Chemometric treatment of vanillin fingerprint chromatograms. Effect of different signal alignments on principal component analysis plots.

This study describes the chemometric treatment of vanillin fingerprint chromatograms to distinguish vanillin from different sources. Prior to principal component analysis, which is used to discriminate vanillin from different origins, the fingerprints are aligned. Three alignment algorithms are tested, correlation optimized warping (COW), target peak alignment (TPA) and semi-parametric time warping (STW). The performance of the three algorithms is evaluated and the effect of the different alignments on the PCA score plots is investigated. The alignment obtained with STW differs somewhat from that with COW and TPA. However, equivalent score plots were obtained regarding the different vanillin groups.

Novel approaches to the biosynthesis of vanillin.

Microorganisms able to produce vanillin in excess of 6g/l from ferulic acid have now been isolated. In Pseudomonas strains, the metabolic pathway from eugenol via ferulic acid to vanillin has been characterised at the enzymic and molecular genetic levels. Attempts to introduce vanillin production into other organisms by genetic engineering have begun.

An opsonised microelectrode. Observation of the respiratory burst of a single human neutrophil.

A 10 micron diameter gold microvoltammetric electrode, opsonised with human IgG, was used to study the respiratory burst of a single human neutrophil. The electrode oxidised superoxide produced near its surface by the neutrophil back to dioxygen. It is suggested that the current so detected is proportional to the rate of superoxide production by the NADPH oxidase of a single cell. In all cases the response consisted of a relatively rapid rise in current after cell addition, followed by a 2-phase decay. It is further suggested that this complex decay results from the production of superoxide being rate-limited initially by the NADPH concentration and later by the coupled metabolism of the hexose monophosphate shunt.

Electrochemically driven respiration in mitochondria and Paracoccus denitrificans. The coupling of the electrochemistry of horse heart cytochrome c with respiration in mitochondria and a model thereof, Paracoccus denitrificans.

By exploiting the rapid, direct electrochemistry of horse heart cytochrome c at a modified gold electrode it has been possible to couple the electrode reaction with respiration in rat liver mitochondria and in protoplasts of Paracoccus denitrificans, but not in protoplasts from E. coli. Oxidation of endogenous and exogenous sources of reducing equivalents via cytochrome c is also observed.

An opsonised electrode. The direct electrochemical detection of superoxide generated by human neutrophils.

A pyrolytic graphite electrode was surface modified with human IgG and used as a stimulus to elicit a respiratory burst from human neutrophils. The oxidation current observed was shown to be due to re-oxidation of superoxide produced by the neutrophils. Both superoxide dismutase and N-ethylmaleimide were effective inhibitors of the oxidation current.

Specificities of the enzymes of N-alkyltropane biosynthesis in Brugmansia and Datura.

The enzymes N-methylputrescine oxidase (MPO), the tropine-forming tropinone reductase (TRI), the pseudotropine-forming tropinone reductase (TRII), the tropine:acyl-CoA transferase (TAT) and the pseudotropine:acyl-CoA transferase (PAT) extracted from transformed root cultures of Datura stramonium and a Brugmansia candida x aurea hybrid were tested for their ability to accept a range of alternative substrates. MPO activity was tested with N-alkylputrescines and N-alkylcadaverines as substrates. TRI and TRII reduction was tested against a series of N-alkylnortropinones, N-alkylnorpelletierines and structurally related ketones as substrates. TAT and PAT esterification tests used a series of N-substituted tropines, pseudotropines, pelletierinols and pseudopelletierinols as substrates to assess the formation of their respective acetyl and tigloyl esters. The results generally show that these enzymes will accept alien substrates to varying degrees. Such studies may shed some light on the overall topology of the active sites of the enzymes concerned.

The measurement of changes in pH associated with electrochemically driven respiration in rat liver mitochondria.

Using an optically transparant thin layer electrode, it has been possible to measure the pH changes associated with the electrochemical turnover of horse heart cytochrome c in the presence of rat liver mitochondria and oxygen. Direct electrochemistry of cytochrome c at a gold electrode modified with bis(4-pyridyl)bisulfide allowed electron flux (current) to be measured simultaneously with the differential change in absorbance associated with phenol red, a pH-sensitive dye. Although the alkalinization due to the reduction of oxygen to water was readily observed, any initial acidification associated with proton pumping was not detected. It is suggested that at the high ratios of oxidized-to-reduced cytochrome c present during the steady state attained, proton pumping may be absent or more localized.

Glyoxylate decarboxylation during glycollate oxidation by pea leaf extracts: significance of glyoxylate and extract concentrations.

Hydrogen peroxide-dependent glyoxylate decarboxylation occurring during glycollate oxidation by pea leaf extracts (Pisum sativum L.) has been studied in relation to the effects of glyoxylate and extract concentration. With a saturating concentration of glycollate, decarboxylation was greatly stimulated by raising the glyoxylate concentration; at 30°C and with approx. 0.04 nkat of glycollate oxidase (as leaf extract) in the reaction mixture, CO2 release in the presence of 5 mM glycollate and 5 mM glyoxylate was equal to about 45% of glycollate oxidation. However, CO2 release at these substrate concentrations was not linearly proportional to the amount of extract supplied and was equal to a diminishing proportion of glycollate oxidation as the amount of extract was increased. This was shown to be due to the low affinity of catalase for H2O2, so that the endogenous catalase was able to destroy a larger proportion of the H2O2 generated at higher extract concentrations. It is argued that although at high glycoxylate concentrations (5-10 mM) in vitro, glyoxylate decarboxylation can be made to equal more than a third of the glycollate oxidised, less than 10% of the glyoxylate generated in vivo is likely to be decarboxylated in peroxisomes where high concentrations of glycollate oxidase and catalase are localised and where high concentrations of glyoxylate are unlikely to be maintained.

The effect of cadaverine on the formation of anabasine from lysine in hairy root cultures of Nicotiana hesperis.

Unlabelled cadaverine did not diminish the incorporation into anabasine of (14)C from L-[U-(14)C] lysine supplied to hairy root cultures of Nicotiana nesperis, despite causing a stimulation of anabasine production. The finding is discussed in the context of previous observations indicating that free cadaverine is not an intermediate in the biosynthesis of anabasine from lysine.

Metabolism and decarboxylation of glycollate and serine in leaf peroxisomes.

The linked utilization of glycollate and L-serine has been studied in peroxisomal preparations from leaves of spinach beet (Beta vulgaris L.). The generation of glycine from glycollate was found to be balanced by the production of hydroxypyruvate from serine and similarly by 2-oxoglutarate when L-glutamate was substituted for L-serine. In the presence of L-malate and catalytic quantities of NAD(+), about 40% of the hydroxypyruvate was converted further to glycerate, whereas with substrate quantities of NADH, this conversion was almost quantitative. CO2 was released from the carboxyl groups of both glycollate and serine. Since the decarboxylation of both substrates was greatly in creased by the catalase inhibitor, 3-amino-1,2,4-triazole, and abolished by bovine liver catalase, it was attributed to the nonenzymic attack of H2O2, generated in glycollate oxidation, upon glyoxylate and hydroxypyruvate respectively. At 25-30° C, about 10% of the glyoxylate and hydroxypyruvate accumulated was decarboxylated, and the release of CO2 from each keto-acid was related to the amounts present. It is suggested that hydroxypyruvate decarboxylation might contribute significantly to photorespiration and provide a metabolic route for the complete oxidation of glycollate, the magnitude of this contribution depending upon the concentrations of glyoxylate and hydroxypyruvate in the peroxisomes.

Glutamate and serine as competing donors for amination of glyoxylate in leaf peroxisomes.

When provided with glycollate, peroxisomal extracts of leaves of spinach beet (Beta vulgaris L. cv.) converted L-serine and L-glutamate to hydroxypyruvate and 2-oxoglutarate respectively. When approximately saturating concentrations of each of these amino acids were incubated separately with glycollate, the utilization of serine was greater than that of glutamate. The utilization of glutamate was substantially reduced by the presence of relatively low concentrations of serine in the reaction mixture, whereas even high concentrations of glutamate caused only small reductions in serine utilization. Over the entire range of concentrations of amino acids examined, serine was invariably the preferred amino-group donor, but this preference was abolished at higher concentrations of glyoxylate. Serine not only competed favourably for glyoxylate but also inhibited L-glutamate: glyoxylate aminotransferase (GGAT), the degree of inhibition depending upon the glyoxylate concentration. Studies of L-serine: glyoxylate aminotransferase (SGAT) and GGAT in partially purified extracts from spinach-beet leaves confirmed that serine competitively inhibited GGAT but glutamate did not affect SGAT. Both enzymes were inhibited by high glyoxylate concentrations, the inhibition being relieved by suitably high concentrations of the appropriate amino acid. It is concluded that at the low glyoxylate concentrations likely to occur in vivo, the preferential utilization of serine would ensure flux through the glycollate pathway to glycerate, but at higher concentrations of glyoxylate, both enzymes could be fully active in glyoxylate amination.

Enzymes of serine and glycine metabolism in leaves and non-photosynthetic tissues of Pisum sativum L.

A comparative study is presented of the activities of enzymes of glycine and serine metabolism in leaves, germinated cotyledons and root apices of pea (Pisum sativum L.). Data are given for aminotransferase activities with glyoxylate, hydroxypyruvate and pyruvate, for enzymes associated with serine synthesis from 3-phosphoglycerate and for glycine decarboxylase and serine hydroxymethyltransferase. Aminotransferase activities differ between the tissues in that, firstly, appreciable transamination of serine, hydroxypyruvate and asparagine occurs only in leaf extracts and, secondly, glyoxylate is transaminated more actively than pyruvate in leaf extracts, whereas the converse is true of extracts of cotyledons and root apices. Alanine is the most active amino-group donor to both glyoxylate and hydroxypyruvate. 3-Phosphoglycerate dehydrogenase and glutamate: O-phosphohydroxypyruvate aminotransferase have comparable activities in all three tissues, except germinated cotyledons, in which the aminotransferase appears to be undetectable. Glycollate oxidase is virtually undetectable in the non-photosynthetic tissues and in these tissues the activity of glycerate dehydrogenase is much lower than that of 3-phosphoglycerate dehydrogenase. Glycine decarboxylase activity in leaves, measured in the presence of oxaloacetate, is equal to about 30-40% of the measured rate of CO2 fixation and is therefore adequate to account for the expected rate of photorespiration. The activity of glycine decarboxylase in the non-photosynthetic tissues is calculated to be about 2-5% of the activity in leaves and has the characteristics of a pyridoxal-and tetrahydrofolate-dependent mitochondrial reaction; it is stimulated by oxaloacetate, although not by ADP. In leaves, the measured activity of serine hydroxymethyltransferase is somewhat lower than that of glycine decarboxylase, whereas in root apices it is substantially higher. Differential centrifugation of extracts of root apices suggests that an appreciable proportion of serine hydroxymethyltransferase activity is associated with the plastids.

Effects of illumination and glycollate oxidation in promoting glyoxylate decarboxylation by pea leaf protoplasts.

During glycollate oxidation, glyoxylate was decarboxylated by pea leaf protoplasts. The characteristics of the reaction were similar to those of the reaction in leaf extracts (Walton, 1982, Planta 155, 218-224). Glyoxylate decarboxylation was not promoted by illumination of the protoplasts.

Use of an electrochemical technique to study plasmamembrane redox reactions in cultured cells of Daucus carota L.

By exploiting the electron transfer reactions of ferricyanide-ferrocyanide at a gold electrode, an electrochemical technique was devised and successfully employed for the measurement of extracellular ferricyanide reduction or ferrocyanide oxidation by carrot (Daucus carota L.) cells grown in suspension culture. This technique eliminated problems of cell damage and insensitivity encountered when these activities are measured spectrophotometrically in magnetically stirred cell suspensions. Cells harvested from the mid-exponential phase of culture growth catalysed a rapid reduction of ferricyanide which was accompanied by H(+) extrusion and was stimulated by ethanol. These cells also oxidised ferrocyanide, which was not associated with H(+) extrusion. These observations can be explained on the basis of a plasmamembrane located, H(+)-translocating redox system. The electrochemical technique is a useful method of studying this system.

Enzymes of N-methylputrescine biosynthesis in relation to hyoscyamine formation in transformed root cultures of Datura stramonium and Atropa belladonna.

The activities of enzymes related to the biosynthesis of N-methylputrescine, a precursor of the alkaloid hyoscyamine, have been measured in root cultures of Datura stramonium L. and Atropa belladonna L. transformed with Agrobacterium rhizogenes. Ornithine δ-Nmethyltransferase and δ-N-methylornithine decafboxylase were undetectable, indicating that δ-N-methylornithine is an unlikely intermediate in the formation of N-methylputrescine. The activity of putrescine-N-methyltransferase (EC 2.1.1.53) was comparable to, or greater than, that of arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17). Radiolabel from DL-[5-(14)C]ornithine, L-[U-(14)C]arginine, [U-(14)C]agmaine and [1,4-(14)C]putrescine was incorporated into hyosyamine by Datura cultures. Hyoscyamine production by Datura cultures was substantially inhibited by the arginine-decarboxylase inhibitor, DL-α-difluoromethylarginine, but not by the corresponding ornithine-decarboxylase inhibitor, DL-α-difluoromethylornithine. Together with the demonstration that label was incorporated from [U-(14)C]agmatine, this indicates clearly that arginine is metabolised to hyoscyamine at least in part via decarboxylation to agmatine, even though a high activity of arginase (EC 3.5.3.1) was measurable under optimal conditions. The effect of unlabelled putrescine in diminishing the incorporation into hyoscyamine of label from DL-[ 5-(14)C] ornithine and L-[U-(14)C] arginine does not lend support to the theory that ornithine is metabolised via a bound, asymmetric putrescine intermediate.

Studies on the biosynthesis of tropane alkaloids in Datura stramonium L. transformed root cultures : 2. On the relative contributions of L-arginine and L-ornithine to the formation of the tropane ring.

The relative contributions made by the L-arginine/agmatine/N-carbamoylputrescine/putrescine and the L-ornithine/putrescine pathways to hyoscyamine formation have been investigated in a transformed root culture of Datura stramonium. The activity of either arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17) was suppressed in vivo by using the specific irreversible inhibitors of these activities, DL-α-difluoromethylarginine or DL-α-difluoromethylornithine, respectively. It was found that suppression of arginine decarboxylase resulted in a severe decrease in free and conjugated putrescine and in the putrescine-derived intermediates of hyoscyamine biosynthesis. In contrast, the suppression of ornithine decarboxylase activity stimulated an elevation of arginine decarboxylase and minimal loss of metabolites from the amine and alkaloid pools. The stimulation of arginine decarboxylase was not, however, sufficient to maintain the same potential rate of putrescine biosynthesis as in control tissue. It is concluded that (i) in Datura the two routes by which putrescine may be formed do not act in isolation from one another, (ii) arginine decarboxylase is the more important activity for hyoscyamine formation, and (iii) the formation of polyamines is favoured over the biosynthesis of tropane alkaloids. An interaction between putrescine metabolism and other amines is also indicated from a stimulation of tyramine accumulation seen at high levels of DL-α-difluoromethylornithine.

Toxicity of quinoline alkaloids to cultured Cinchona ledgeriana cells.

The toxicity of Cinchona alkaloids to cell cultures of C. ledgeriana has been studied in relation to alkaloid uptake and possibilities for selecting high-yielding cell lines. The most toxic, quinine, was completely toxic at 5.5 mM. Both quinine and quinidine were more toxic than their unmethoxylated precursors, cinchonidine and cinchonine. The permanently-charged metho-chlorides of quinine and cinchonidine were less toxic than the parent alkaloids, despite showing similar accumulation ratios in 5-day uptake experiments at sub-toxic concentrations (ca 1.7mM). The toxicity of the natural quinoline alkaloids appears to be a non-specific effect which may be caused by intracellular alkalinisation following uptake of the uncharged bases. The use of precursors of quinine and quinidine as toxic agents for the selection of cell lines with enhanced quinine and quinidine production is ruled out by the lower toxicity of these precursors and by the correlation of an apparently non-specific toxicity with uptake.

A spectrophotometric assay for strictosidine synthase.

A spectrophotometric assay for strictosidine synthase is described. Strictosidine is extracted with ethyl acetate and, where high substrate concentrations are used, the organic extract is washed with dilute ammonia to remove coextracted secologanin; after evaporation of the solvent, the residue is heated with 5 M H2SO4 for 45 min and the A348 value is measured. Strictosidine production is calculated from the response of similarly treated standards. A minimum production of 10-25 nmol of strictosidine may be determined. The assay is demonstrated using extracts of cultured Cinchona ledgeriana cells.

Factors regulating tropane-alkaloid production in a transformed root culture of a Datura candida × D. aurea hybrid.

Using in combination an analysis of (i) the levels of enzyme activities present, (ii) the pool sizes of metabolic intermediates and end products and (iii) the effects of feeding metabolic intermediates, the limitations ℴ flux into tropane alkaloids in a Datura root culture have been examined. This culture, produced by transforming a Datura candida × D. aurea hybrid with Agrobacterium rhizogenes, is found to be highly competent in the biosynthesis of both hyoscyamine and scopolamine as well as a wide range of other hygrine-derived alkaloids. It has been found that, of six enzymes which are involved in this pathway, the two initial activities, ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (EC 4.1.1.19), are present at potentially flux-limiting levels, in contrast to those other enzymes assayed which act further down the pathway. An additional limitation to flux, involving the supply of activated acids for condensation with tropine to form the identified tropoyl and tigloyl derivatives, is also indicated from the observed effect of feeding free acids. The relative contribution to flux limitation caused by these two interacting phenomena is inferred from an analysis of the changing relative levels of metabolic intermediates and end products as cultures mature.