinase from Brevibacterium ammoniagenes IFO 12071

Pantothenate kinase (ATP: pantothenate 4′-phosphotransferase, EC 2.7.1.33) was purified about 200-fold from the cell extract of Brevibacterium ammoniagenes IFO 12071 by ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephadex G-150 gel filtration. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular weight was calculated approximately 45,000. The enzyme catalyzed the formation of pantothenic acid 4′-phosphate and ADP from pantothenate and ATP in the presence of Mg²⁺ ATP could be substituted for, partly, by ITP, GTP, and UTP. The enzyme phosphorylated not only pantothenate, but also pantothenoylcysteine, pantetheine, and pantothenyl alcohol. Apparent Km values were 6.7×10^?5 m for pantothenate, 3.5×10^?5 m for ATP, and 10^?3 m for Mg²⁺. The reaction was inhibited by the intermediates of CoA biosynthesis, of which CoA itself was a most effective inhibitor. Other properties of the enzyme were also investigated.     

Purification and properties of UDP-glucose: coniferyl alcohol glucosyltransferase from suspension culturesof Paul's scarlet rose.

UDP-glucose:coniferyl alcohol glucosyltransferase was isolated from 10-day-old, darkgrown cell suspension cultures of Paul's scarlet rose. The enzyme was purified 120-fold by (NH₄)₂SO₄ fractionation and chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-100. The enzyme has a pH optimum of 7.5 in Tris-HCl buffer, required an -SH group for activity, and is inhibited by ?-chloromercuribenzoate and EDTA. Its molecular weight is estimated to be 52,000. The enzyme is specific for the glucosylation of coniferyl alcohol (K_m 3.3 × 10^?6 M) and sinapyl alcohol (K_m 5.6 × 10^?6 M). With coniferyl alcohol as substrate the apparent K_m value for UDP-glucose is 2 × 10^?6m. The enzyme activity can be detected in a number of callus-tissue and cell-suspension cultures. The role of this enzyme is believed to be to catalyze the transfer of glucose from UDPG to coniferyl (or sinapyl) alcohol as storage intermediates in lignin biosynthesis.     

EDTA activation of H2 photoproduction by Rhodospirillum rubrum

Summary The effect of trace elements (Fe, Ni) and chelating compounds on the activity of hydrogen (H₂) uptake (Hup) hydrogenase, nitrogenase and rate and yield of H₂ photoproduction from l-lactate in photosynthetic cultures of Rhodospirillum rubrum was investigated. Hup activity depended on the availability of Ni²⁺ and was inhibited by EDTA (0.3–0.5 mm ethylenedinitrilotetraacetic acid). Addition of EDTA (0.5 mm) to the culture medium caused a nearly complete inactivation of Hup activity and activation of nitrogenase, which was paralleled by a threefold increase in total H₂ photoproduced from lactate. Hup^– mutants, isolated by transposon Tn5 mutagenesis, produced maximally twofold more H₂ than the wild-type. Experiments with different chelating agents [EDTA, NTA (nitrilotriacetic acid), citrate, isocitrate] and varying concentrations of Fe²⁺ and Fe³⁺ showed that photosynthetic growth and nitrogenase activity of R. rubrum were strongly influenced by the iron supply. It is concluded that EDTA enhanced H₂ photoproduction by (I) inhibition of biosynthesis of Hup hydrogenase and (II) mobilization of iron, thereby activating the biosynthesis of the nitrogenase complex. Correspondence to: M. Kern     

Nitrogen regulation of gibberellin biosynthesis in Gibberella fujikuroi

Summary In Gibberella fujikuroi, ammonium (NH₄ ⁺) interfered with the production of gibberellic acid (GA₃). Optimal production occurred at 19 mm (NH₄)₂SO₄ and the synthesis of GA₃ was reduced threefold in a medium with 38 mm (NH₄)₂SO₄. Using a resting cell system with mycelia previously grown on two concentrations (19 mm and 38 mm) of (NH₄)₂SO₄, it was found that NH₄ ⁺ depressed synthesis of the gibberellin-synthesizing enzymes. Furthermore, addition of NH₄ ⁺ to a producing system shut off gibberellin formation, indicating that the negative effect of NH₄ ⁺ ions is also due to inhibition of one or more enzymes in the gibberellin biosynthesis pathway. The onset of gibberellin biosynthesis in media with high (38 mm) and low (19 mm) concentrations of (NH₄)₂SO₄ was studied by addition of cycloheximide to batch cultures of various ages. Offprint requests to: B. Brückner     

Calcium-dependent Properties of Peptidylarginine Deiminase from Rabbit Skeletal Muscle

Peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, required Ca²⁺ as an essential cofactor and the half-maximal activity was attained at 40—60 μm Ca²⁺. Other divalent cations were practically inactive except for Sr²⁺, which was about 50% as active as Ca²⁺ when tested at 10 mm. However, Sr²⁺ at less than the concentration of 100 μm had little or no activity. The direct Ca²⁺-binding for the enzyme showed a sigmoidal curve with a transition midpoint of about 110 μm, indicating that the binding is cooperative. Analysis of Hill plots of the data revealed that the enzyme binds 3 mol of Ca²⁺/mol of protein with an apparent dissociation constant of llO μm. A conformational change upon Ca²⁺-binding was also described for the enzyme using UV-difference spectra. The alteration could be attributed to an increased exposure of the aromatic residues to a more aqueous environment, as has been described for Ca²⁺-binding proteins such as calmodulin. Phosphatidylserine enhanced the reaction velocity and concomitantly reduced the Ca²⁺-requirement for the enzyme. These effects were stimulated by the addition of diacylglycerol. Diacylglycerol alone had little or no effect. On the other hand, calmodulin had no effect on the enzymatic activity over a wide range of Ca²⁺ concentrations. These suggest that the activity and Ca²⁺-sensitivity of peptidylarginine deiminase is increased at the cell membrane.     

Biochemical Characterization of the Pneumococcal Glucose 1-Phosphate Uridylyltransferase (GalU) Essential for Capsule Biosynthesis

The glucose 1-phosphate uridylyltransferase (GalU) is absolutely required for the biosynthesis of capsular polysaccharide, the sine qua non virulence factor of Streptococcus pneumoniae. The pneumococcal GalU protein was overexpressed in Escherichia coli, and purified. GalU showed a pI of 4.23, and catalyzed the reversible formation of UDP-glucose and pyrophosphate from UTP and glucose 1-phosphate with K_m values of 0.4 mM for UDP-glucose, 0.26 mM for pyrophosphate, 0.19 mM for glucose 1-phosphate, and 0.24 mM for UTP. GalU has an optimum pH of 8–8.5, and requires Mg²⁺ for activity. Neither ADP-glucose nor TDP-glucose is utilized as substrates in vitro. The purification of GalU represents a fundamental step to provide insights on drug design to control the biosynthesis of the main pneumococcal virulence factor.     

PAR modulation of the UV-dependent levels of flavonoid metabolites in Arabidopsis thaliana (L.) Heynh. leaf rosettes: cumulative effects after a whole vegetative growth period

Long-term effects of ultraviolet (UV) radiation on flavonoid biosynthesis were investigated in Arabidopsis thaliana using the sun simulators of the Helmholtz Zentrum München. The plants, which are widely used as a model system, were grown (1) at high photosynthetically active radiation (PAR; 1,310 µmol m^?2?s^?1) and high biologically effective UV irradiation (UV-B_BE 180 mW m^?2) during a whole vegetative growth period. Under this irradiation regime, the levels of quercetin products were distinctively elevated with increasing UV-B irradiance. (2) Cultivation at high PAR (1,270 µmol m^?2?s^?1) and low UV-B (UV-B_BE 25 mW m^?2) resulted in somewhat lower levels of quercetin products compared to the high-UV-B_BE conditions, and only a slight increase with increasing UV-B irradiance was observed. On the other hand, when the plants were grown (3) at low PAR (540 µmol m^?2?s^?1) and high UV-B (UV-B_BE 180 mW m^?2), the accumulation of quercetin products strongly increased from very low levels with increasing amounts of UV-B but the accumulation of kaempferol derivatives and sinapoyl glucose was less pronounced. We conclude (4) that the accumulation of quercetin products triggered by PAR leads to a basic UV protection that is further increased by UV-B radiation. Based on our data, (5) a combined effect of PAR and different spectral sections of UV radiation is satisfactorily described by a biological weighting function, which again emphasizes the additional role of UV-A (315–400 nm) in UV action on A. thaliana.     

Application of an airlift bioreactor to the nystatin biosynthesis

Pilot plant studies were performed using a concentric-tube airlift bioreactor of 2.5 m³ fermentation volume. The results have proven the relative merits of such a system in the biosynthesis of nystatin, produced by Streptomyces noursei, in submerged aerobic cultivation and batch operation mode. The results were compared to those obtained in a pilot-scale stirred tank bioreactor of 3.5 m³ fermentation volume. The fermentation processes in the two fermentation devices were similar with respect to substrate utilization, biomass production and nystatin biosynthesis. In the riser section, the dissolved oxygen concentration was higher than that in the downcomer. The volumetric oxygen mass transfer coefficient was dependent on the rheological behaviour of the biosynthesis liquids, which was not constant during the fermentation process. The total energy consumption for nystatin production in the airlift bioreactor was 56% of that in the stirred tank, while the operating costs represented 78% of those in the stirred tank bioreactor.     

Tryptophan decarboxylase from Catharanthus roseus cell suspension cultures: purification,molecular and kinetic data of the homogenous protein

The purification of tryptophan decarboxylase from Catharanthus roseus (TDC, E.C.:4.1.1.27), to apparent homogeneity, is described. The enzyme represents a soluble protein with a molecular weight of 115 000±3 000, consisting of 2 identical subunits of 54 000±1 000. The pI was estimated to be 5.9 and the K_m for L-tryptophan was found to be 7.5×10^-5 M. Phenylalanine, tyrosine and DOPA were not decarboxylated by tryptophan decarboxylase from Catharanthus cells. Similar to the aromatic amino acid decarboxylase from hog kidney the enzyme does not appear to be obligatorily dependent on exogenously supplied pyridoxal phosphate, as it seems to contain a certain amount of this cofactor. The average percentage of TDC in the cells was found to be 0.002% in the growth medium while the level increased up to 0.03% when indole alkaloid biosynthesis was induced. The role of the protein as a bottleneck enzyme of indole alkaloid biosynthesis is discussed.     

Regulation of hormone biosynthesis in cultured islet cells from anglerfish

Summary The effects of glucose and arginine on islet hormone biosynthesis were investigated using primary cell cultures prepared from islets of the anglerfish (Lophius americanus). After dispersion under sterile conditions, islet cells were maintained at 23° C in medium containing RPMI 1640 with Hanks' buffer, pH 7.5, modified by the adjustment of glucose (to 0.56 or 5.6 mM) and arginine (to 0.1, 1.15, or 10 mM) with the addition of 10% fetal bovine serum (dialyzed, heat inactivated) and penicillin/streptomycin. After 48 h, media were replaced by incorporation media containing [¹⁴C]isoleucine and [³H]tryptophan and incubated for an additional 8 h under otherwise identical conditions. Culture samples (cells plus media) were extracted, desalted, and gel filtered to identify and quantitate [¹⁴C]insulin, [³H]glucagon(s) plus [³H]somatostatin-28, and [³H]somatostatin-14 were In some experiments, [¹⁴C]insulin, [³H]glucagon(s), [³H]somatostatin-28, and [³H]somatostatin-14 were separated by high performance liquid chromatography. Raising the medium glucose from 0.56 (control) to 5.6 mM resulted in an augmentation in incorporation of [¹⁴C]isoleucine into insulin and an augmentation of [³H]tryptophan into glucagon(s) and somatostatin-14, but no change in incorporation of [³H]tryptophan into somatostatin-28. Raising the concentration of arginine from 0.1 to 1.15 or 10 mM resulted in a dose-dependent inhibition of labeled amino acid incorporation into all hormones except somatostatin-28. The results demonstrate the usefulness of the culture system for studying the modulation of hormone biosynthesis in anglerfish islet cells. This work was supported by Grants AM 16921 and AM 26378 from the National Institutes of Health, Bethesda, MD.     

Two forms of phosphoinositol kinase from germinating mung bean seeds

Phosphoinositol kinase, the key enzyme responsible for the biosynthesis of higher inositol phosphates has been isolated from the cotyledons of mung beans germinated for 24 hr and has been resolved into two different forms, phosphoinositol kinase A and phosphoinositol kinase B. Both forms were purified to homogeneity and characterized. The K_m values for ATP with phosphoinositol kinase A (1.78 × 10^?4 M) and phosphoinositol kinase B (3.12 × 10 ^?5 M) showed that phosphoinositol kinase B had a greater affinity for ATP. ATP could be partially replaced as phosphate donor by UTP and phosphoenolpyruvate in the case of phosphoinositol kinase A but not in the case of phosphoinositol kinase B.     

Enhancement of phenylpropanoid enzymes and lignin in Phalaenopsis orchid and their influence on plant acclimatisation at different levels of photosynthetic photon flux

Effects of three levels of photosynthetic photon flux (PPF: 60, 160 and 300 μmol m⁻²s⁻¹) were investigated in one-month-old Phalaenopsis plantlets acclimatised ex vitro. Optimal growth, chlorophyll and carotenoid concentations, and a high carotenoid:chlorophyll a ratio were obtained at 160 μmol m⁻²s⁻¹, while net CO₂ assimilation (A), stomatal conductance (g), transpiration rate (E) and leaf temperature peaked at 300 μmol m⁻²s⁻¹, indicating the ability of the plants to grow ex vitro. Adverse effects of the highest PPF were reflected in loss of chlorophyll, biomass, non-protein thiol and cysteine, but increased proline. After acclimatisation, glucose-6-phosphate dehydrogenase, shikimate dehydrogenase, phenylalanine ammonia-lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD) increased, as did lignin. Peroxidases (POD), which play an important role in lignin synthesis, were induced in acclimatised plants. Polyphenol oxidase (PPO) and β-glucosidase (β-GS) activities increased to a maximum in acclimatised plants at 300 μmol m⁻²s⁻¹. A positive correlation between PAL, CAD activity and lignin concentration was observed, especially at 160 and 300 μmol m⁻²s⁻¹. The study concludes that enhancement of lignin biosynthesis probably not only adds rigidity to plant cell walls but also induces defence against radiation stress. A PPF of 160 μmol m⁻²s⁻¹was suitable for acclimatisation when plants were transferred from in vitro conditions.     

Use of carbon-11 in Populus shows that exogenous jasmonic acid increases biosynthesis of isoprene from recently fixed carbon

A new approach for pulse labelling of plants using the short-lived positron emitting radioisotope carbon-11 (half-life: 20.4 min) as ¹¹CO₂ is reported together with its application to measuring [¹¹C]isoprene emissions from intact leaves capturing information associated with: (1) rate of emission; (2) the relative contribution of recently fixed carbon to isoprene biosynthesis; and (3) the transit time for tracer movement through the leaf and biochemical pathways associated with isoprene biosynthesis. This approach was applied to study the response of certain Populus species to exogenous treatments of jasmonic acid (JA), a plant hormone implicated in signal transduction linked to defence response against herbivory. Twelve hours after treatment of single intact leaves of aspen (Populus tremuloides) with a 1 m m JA spray, isoprene emissions from those leaves increased 1.5 times the controls from 35.4 ± 2.2 to 53.1 ± 4.8 nmol m⁻² s⁻¹. [¹¹C]Isoprene emissions from the same leaves, reflecting the isoprene that was derived from recently fixed carbon, increased much more, to 2.2 times the controls. This increase coincided with a change in emitted [¹¹C]isoprene from 0.31 to 0.68% of ¹¹C fixed in the leaf tissue, while the tracer transit time remained constant at 12.5 min. These results suggest that JA had no effect on enzyme kinetics involved in isoprene biosynthesis, but did impact the source of recent carbon feeding that pathway. Studies with poplar (Populus nigra clone NC 5271) showed similar trends in systemic emissions (from an untreated leaf on the same plant).     

Enzymic coupling of acylhydrolase and prostaglandin synthase activities in subcellular fractions from rabbit renal medulla

We have recently shown that mitochondrial and plasma-membrane fractions from kidney medulla possess Ca²⁺-stimulated acylhydrolase and prostaglandin synthase activities. The nature of the enzymic coupling between the Ca²⁺-stimulated arachidonic acid release and its subsequent conversion into prostaglandins was investigated in subcellular fractions from rabbit kidney medulla. Plasma-membrane, mitochondrial and microsomal fractions were found to have similar apparent K_m values for conversion of added exogenous arachidonate into prostaglandins. The rate of prostaglandin biosynthesis (V_max.) from added arachidonic acid in the microsomal fraction was approx. 2-fold higher than in the other subcellular fractions. In contrast, prostaglandin E₂ synthesis from endogenous arachidonate in plasma-membrane and mitochondrial fractions was 3–4-fold higher than in microsomes. Furthermore, Ca²⁺ stimulated endogenous arachidonate deacylation and prostaglandin E₂ generation in the former two fractions but not in microsomes. In mitochondrial or crude plasma-membrane fractions, in which prostaglandin biosynthesis was inhibited with aspirin, arachidonate released from these fractions was converted into prostaglandins by the microsomal prostaglandin synthase. Thus an intracellular prostaglandin generation process that involves inter-fraction transfer of arachidonic acid can operate. Prostaglandin generation by such an inter-fraction process is, however, less efficient than by an intra-fraction process, where arachidonic acid released by mitochondria or crude plasma membranes is converted into prostaglandins by prostaglandin synthase present in the same fraction. This demonstrates the presence of a tight intra-fraction enzymic coupling between Ca²⁺-stimulated acylhydrolase and prostaglandin synthase enzyme systems in both mitochondrial and plasma-membrane fractions.