The carbon assimilation pathways of Methylococcus capsulatus, Pseudomonas methanica and Methylosinus trichosporium (OB3B) during growth on methane

d-arabino-3-Hexulose 6-phosphate was prepared by condensation of formaldehyde with ribulose 5-phosphate in the presence of 3-hexulose phosphate synthase from methane-grown Methylococcus capsulatus. The 3-hexulose phosphate was unstable in solutions of pH greater than 3, giving a mixture of products in which, after dephosphorylation, allulose and fructose were detected. A complete conversion of d-ribulose 5-phosphate and formaldehyde into d-fructose 6-phosphate was demonstrated in the presence of 3-hexulose phosphate synthase and phospho-3-hexuloisomerase (prepared from methane-grown M. capsulatus). d-Allulose 6-phosphate was prepared from d-allose by way of d-allose 6-phosphate. No evidence was found for its metabolism by extracts of M. capsulatus, thus eliminating it as an intermediate in the carbon assimilation process of this organism. A survey was made of the enzymes involved in the regeneration of pentose phosphate during C(1) assimilation via a modified pentose phosphate cycle. On the basis of the presence of the necessary enzymes, two alternative routes for cleavage of fructose 6-phosphate are suggested, one route involves fructose diphosphate aldolase and the other 6-phospho-2-keto-3-deoxygluconate aldolase. A detailed formulation of the complete ribulose monophosphate cycle of formaldehyde fixation is presented. The energy requirements for carbon assimilation by this cycle are compared with those for the serine pathway and the ribulose diphosphate cycle of carbon dioxide fixation. A cyclic scheme for oxidation of formaldehyde via 6-phosphogluconate is suggested.     

Microbial growth on C1 compounds: Uptake of [14C]formaldehyde and [14C]formate by methane-grown Pseudomonas methanica and determination of the hexose labelling pattern after brief incubation with [14C]methanol

1. A study has been made of the incorporation of carbon from [¹⁴C]formaldehyde and [¹⁴C]formate by cultures of Pseudomonas methanica growing on methane. 2. The distribution of radioactivity within the non-volatile constituents of the ethanol-soluble fractions of the cells, after incubation with labelled compounds for periods of up to 1min., has been analysed by chromatography and radioautography. 3. Radioactivity was fixed from [¹⁴C]formaldehyde mainly into the phosphates of the sugars, glucose, fructose, sedoheptulose and allulose. 4. Very little radioactivity was fixed from [¹⁴C]formate; after 1min. the only products identified were serine and malate. 5. The distribution of radioactivity within the carbon skeleton of glucose, obtained from short-term incubations with [¹⁴C]methanol of Pseudomonas methanica growing on methane, has been investigated. At the earliest time of sampling over 70% of the radioactivity was located in C-1; as the time increased the radioactivity spread throughout the molecule. 6. The results have been interpreted in terms of a variant of the pentose phosphate cycle, involving the condensation of formaldehyde with C-1 of ribose 5-phosphate to give allulose phosphate.     

Enzymological aspects of the pathways for trimethylamine oxidation and C1 assimilation of obligate methylotrophs and restricted facultative methylotrophs.

Extracts of trimethylamine-grown W6A and W3A1 (type M restricted facultative methylotrophs) contain trimethylamine dehydrogenase whereas similar extracts of Bacillus PM6 and Bacillus S2A1 (type L restricted facultative methylotrophs) contain trimethylamine mono-oxygenase and trimethylamine N-oxide demethylase but no trimethylamine dehydrogenase. Extracts of the restricted facultatives and of the obligate methylotroph C2A1 contain hexulose phosphate synthase-hexulose phosphate isomerase activity; hydroxypyruvate reductase was not detected. Neither the restricted facultatives nor the obligates 4B6 and C2A1 contain all the enzymes of the hexulose phosphate cycle of formaldehyde assimilation as originally proposed by Kemp & Quayle (1967). Organisms PM6 and S2A1 lack transaldolase and use a modified cycle involving sedoheptulose 1,7-diphosphate and sedoheptulose diphosphatase. The obligates 4B6 and C2A1, and the type M organisms W6A and W3A1, use a different modification of the assimilatory hexulose phosphate cycle involving the Entner-Doudoroff-pathway enzymes phosphogluconate dehydratase and phospho-2-keto-3-deoxygluconate aldolase. The lack of fructose diphosphate aldolase and hexose diphosphatase in these organisms may be a partial explanation of their restricted growth-substrate range. Enzymological evidence suggests that all the obligates and the restricted facultatives use a dissimilatory hexulose phosphate cycle to accomplish the complete oxidation of formaldehyde to CO2 and water.     

Metabolism of [1-14C]glyoxylate, [1-14C]-glycollate, [1-14C]glycine and [2-14C]-glycine by homogenates of kidney and liver tissue from hyperoxaluric and control subjects

1. The metabolism of [1-(14)C]glyoxylate to carbon dioxide, glycine, oxalate, serine, formate and glycollate was investigated in hyperoxaluric and control subjects' kidney and liver tissue in vitro. 2. Only glycine and carbon dioxide became significantly labelled with (14)C, and this was less in the hyperoxaluric patients' kidney tissue than in the control tissue. 3. Liver did not show this difference. 4. The metabolism of [1-(14)C]glycollate was also studied in the liver tissue; glyoxylate formation was demonstrated and the formation of (14)CO(2) from this substrate was likewise unimpaired in the hyperoxaluric patients' liver tissue in these experiments. 5. Glycine was not metabolized by human kidney, liver or blood cells under the conditions used. 6. These observations show that glyoxylate metabolism by the kidney is impaired in primary hyperoxaluria.     

In vitro oxidation of [U-14C] palmitate, [1-14C] and [6-14C] glucose in newborn and adult rats and pigs

Studies have been made on the intensity of oxidation of [U-14C]-palmitate, [1-14C]- and [6-14C]-glucose by slices of the liver and skeletal muscles of new-born, 1-day, 5-day and adult Wistar rats and domestic pigs. It was found that the level of 14CO2 production from these substrates is higher in tissues of rats than in those of pigs. At early stages of ontogenesis, in tissues of both species intensive oxidation of glucose is observed together with oxidation of fatty acids. In the course of ontogenetic development, the intensity of glucose utilization significantly decreases, whereas the level of fatty acid catabolism remains relatively unaffected.     

Distribution of Methylomonas methanica and Methylosinus trichosporium in Cleveland Harbor as Determined by an Indirect Fluorescent Antibody-Membrane Filter Technique

An indirect fluorescent antibody-membrane filter staining technique, which permitted the autecological study of Methylomonas methanica and Methylosinus trichosporium, was developed. This technique was used to assay the numbers of these two organisms in Cleveland Harbor. The concentrations of M. methanica and M. trichosporium were found to be inversely proportional to the sampling depth, with the highest cell counts observed in the sediments. M. methanica was observed at every sampling station, whereas M. trichosporium was found at only two of the stations.     

Oxidation of [1-14C]palmitoyl-CoA, [1-14C]acetyl-CoA and [2-14C]pyruvate in rabbit adrenal, liver and heart mitochondria under normal conditions and in acute stress

The acute immobilized stress was studied for its effect on oxidation rate of [1-14C]palmitoyl-CoA, [1-14C]acetyl-CoA and [2-14C]pyruvate in mitochondria of the adrenals, liver and heart of rabbits. The stress effect on the energy metabolism of adrenals is associated with an increase of the rate of CO2 formation from pyruvate and with a decrease of the rate of CO2 formation from palmitoyl-CoA. Intensified oxidation of all substrates is observed in the heart mitochondria. The processes of beta-oxidation are more active in the liver. The data obtained evidence for differences in the mechanisms of energy metabolism reconstruction under acute stress in tissues with different functional specialization.     

Biosynthesis of [14C]zearalenone from [1-14C]acetate by Fusarium roseum 'Gibbosum'.

Addition of [1-14C]acetate or [1,2-14C]acetate to actively growing cultures of Fusarium roseum 'Gibbosum' on rice yielded zearalenone with a specific activity ranging between 1.63 and 46.5 microCi/mmol.     

Respiration of 14CO2 by intact animals of various species given l-[1-14C]fucose or d-[1-14C]arabinose

The production of ¹⁴CO₂ from l-[1-¹⁴C]fucose and d-[1-¹⁴C]arabinose has been studied in five mammalian species.Cats, guinea pigs, mice, and rabbits respired about 22% of the label of l[1-¹⁴C]fucose or of d-[1-¹⁴C]arabinose within 6 h after intraperitoneal injection of the sugar. Rats respired only 1.5% of the l-fucose label and 5% of the d-arabinose label in the same time period.Liver homogenates from cat, guinea pig, and rabbit produced significantly more ¹⁴CO₂ from l-[1-¹⁴C]fucose or d-[1-¹⁴C]arabinose than mouse or rat liver homogenates. Unlike those of the other species, guinea pig liver homogenates had very low l-fucose dehydrogenase activity.The results suggest that substantial catabolism of l-fucose and d-arabinose occurs in the tissues of some animal species. Investigators wishing to employ l-fucose as a tracer of glycoprotein metabolism must, therefore, ensure that the species that they employ does not metabolize l-fucose to products interfering with their studies.     

Metabolism of ent-kaurenol-[17-14C], ent-kaurenal-[17-14C] and ent-kaurenoic acid-[17-14C] by germinating Hordeum distichon grains

Subcellular fractions from germinated barley embryos, chloroplast preparations and whole germinating barley grains are able to carry out the conversions ent-kaurenol → ent-kaurenal → ent-kaurenoic acid → ent-hydroxykaurenoic acid, the initial steps of the biosynthetic pathway to gibberellins. Whole grains, and chloroplasts to a slight extent, incorporate radioactivity from ent-kaurenol-[17-¹⁴C] and ent-kaurenoic acid-[17-¹⁴C] into materials with similar but distinct properties from the gibberellins GA₁, GA₃, GA₄ and GA₇.     

[14C]Acetate incorporation by cultured normal,familial hypercholesterolemia and Down's syndrome fibroblasts

Fibroblasts from patients with homozygous familial hypercholesterolemia (FH), a disease characterized by accelerated atherogenesis, are known to lack functional low-density-lipoprotein receptors, which ultimately results in increased cholesterol biosynthesis in the cultured cells. [¹⁴C]Acetate incorporation in these cells was compared to that of normal fibroblasts and to fibroblasts from patients with Down's syndrome, a disease in which atherosclerosis is rare. Total [¹⁴C]acetate incorporation did not differ significantly between normal and Down's fibroblasts, nor did its partitioning into the hexane-extractable and aqueous fractions of the cell hydrolysates. [¹⁴C]Acetate incorporation was much greater in FH cells in both the aqueous and hexane-extractable fractions. Preincubation in fetal bovine serum increased acetate incorporation only by FH cells, while 50 μg low-density lipoprotein/ml medium depressed acetate incorporation in all three groups. A C₂₇ sterol, identified by gas chromatography-mass spectrometry as a probable isomer of cholesterol, was present in small amounts in FH fibroblasts, but was not detectable in the normal or Down's cells. The absolute amounts of [¹⁴C]acetate incorporated into the non-sterol lipids were greater in the FH fibroblasts, indicating that these cells may have to synthesize, in addition to cholesterol, other required cellular lipids which are delivered to the normal cells by low-density lipoproteins.     

Isoprenoid biosynthesis in a marine sponge of the Amphimedon genus: incorporation studies with [1-14C] acetate, [4-14C] cholesterol and [2-14C] mevalonate

1. We present quantitative evidence from incorporation of [1-14C] acetate that the enzymes to synthesise isoprenoids are present in the marine sponge Amphimedon sp. and that efficient carotenoid synthesis takes place. 2. The de novo synthesis of b,b-carotene and (3R,3'R)-zeaxanthin may occur in a chlorophyll a-producing microalgal symbiont with subsequent aromatisation to (3R)-isoagelaxanthin by the sponge itself. 3. Amphimedon sp. contains nuclear-modified sterols derived by modification of conventional dietary sterols.