Iain M. Campbell

Incorporation and dilution values—Their calculation in mass spectrally assayed stable isotope labeling experiments

Abstract The significance and validity of incorporation and dilution values in radioisotope tracer work are reviewed. The parallels between specific activity and enrichment factor are drawn. Methods of obtaining enrichment factors from mass spectral data are examined, and examples are given. Natural-abundance correction procedures are documented.

Mass spectral identification of melatonin in blood

Summary Although the pineal body is generally considered to be an endocrine organ, and melatonin is thought by many to be its secretion, identification of this substance in the circulation has never been accomplished. In chicken blood we have identified melatonin using mass spectroscopy and also have shown that melatonin detected by the Rana pipiens bioassay in serum extracts disappears following pinealectomy. These data support the notion that melatonin is released into the circulation and that it may be a pineal hormone.

Binding of trichodermin to mammalian ribosomes and its inhibition by other 12,13-epoxytrichothecenes

SummaryTrichodermin binds to ribosomes from rabbit reticulocyte polysomes with an apparent Ka of 9.2 × 105 and with a maximum binding ratio of 0.44 molecules per ribosome. Trichodermin also binds firmly to HeLa cell polysomes under conditions where it is inhibiting protein synthesis. Purified HeLa 60S ribosomal subunits showed only a slight ability to bind trichodermin, and no binding was found with 40S subunits. Seventeen 12,13-epoxytrichothecenes were tested for their ability to interfere with [3H] trichodermin binding to reticulocyte ribosomes. All compounds known to inhibit ribosomal functions required for elongation-termination or initiation processes exhibited significant positive interference with the binding of trichodermin.

Glutamate — A precursor for the naphthalene nucleus of bacterial menaquinones

Carbon atoms 2, 3 and 4(1) of the naphthalene nucleus of the menaquinones of E,coli and M,phlei are derived from glutamate-U-14C; C-4(1) is specifically derived from glutamate-2-14C. Menaquinone biosynthesis is postulated to involve condensation of shikimate with 2-ketoglutarate or a derivative thereof, both carboxyl groups of 2-ketoglutarate being removed during biosynthesis.

Secondary Metabolism and Microbial Physiology

Publisher Summary This chapter discusses the biochemical similarities and differences between primary and secondary metabolism. The known secondary metabolites are divided into three categories according to the perceived source of the carbon from which their skeletons are derived. These include metabolites derived from acetate, metabolites derived from amino acids, and metabolites derived from components of sugar metabolism. Divisions of secondary metabolites into groups according to the origin of their skeletal carbon atom have many advantages. It allows an impression to be gained of the commitment various taxa have to the use of acetate, amino acids, and sugars as a fuel for secondary metabolism. The chapter concludes that primary metabolism and secondary metabolism are co-extensive. Both processes draw carbon from the same sources and, in many cases, use the same chemical compounds to build that carbon into end products. In several aspects, secondary metabolism is more biochemically sophisticated than primary metabolism. In the areas of polyketide and terpene biosynthesis, there is an inherent biochemical complexity present in secondary metabolism, which is absent from primary metabolism. Problems of considerable logistical involvement arise in secondary metabolism and have been solved satisfactorily.

A combined radiogas chromatograph/mass spectrometer detects intermediates in mycophenollc acid biosynthesis

Abstract The fusion of a gas-flow proportional counter to a combined gas chromatograph/mass spectrometer is described. Provided components of a tissue extract are volatile in their own right, or can be rendered so by derivatization, the combined unit can separate them, identify each, and provide assays of mass and radioactivity. The counting module has an efficiency of circa 65% for 14 C. To demonstrate the use and versatility of the radiogas chromatograph/mass spectrometer, three biosynthetic intermediates between acetate and mycophenolic acid were identified in extracts of Penicillium brevicompactum . The three substances, 4,6-dihydroxy-2,3-dimethylbenzoic acid; 5,7-dihydroxy-4-methylphthalide, and 6-farnesyl-5,7-dihydroxy-4-methylphthalide, had been implicated previously in the biosynthetic pathway by direct feeding and swamping experiments. A tentative structure for a fourth and previously unencountered intermediate is proposed.

Intermediate symmetry in lawsone biosynthesis

Abstract Feeding experiments in Impatiens balsamina have established that 2- 14 C-acetate predominantly labels C-2 of lawsone. This finding confirms previous biosynthetic postulates, indicates that no symmetrical intermediate is involved in lawsone biosynthesis, and sheds light on the final steps in that process. The experimental methods that have been employed in this series of studies are also described in detail.

4-(2′-Carboxyphenyl)-4-oxobutyrate: An obligatory intermediate in lawsone biosynthesis

Abstract 4-(2′-Carboxyphenyl)-4-oxobutyric acid ( 6 ) has been detected in cuttings of Impatiens balsamina . It is labelled under conditions where activity from U- 14 C-glutamate is incorporated effectively into lawsone ( 1 ). 3-(2′-Carboxyphenyl)-3-oxopropionic acid ( 7 ) has also been encountered in the cuttings.

Occurrence and biosynthesis of asperphenamate in solid cultures of Penicillium brevicompactum

Abstract The ester of N-benzoylphenylalanine and N-benzoylphenylalaninol, asperphenamate, was isolated from solid cultures of Penicillium brevicompactum. Isotope from l -[U-14C] phenylalanine was well incorporated into both benzoyl groups and into the phenylalanine and phenylalaninol moieties. Isotope from [U-14C]benzoic acid was also well incorporated into asperphenamate.

Isolation and Characterization of Endotoxin from Cyanobacteria (Blue-Green Algae)

A Sewickley, Pennsylvania, epidemic of water-borne gastroenteritis of unknown etiology which occurred in 1975 was characterized by high concentration of Schizothrix calcicola (Cyanobacteria) in the corresponding drinking water system. As a consequence, the most common cyanobacterial contaminants of drinking water were tested for endotoxin (lipopolysaccharide - LPS) content. LPS was isolated and characterized from Schizothrix calcicola, Anabaena flos-aquae (UTEX 1444), Oscillatoria tenuis, and Oscillatoria brevis. The isolation was performed by using Westphal’s modified phenol-water extraction method. Subsequently, the glucan contaminating LPS was eliminated by enzymatic hydrolysis by cellulase. The resulting macromolecular substances were composed of a polysaccharide moiety and a lipid part. Contrary to Wang and Hill’s report that Anabaena flos-aquae A37 contains a polysaccharide without covalently bound lipid, Anabaena flos-aquae (UTEX 1444) contained a true LPS. No endotoxin could be isolated from the toxic strain Anabaena flos-aquae NRC-44-1 and preliminarily from Anabaena cylindrica.