Radiorespirometry evidence for the discrimination between 13C-enriched glucose and unlabelled glucose molecules by Paracoccus denitrificans.

Paracoccus denitrificans was grown on either unlabelled glucose, [1-13C]glucose or [6-13C]glucose as the sole carbon source for growth. The cells were then incubated with a range of 14C-glucose substrates to compare the 14CO2-evolution rates between cells grown on the glucose and the 13C-labelled glucose. Cells grown on 13C-glucose had significantly faster rates of 14CO2-evolution than those grown on unlabelled glucose. The % yields of 14CO2, per [1-14C]-, [6-14C]- and [U-14C]glucose supplied were also substantially greater than those measured for cells grown on unlabelled glucose. The data indicated that growth of Paracoccus on 13C-enriched glucose substrates resulted in cells with notably different 14C-glucose oxidation metabolism compared to that observed in cells grown on unlabelled glucose.

Populus candicans and the Balm of Gilead.

Bud exudates of some poplars are compared using GC-MS. P. candicans resembles P. balsamifera. A sample of "Balm of Gilead" purchased in Oxford proved not to resemble its supposed source, P. candicans. Instead it was similar to P. nigra, from which it was presumably collected in Europe.

Metabolic changes during development of Phytophthora palmivora examined by gas chromatography/mass spectrometry.

Metabolic profiles from four stages of differentiation of the fungus Phytophthora palmivora were obtained by gas chromatography/mass spectrometry. The profiles showed the presence of sterols in the asexual reproduction stage of the organism, and confirmed their virtual absence from the mycelial stages. The zoospore stage was characterized by the presence of polyunsaturated fatty acids of C20 and C22 chain length. The transition from zoospore to cyst was also marked by the appearance of disaccharides and by a decrease in the amount of phosphate present. There were also distinctive shifts in the proportions and the total amounts of amino acids present, with gamma-aminobutyrate and alanine increasing as germination took place. These distinctive profiles identify some of the metabolic changes which accompany differentiation in this fungus.

Stable isotope evidence for the localisation of some metabolic pathways in the bacterium Paracoccus denitrificans.

Paracoccus denitrificans was grown on [6-13C]-glucose as the sole carbon source for growth and the extracts were fractionated and analysed by gas chromatography-mass spectrometry. The 13C-enrichments of some metabolites indicated that the "hydrolysate pools" of these metabolites were not in isotopic equilibrium with the water soluble "free pools". It was concluded that localisation of some metabolic pathways had occurred in Paracoccus during growth on [6-13C]-glucose.

Stable isotope evidence for the localisation of phenylalanine biosynthesis in the bacterium Paracoccus denitrificans.

Paracoccus denitrificans was grown on [6-13C]-glucose as the sole carbon source for growth and the extracts were fractionated and analysed by gas chromatography-mass spectrometry. The 13C-labelling pattern observed in phenylalanine indicated that the biosynthetic sequences of enzymes for phenylalanine production were unequally distributed within the cell and that there are at least 2 separate loci of phenylalanine biosynthesis. The principal locus of phenylalanine production was associated with the Entner-Doudoroff and/or the pentose phosphate pathways and it was responsible for producing 3/4 of the bacterium's phenylalanine. A second locus was associated with the G6 oxidation pathway and was responsible for producing the remaining 1/4 of the cell's phenylalanine.

Growth of Paracoccus denitrificans on [2,3-13C]succinate and [1,4-13C]succinate. III. Biosynthetic pathways.

The biosynthesis in vivo of a number of amino acids, sugars, and purines in Paracoccus denitrificans grown on either [2,3-13C]succinate or [1,4-13C]succinate was investigated by using gas chromatography-mass spectrometry. The distribution of label in the TCA-cycle-related amino acids indicated that carbon intermediates of energy metabolism were utilized as precursors for the biosynthesis of these amino acids in vivo. The biosynthesis of glycine, serine, phenylalanine and glycerol from labelled succinate in vivo were consistent with phosphoenol pyruvate as an intermediate. A mechanism for the formation of C4, C5 and C6 sugars without the use of fructose-1,6-bisphosphate aldolase (which has not been detected in P. denitrificans) is proposed. The 13C-enrichments of ribose in the bacterium indicate that there are at least three routes of ribose biosynthesis operating during growth on labelled succinate. The probability distribution of labelled purine molecules was successfully predicted for adenine, guanine and adenosine, thus confirming their generally accepted route of biosynthesis in vivo.

Growth of Paracoccus denitrificans on [2,3-13C]succinate and [1,4-13C]succinate. I. The flux of carbon in energy metabolism and the operation of the TCA cycle.

The metabolism of Paracoccus denitrificans, grown on either [2,3-13C]succinate or [1,4-13C]succinate, was investigated by using gas chromatography-mass spectrometry. The distribution of label in a group of metabolites closely related to the TCA-cycle intermediates showed that the flux of carbon from succinate in energy metabolism in vivo was via pyruvate (malic enzyme) and acetyl CoA. The labelling pattern of the carboxyl groups showed that one fifth of the succinate pool was formed by the regeneration of succinate via the TCA cycle, and four fifths was supplied externally as substrate from the medium.

Growth of Paracoccus denitrificans on [2,3-13C]succinate and [1,4-13C]succinate. II. Isoleucine biosynthesis.

Paracoccus denitrificans was grown on either [2,3-13C]succinate or [1,4-13C]succinate, and extracts were analysed by using gas chromatography-mass spectrometry. The distribution of label in isoleucine indicated that the 2-ketobutyrate required for isoleucine biosynthesis was mainly produced from pyruvate by 2-keto-acid chain elongation (i.e. the 'pyruvate elongation pathway'). Approximately 10% of isoleucine was produced by a second pathway involving propionyl CoA. Threonine and glutamate were not utilized by P. denitrificans as a source of 2-ketobutyrate production for isoleucine biosynthesis under the growth conditions used.

WHEN IS A CHROMOPLAST?

Chromoplasts are heterogeneous organelles and their carotenoids can be associated with several different structural elements including globules, tubules and membranes. In the flower petals of some species, carotenoids are absent or present only in trace amounts. Nevertheless, the plastids in such flowers can closely resemble or even be indistinguishable in structure from chromoplasts.

Conditions Required for the Rapid Activation In Vitro of the Chloroplast Fructose-1,6-bisphosphatase.

Conditions required for the reductive activation of purified, spinach chloroplast fructose-1,6-bisphosphatase (EC 3.1.3.11) have been determined in vitro. Full reductive activation was observed only when fructose-1,6-bisphosphate and Mg(2+) were present at the same time as the reducing agent (dithiothreitol). Reduction in the absence either of fructose-1,6-bisphosphate or of Mg(2+) slowly and irreversibly inactivated the enzyme. The concentration of fructose-1,6-bisphosphate that must be present during reduction for maximum activation depends upon the divalent cation present: it is highest with Mg(2+), lower with Ca(2+), and lowest when both Mg(2+) and Ca(2+) are present. A scheme for the reductive activation and inactivation of the enzyme is presented.

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.

Crystallisation and preliminary X-ray data of ribulose-1,5-bisphosphate carboxylase from spinach.

Crystals of a tertiary complex of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase with the activators Mg and CO(2) have been grown. These crystals diffract strongly to 1.6 A resolution. The spacegroup is C222(1) with unit cell dimensions a = 158.6 A, b = 158.6 A, c = 203.4 A. Additional local symmetry is apparent in the pattern of absences and the intensity distribution of the X-ray precession photographs. The photographs have been interpreted in terms of a molecule (consisting of eight large and eight small subunits, L(8)S(8)) with 222 symmetry and a molecular centre shifted 2 A in the x direction from the origin of the unit cell. The asymmetric unit contains half the L(8)S(8) molecule. The intensity distribution suggests that the molecular symmetry does not deviate far from 422. These crystals are compared with other crystalline forms of the enzyme and the implications of these results are discussed.

The influence of secretion elicitors and external pH on the kinetics of D-alanine uptake by the trap lobes of Dionaea muscipula Ellis (Venus's Flytrap).

Simple kinetic techniques were used to examine the mechanism of D-alanine uptake by the adaxial surfaces of the trap lobes of Dionaea muscipula Ellis (Venus's Flytrap.) On the basis of these analyses, the uptake of D-alanine was found to depend on the time during which the trap lobes were inoculated with elicitors of secretion before excision and measurement of uptake. Disks taken from traps that had not been subjected to a preceding period of inoculation with secretion elicitors showed a low basal rate of uptake which was neither pH-dependent nor exhibited saturation with respect to external D-alanine concentration. Disks from preinoculated traps, on the other hand, displayed an enhanced rate of uptake which showed both pH-dependence and saturation with respect to external D-alanine concentration. The capacity for enhanced uptake was lost upon prolonged inoculation or when inoculation was stopped. Of the compounds tested, only elicitors of secretion caused an enhancement of uptake. The enhanced rate of D-alanine uptake is temperature-sensitive with a Q10 characteristic of a mediated process. Uncouplers cause an instantaneous abolition of uptake whereas the effects of terminal-oxidase inhibitors are time-dependent. The pH-dependence of uptake is inferred to result from an increased affinity of the carrier system for D-alanine at low pH values. Although the ionic state of D-alanine is relatively unaffected over the pH range examined, a decrease in the external pH from 6.0 to 3.8 decreases the apparent K m for uptake by four-fold but increases V max by only 30%. It is concluded that the acid secreted by the digestive glands of Dionaea plays a direct role in facilitating the uptake of amino acids from the trap cavity.

Effect of photoinhibitory treatments on the activity of light-activated enzymes of c(3) and c(4) photosynthetic carbon metabolism.

Exposure of maize leaves to a 3-hour photoinhibitory treatment (photon flux rate of 2,000 microeinsteins meter(-2) second(-1), CO(2)-free air) resulted in lower activities of the light-activated enzymes NADP malate dehydrogenase, pyruvate, Pi dikinase, and ribulose-5-phosphate kinase. The activities could be recovered partially either by incubating enzyme extracts with dithiothreitol or by illuminating the treated leaf in air. Several enzymes which are not light-activated were not affected by the treatment. Ribulose-5-phosphate kinase activity was also reduced when bean plants grown in low light were subjected to a similar photoinhibitory treatment.It is suggested that, although the reactivation of these enzymes may be correlated with the short term increase of CO(2) uptake capacity observed when photoinhibited leaves are returned to illumination in air, inactivation of these enzymes does not contribute significantly to the long term in vivo expression of photoinhibition observed after 2 to 4 hours.The results provide an example of partial inactivation of light-activated enzymes under illumination equivalent to full sunlight.

Uncouplers stimulate photosynthesis in intact chloroplasts by enhancing light-activation of enzymes regulated by the ferredoxin-thioredoxin system.

Some uncouplers stimulate CO(2)-dependent O(2) evolution by intact spinach chloroplasts at pH 8.6. This effect is not due to alkalinization of the stroma. The stimulation is observed only when photosynthesis has been partly inhibited by the presence of H(2)O(2), generated in a Mehler-type reaction by the broken chloroplasts which always contaminate the intact chloroplast preparations. The addition of methyl viologen increases the Mehler-type reaction and results in greater inhibition of photosynthesis. The addition of excess catalase stimulates photosynthesis by preventing accumulation of H(2)O(2). The uncouplers stimulate photosynthesis primarily by enhancing the light-activation of enzymes that are regulated by the ferredoxin-thioredoxin system, and this effect results from the influence of the uncouplers on the redox poising of the ferredoxin in the intact chloroplasts.

The establishment of mitochondria: Paracoccus and Rhodopseudomonas.

Many aerobic bacteria (both facultative and obligate) possess a number of those biochemical features of mitochondria which are concerned with energy metabolism. However, only restricted number, notably Paracoccus denitrificans and Rhodopseudomonas spheroides, have the majority of these features. The theory of endosymbiosis proposes that a primitive eukaryote took up bacteria to yield mitochondria. The present-day Paracoccus then resembles the ancestral bacterium in many respects the primitive amoeba, Pelomyxa palustris, which lacks mitochondria but contains a permanent population of unique symbiotic bacteria, has many of the characteristics of a present-day transitional form. The evolution of mitochondria from endosymbiotic bacteria would involve their integration with the host cell both biochemically and structurally: a number of the intermediate steps are discussed. Attention is drawn to the existence in some ciliates of hydrogenosomes, which function as anaerobic mitochondria.

From extracellular to intracellular: the establishment of mitochondria and chloroplasts.

Paracoccus and Rhodopseudomonas are unusual among bacteria in having a majority of the biochemical features of mitochondria; blue-green algae have many of the features of chloroplasts. The theory of serial endosymbiosis proposes that a primitive eukaryote successively took up bacteria and blue-green algae to yield mitochondria and chloroplasts respectively. Possible characteristics of transitional forms are indicated both by the primitive amoeba, Pelomyxa, which lacks mitochondria but contains a permanent population of endosymbiotic bacteria, and by several anomalous eukaryotic algae, e.g. Cyanophora, which contain cyanelles instead of chloroplasts. Blue-green algae appear to be obvious precursors of red algal chloroplasts but the ancestry of other chloroplasts is less certain, though the epizoic symbiont, Prochloron, may resemble the ancestral green algal chloroplast. We speculate that the chloroplasts of the remaining algae may have been a eukaryotic origin. The evolution or organelles from endosymbiotic precursors would involve their integration with the host cell biochemically, structurally and numerically.

Effects of inhibitors of catalase on photosynthesis and on catalase activity in unwashed preparations of intact chloroplasts.

The catalase activity of unwashed preparations containing intact spinach (Spinacia oleracea L.) chloroplasts is inhibited both by cyanide and by azide at concentrations which also cause inhibition of photosynthetic CO(2)- dependent O(2) evolution.Aminotriazole can also be used to inhibit this contaminant catalase, and in this case inhibition of catalase can be achieved at aminotriazole concentrations which have little effect on the rate of photosynthetic CO(2) fixation. Aminotriazole may be used as a specific inhibitor of catalase in order to demonstrate inhibition of photosynthesis by added H(2)O(2).It is therefore concluded that inhibition of photosynthesis by cyanide and azide does not necessarily result from inhibition of catalase in the chloroplast preparation, and that intact chloroplasts do not produce inhibitory concentrations of H(2)O(2) under the best experimental conditions for CO(2) fixation.