W. Greenaway

The analysis of bud exudate of Populus x euramericana, and of propolis, by gas chromatography-mass spectrometry

Propolis and bud exudate of Populus x euramericana have been analysed by high-resolution gas chromatography–mass spectrometry; 104 compounds have been identified, including 26 not previously identified in propolis or bud exudate. The compounds identified in propolis are derived from three sources: plant exudate collected by bees, wax secreted by bees, and materials such as sugars, which are probably introduced accidentally either during propolis manufacture or during subsequent passage of bees over the propolis. Previous observations that bees use bud exudate from Populus as a primary source for propolis manufacture are confirmed.

The metabolism of [1-13C]-glucose by Paracoccus denitrificans, investigated by gas chromatography-mass spectrometry

The metabolism of Paracoccus denitrificans, grown on [1-13C]-glucose as the sole carbon source, was investigated by using gas chromatographymass spectrometry and metabolic profiling. The distribution of label in a wide range of metabolites (42 in total) showed that the EmbdenMeyerhoff pathway did not operate in vivo whereas both the EntnerDoudoroff and pentose phosphate pathways were operative under the specified growth conditions. It was calculated that 31% of the [1-13C]-glucose was metabolized by the Entner-Doudoroff pathway and 69% by the pentose phosphate pathway.

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. 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 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.

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.

Growth of Paracoccus denitrificanson [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.