Role of acetate in sporogenesis of Bacillus cereus

Nakata, H. M. (Washington State University, Pullman). Role of acetate in sporogenesis of Bacillus cereus. J. Bacteriol. 91:784-788. 1966.-The distribution of radioactivity associated initially with acetate-2-C(14) was followed during sporogenesis of Bacillus cereus strain T. This was accomplished by replacing cells committed to sporulation into a chemically defined sporulation medium. It was observed that 65 to 70% of the initial radioactivity was incorporated into poly-beta-hydroxybutyrate, whereas 20 to 25% was found in other cellular constituents. Virtually no radioactivity was lost as C(14)O(2) during the first 5 to 6 hr after replacement. Then, a gradual evolution of C(14)O(2) coincident with poly-beta-hydroxybutyrate degradation, was observed until about the ninth hour. By this time, the polymer was essentially depleted, and the first spore structures were observed in stained preparations. The total amount of radioactivity lost as C(14)O(2) was 20 to 25%. The major portion of products derived from poly-beta-hydroxybutyrate was incorporated into the spores. As much as 17% of the radioactivity associated with the spores was found in dipicolinic acid. More than 50% was located in spore proteins, 20 to 25% in the hot 5% trichloroacetic acid-soluble fraction, 4 to 5% in the lipid fraction, and 15 to 20% in the cold 5% trichloroacetic acid-soluble fraction. These data, accounting for 70 to 75% of the initial radioactivity, confirmed the hypothesis that the major role of acetate, and subsequently of poly-beta-hydroxybutyrate, in sporulation of B. cereus T is to provide carbon precursors and energy for sporogenesis.     

Biosynthesis of streptococcal cell walls: N-acetyl-D-muramic acid

Glucose-1-(14)C and acetylglucosamine-1-(14)C were added singly and together with equal amounts of the unlabeled reciprocal to Brain Heart Infusion and used for the culture of Streptococcus pyogenes. The labeling pattern of the rhamnose, glucosamine, and muramic acid in the cell wall supported an intermediary role for acetylglucosamine in providing the C1-C6 moiety of muramic acid. Although radioactivity in the C2-C9 portion of muramic acid suggested that some of the lactyl group (C7-C9) came from glycolytic products, there was also considerable contribution to it from noncarbohydrate sources. Using cell-free extracts, we were unable to demonstrate biosynthesis of acetylmuramic acid, either free or nucleotide-bound, while glycolysis was occurring. The formation of uridine diphosphoacetylmuramic acid has been reported by others who used uridine diphospho-N-acetyl-d-glucosamine, phosphoenolpyruvate, reduced nicotinamide adenine dinucleotide, and reduced nicotinamide adenine dinucleotide phosphate. However, we did not detect the formation of this compound.     

Correlation of Carbohydrate Catabolism and Synthesis of Macromolecules During Enzyme Synthesis in Pseudomonas fluorescens.

Kirkland, Jerry J. (Oklahoma State University, Stillwater), and Norman N. Durham. Correlation of carbohydrate catabolism and synthesis of macromolecules during enzyme synthesis in Pseudomonas fluorescens. J. Bacteriol. 90: 23-28. 1965.-Glucose, ribose, and fructose shorten the lag period required for synthesis of protocatechuate oxygenase. Radioactivity from uracil-2-C(14) is incorporated into the hot trichloroacetic acid-soluble fraction after a lag period of approximately 20 min after addition of protocatechuic acid. Addition of glucose or ribose simultaneously with the inducer shortens the lag period to approximately 5 min and increases the rate of uracil incorporation. The inducer must be present to initiate incorporation of radioactivity, and the exogenous carbon source accelerates incorporation but is not sufficient to initiate synthesis by itself. The addition of protocatechuic acid increases the rate and total incorporation of radioactivity from uniformly labeled glucose or ribose-1-C(14) into the hot trichloroacetic acid-soluble fraction. Ribose decreases the incorporation of radioactivity from uniformly labeled glucose into the hot trichloroacetic acid-soluble fraction, and glucose shows a similar effect on incorporation of radioactivity from ribose-1-C(14), indicating the two sugars are serving in the same capacity to enhance enzyme synthesis. Radioactivity from glucose-1-C(14) is not incorporated into the hot trichloroacetic acid-soluble fraction. The results suggest that glucose and ribose shorten the lag period for inducible enzyme formation by serving as a "specific" carbon source for synthesis of macromolecules such as ribonucleic acid.     

Incorporation of 14C-labelled fatty acids into the mammary-gland lipids of the lactating rabbit

1. [1-(14)C]Stearic acid, [9-(14)C]stearic acid, [9-(14)C]palmitic acid and [9-(14)C]decanoic acid were fed to lactating rabbits, and the fatty acids of the mammary gland were separated and partially degraded. 2. Most of the (14)C recovered was located in the acids fed. Stearic acid was least efficiently absorbed; decanoic acid was the most extensively metabolized. 3. Resynthesis after degradation to C(2) units led to uniform alternate labelling in the C(2)-C(10) acids, whereas C(12)-C(18) acids had excess of (14)C at the carboxyl end. 4. Acids formed by beta-oxidation down to C(12), but not below, were also present in the mammary-gland lipids. Desaturation of the administered acids was a very minor reaction.     

Biosynthetis of phytoquinones. Biosynthetic origins of the nuclei and satellite methyl groups of plastoquinone, tocopherols and tocopherolquinones in maize shoots, bean shoots and ivy leaves

1. By using dl-[ring-(14)C]phenylalanine, dl-[beta-(14)C]phenylalanine, dl-[alpha-(14)C]-tyrosine and dl-[beta-(14)C]tyrosine it was shown that in maize shoots (Zea mays) the nucleus and one nuclear methyl group of each of the following compounds, plastoquinone, gamma-tocopherol (aromatic nucleus) and alpha-tocopherolquinone, are formed from the nuclear carbon atoms and beta-carbon atom respectively of either exogenous phenylalanine or exogenous tyrosine. With ubiquinone only the aromatic ring of the amino acid is used in the synthesis of the quinone nucleus. Chemical degradation of plastoquinone and gamma-tocopherol molecules labelled from l-[U-(14)C]tyrosine established that a C(6)-C(1) unit directly derived from the amino acid is involved in the synthesis of these compounds. Radioactivity from [beta-(14)C]cinnamic acid is not incorporated into plastoquinone, tocopherols or tocopherolquinones, demonstrating that the C(6)-C(1) unit is not formed from any of the C(6)-C(1) phenolic acids associated with the metabolism of this compound. 2. The incorporation of radioactivity from l-[U-(14)C]tyrosine, dl-[beta-(14)C]tyrosine and dl-[U-(14)C]phenylalanine into bean shoots (Phaseolus vulgaris) and dl-[beta-(14)C]tyrosine and l-[Me-(14)C]methionine into ivy leaves (Hedera helix) was also investigated. Similar results were obtained to those reported for maize, except that in beans phenylalanine is only used for ubiquinone biosynthesis. This is attributed to the absence of phenylalanine hydroxylase from these tissues. In ivy leaves it is found that the beta-carbon atom of tyrosine gives rise to the 8-methyl group of delta-tocopherol, and it is suggested that for all other compounds examined it will give rise to the nuclear methyl group meta to the polyprenyl unit. 3. Preliminary investigations with the alga Euglena gracilis showed that in this organism ring-opening of tyrosine occurs to such an extent that the incorporation data from radiochemical experiments are meaningless. 4. The above results, coupled with previous observations, are interpreted as showing that in higher plants the nucleus of ubiquinone can be formed from either phenylalanine or tyrosine by a pathway involving as intermediates p-coumaric acid and p-hydroxybenzoic acid. Plastoquinone, tocopherols and alpha-tocopherolquinone are formed from p-hydroxyphenylpyruvate by a pathway in which the aromatic ring and C-3 of the side chain give rise respectively to the nucleus and to one nuclear methyl group. 5. Dilution experiments provided evidence that in maize shoots p-hydroxyphenylpyruvic acid and homogentisic acid (produced from p-hydroxyphenylpyruvic acid) are involved in plastoquinone biosynthesis, and presumably the biosynthesis of related compounds: however, other possible intermediates in the conversion including toluquinol (the aglycone of the proposed key intermediate) showed no dilution effects. Further, radioactivity from [Me-(14)C]toluquinol is not incorporated into any of the compounds examined. 6. Dilution experiments with 3,4-dihydroxybenzaldehyde and radioactive-labelling experiments with 3,4-dihydroxy[U-(14)C]benzoic acid demonstrated that these compounds are not involved in the biosynthesis of either ubiquinone or phylloquinone in maize shoots. 7. Evidence is also presented to show that in maize shoots ring-opening of the aromatic amino acids takes place. The suggestion is offered that this may take place via homogentisic acid, as in animals and some micro-organisms.     

Identification of [1-14C]pantothenic-acid-mediated modified mitochondrial proteins

The in vivo administration of [1-14C]pantothenic acid, which is the precursor of coenzyme A, resulted in the radioactive labelling of several mitochondrial proteins in rat liver. The incorporated radioactivity could be released by glutathione or 2-mercaptoethanol. Two mitochondrial matrix proteins acetyl-CoA acetyltransferase (liver and heart), an enzyme involved in the biosynthesis or degradation of ketone bodies, and 3-oxoacyl-CoA thiolase (liver), a protein participating in fatty acid oxidation were identified as modified proteins. The radioactivity was localized exclusively in forms A1 and A2 indicating that these forms represent the modified states of the acetyl-CoA acetyltransferase protein. Kinetics of incorporation of radioactivity revealed an accumulation of the modified forms. The ratio of specific radioactivities of A2 compared to A1 was 2.41 +/- 0.15 (n = 10). After in vivo labelling with [14C]leucine, the specific radioactivity of acetyl-CoA acetyltransferase depended on the state of the enzyme protein. The unmodified enzyme exhibited a lower specific radioactivity than its modified forms suggesting different turnover rates of these proteins.     

Dipicolinic acid synthesis in Penicillium citreo-viride

Hodson, Phillip H. (University of Texas, Austin), and J. W. Foster. Dipicolinic acid synthesis in Penicillium citreo-viride. J. Bacteriol. 91:562-569. 1966.-Dipicolinic acid (DPA) accumulation in culture filtrates of the mold Penicillium citreo-viride was studied in surface and submerged cultures. Good DPA yields were obtained in suspensions of washed, submerged mycelium in the presence of a carbon and a nitrogen source but in the absence of other minerals essential for growth. Fumaric acid was the only other acid formed in significant amounts. Glucose and glycerol were superior to various salts of organic acids as carbon sources, and certain amino acids were excellent nitrogen sources. l-Leucine, l-norvaline, l-tyrosine, and l-histidine were superior to urea, NH(4)Cl, or NaNO(3) as nitrogen precursors for DPA production. d-Norvaline was useless for DPA production. Glycerol-2-C(14) and -1-C(14), C(14)O(2), and l-leucine-C(14), l-tyrosine-C(14), and l-histidine-C(14) were tested as precursors in conjunction with suitable carbon and nitrogen sources. The DPA was decarboxylated chemically, and the distribution of C(14) was determined in the pyridine-C and in the carboxyl-C. The data are consistent with Martin and Foster's suggestion for bacteria that the DPA molecule is formed by a condensation of C(3) plus C(4) precursors, the resulting 2-keto, 6-aminopimelic acid derivate undergoing ring closure to form a heterocyclic precursor of DPA. The C(14)O(2) experiments indicate that oxaloacetate is formed by beta-carboxylation of pyruvate, this in turn probably becoming aspartic acid beta-semialdehyde, the C(4) compound which condenses with a second pyruvate. The enhancement of DPA formation by l-norvaline, l-leucine, and l-histidine is not ascribable to their functioning either as a source of nitrogen or carbon. l-Tyrosine, in a glycerol medium, contributed nearly 40% of the DPA carbon. The mechanism of biosynthesis of C(7) straight-chain and cyclic compounds is discussed.     

A method for the degradation of radioactive nicotinic acid

A chemical degradation scheme is reported, which permits the measurement of the radioactivity of each carbon atom of nicotinic acid. Nicotinic acid is decarboxylated by heating with copper chromite to give carbon dioxide (C-7) and pyridine. The pyridine is converted into 4-nitropyridine 1-oxide, which is heated with aqueous calcium hypobromite to give tribromonitromethane. Combustion of the latter gives carbon dioxide derived from C-4 of the nicotinic acid. Nicotinic acid is also reduced to nipecotic acid, which is oxidized to succinic acid by acidic potassium permanganate. Stepwise degradation of the succinic acid by standard procedures gives two samples of carbon dioxide, which correspond to C-3, C-6 and C-4, C-5 of the nicotinic acid. Benzoylation of the nipecotic acid, followed by oxidation with permanganate at pH7, gives 5-amino-4-carboxyvaleric acid; this is converted into 2-methyleneglutaric acid by the action of nitrous acid. Hydrogenation of the 2-methyleneglutaric acid over rhodium in methanol gives 2-methylglutaric acid, which is oxidized with dilute chromic acid to acetic acid. Stepwise degradation of the acetic acid by standard procedures gives two samples of carbon dioxide, which correspond to C-2 and C-3 of the nicotinic acid. Thus the radioactivities of C-2, C-3, C-4 and C-7 are determined directly and those of C-5 and C-6 by difference. The method was shown to be isotopically valid for [2,3,7-¹⁴C]-, [4,6-¹⁴C₂]- and [5-¹⁴C]-nicotinic acid.     

The pattern of fatty acid synthesis in lactating rabbit mammary gland studied in vivo

The biosynthesis of fatty acids has been studied in lactating rabbits at 6h after intravenous injection of sodium [1-(14)C]acetate. The specific radioactivities of the individual fatty acids (C(6:0) to C(14:0)) and the proportions of these fatty acids synthesized were similar in mammary tissue and milk. Hexanoic acid had the highest specific radioactivity, and the C(8:0)-C(14:0) fatty acids had similar specific radioactivities, which were about five times those of C(16) and C(18) acids. No radioactivity was detected in fatty acids of chain length C(14) in these tissues were similar to those of the long-chain fatty acids in the milk and mammary gland. The results show that the C(4:0)-C(14:0) fatty acids are synthesized within the mammary gland rather than by fatty acid uptake from circulating blood or by oxidation of long-chain fatty acids within the gland. We conclude that de novo synthesis of esterified fatty acids in vivo by this tissue has a high degree of chain-length specificity.     

Sterol biosynthesis in the echinoderm Asterias rubens.

1. [2(-14)C]Mevalonic acid injected into the echinoderm Asterias rubens (Class Asteroidea) was effectively incorporated into the non-saponifiable lipid. 2. The most extensively labelled compounds were squalene and the 4,4-dimethyl sterols with much lower incorporations into the 4alpha-monomethyl and 4-demethyl sterol fractions. 3. Labelled compounds identified were squalene, lanosterol, 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol and 4alpha-methyl-5alpha-cholest-7-en-3beta-ol; these are all intermediates in sterol biosynthesis. 4. The major sterol in A. rubens, 5alpha-cholest-7-en-3beta-ol, was also labelled showing that this echinoderm is capable of sterol biosynthesis de novo. 5. No evidence was obtained for the incorporation of [2(-14)C]mevalonic acid into the C28 and C29 components of the 4-demethyl sterols or 9beta,19-cyclopropane sterols found in A. rubens and it is assumed that these sterols are of dietary origin. 6. Another starfish Henricia sanguinolenta also incorporated [2(-14)C]mevalonic acid into squalene and lanosterol. 7. Various isolated tissues of A. rubens were all capable of incorporation of [2(-14)C]mevalonic acid into the nonsaponifiable lipid. With the body-wall and stomach tissues radioactivity accumulated in squalene and the 4,4-dimethyl sterols, but with the gonads and pyloric caecae there was a more efficient incorporation of radioactivity into the 4-demethyl sterols, principally 5alpha-cholest-7-en-3beta-ol.     

Biosynthesis of the pyrimidinyl amino acid lathyrine by Lathyrus tingitanus L.

The biosynthesis of the pyrimidinyl amino acid lathyrine by seedlings of Lathyrus tingitanus L. was shown to be stimulated by uracil. [6(-14)C]Orotate, [2(-14)C]uracil and [3(-14)C]serine were incorporated into lathyrine; the incorporation of [6(-14)C]orotate was substantially decreased in the presence of uracil. Chemical degradation to locate the 14C incorporated from labelled precursors showed that 90% of the radioactivity incorporated into lathyrine from [3(-14)C]serine could be recovered in the alanine side chain. Over 80% of the radioactivity incorporated from [2(-14)C]uracil was shown to be located in C-2 of lathyrine. It is concluded that under the conditions studied, lathyrine arises from a preformed pyrimidine arising via the orotate pathway. Paradoxically, it was also possible to confirm previous reports that radioactivity from L-[guanidino-14C]homoarginine is incorporated into lathyrine and gamma-hydroxyhomoarginine. However, as homoarginine and gamma-hydroxyhomoarginine are also both labelled by [2(-14)C]uracil, it is suggested that they are products of the ring-opening of lathyrine and that reversibility of this process accounts, at least in part, for their observed experimental incorporation into lathyrine.     

Measurement of the time between biosynthesis and surface excretion of sebaceous lipids in the horse

The time between the biosynthesis and excretion of sebum to the skin surface of the horse was examined by in vivo intradermal injection of [1-14C]acetate followed by periodic surface lipid collections. The radiolabelling of the major neutral lipid classes, equolides (giant ring omega-lactones, C32-C36) and cholesteryl esters, was evaluated by thin-layer chromatography and autoradiography. The distribution of radioactivity within the monounsaturated equolides was examined by oxidative fragmentation and evaluation of the products. A peak of radioactivity in the equolides and cholesteryl esters occurred 15-21 days and 10-16 days, respectively, after injection. The time-courses of specific radioactivity of the two types of equolide oxidation fragments were also found to be dissimilar. The results are interpreted as indicating that in the biosynthesis of a large proportion of the equolides, de novo fatty acid synthesis was not followed immediately by fatty acid chain elongation. The phospholipids of the sebaceous cells are proposed as the long-term intermediate in which fatty acids reside between these two biosynthetic processes.     

Dipicolinic acid prevents the copper-dependent oxidation of low density lipoprotein

Effect of dipicolinic acid (pyridine 2,6-dicarboxylic acid) and pyridine compounds on the copper-dependent oxidation of human low density lipoprotein was analyzed in relation to the inhibition of copper reduction. Dipicolinic acid inhibited copper-dependent LDL oxidation completely, but the LDL oxidation was slightly inhibited by pyridine compounds with one carboxyl group at 2 or 6-position. Reduction of copper by LDL itself and ascorbate was inhibited completely by dipicolinic acid, but only partially by picolinic acid, quinolinic acid and isocinchomeronic acid with 2- or 6-carboxylic group. Pyridine compounds without 2- or 6-carboxyl group did not show any inhibitory effect on the LDL oxidation and the copper reduction. Protective effect of dipicolinic acid on the LDL oxidation was closely correlated with the copper-reducing activity. Dipicolinic acid shows an antioxidant action by the formation of a chelation complex with copper. This may have implications in understanding mechanisms of preventing LDL oxidation during the early phase of atherosclerosis.     

The role of peroxisomal fatty acyl-CoA beta-oxidation in bile acid biosynthesis

The physiological role of the peroxisomal fatty acyl-CoA beta-oxidizing system (FAOS) is not yet established. We speculated that there might be a relationship between peroxisomal degradation of long-chain fatty acids in the liver and the biosynthesis of bile acids. This was investigated using [1-14C]butyric acid and [1-14C]lignoceric acid as substrates of FAOS in mitochondria and peroxisomes, respectively. The incorporation of [14C]lignoceric acid into primary bile acids was approximately four times higher than that of [14C]butyric acid (in terms of C-2 units). The pools of these two fatty acids in the liver were exceedingly small. The incorporations of radioactivity into the primary bile acids were strongly inhibited by administration of aminotriazole, which is a specific inhibitor of peroxisomal FAOS in vivo [F. Hashimoto and H. Hayashi (1987) Biochim. Biophys. Acta 921, 142-150]. Aminotriazole inhibited preferentially the formation of cholate, the major primary bile acid, from both [14C]lignoceric acid and [14C]butyric acid, rather than the formation of chenodeoxycholate. The former inhibition was about 70% and the latter was approximately 40-50%. In view of reports that cholate is biosynthesized from endogenous cholesterol, the above results indicate that peroxisomal FAOS may have an anabolic function, supplying acetyl CoA for bile acid biosynthesis.     

The catabolism of cholesterol in vitro. Formation of 3α,7α,12α-trihydroxy-5β-cholestanoic acid from cholesterol by rat liver

1. Both 25-d- and 25-l-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid were isolated from the gall-bladder bile of Crocodylus niloticus. 2. The catabolism of cholesterol to 25-d- and 25-l-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid respectively was studied by using a rat liver preparation in vitro. The results show that rat liver can metabolize cholesterol to both forms of 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid. However, a preference was noted for the formation from [4-(14)C]cholesterol of 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid (25-d), which was isolated from the incubations with a specific radioactivity about four times that of 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid (25-l). 3. The results indicate that 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid is a normal intermediate in the biosynthesis of bile acids from cholesterol in the rat.     

The effect of carbon monoxide on the nature of the accumulated 4,4-dimethyl sterol precursors of cholesterol during its biosynthesis from [2-14C]mevalonic acid in vitro

Cholesterol biosynthesis was studied in rat liver subcellular fractions incubated with dl-[2-(14)C]mevalonic acid under gas phases consisting of either N(2)+O(2) (90:10) or CO+O(2) (90:10). CO inhibits cholesterol biosynthesis from [2-(14)C]mevalonic acid and results in a large accumulation of radioactive 4,4-dimethyl sterols. Separation of the components of the 4,4-dimethyl sterol fraction showed that lanosterol and dihydrolanosterol are the major components that accumulate during cholesterol biosynthesis in an atmosphere containing CO, whereas 14-demethyl-lanosterol and 14-demethyldihydrolanosterol are the major components of the much less intensely radioactive 4,4-dimethyl sterol fraction isolated from incubations with N(2)+O(2) as the gas phase. The identities of lanosterol, dihydrolanosterol and 14-demethyldihydrolanosterol were confirmed by both radiochemical and physicochemical methods, including g.l.c. and combined g.l.c.-mass spectrometry. CO therefore results in a qualitative as well as a quantitative difference in the 4,4-dimethyl sterol fraction which arises during cholesterol biosynthesis from mevalonic acid. The specific radioactivity of the [(14)C]lanosterol biosynthesized in the presence of CO was lower than that of its companion, [(14)C]dihydrolanosterol. The relative amounts of 4,4-dimethyl-Delta(24)-sterols and 4,4-dimethyl-24,25-dihydrosterols present in each type of incubation suggest that enzymic reduction of the sterol side chain occurs predominantly at a stage after that of lanosterol.     

Effect of short-term salt stress on the metabolic profiles of pyrimidine, purine and pyridine nucleotides in cultured cells of the mangrove tree, Bruguiera sexangula

To investigate the short‐term (3 h) effect of salt on the metabolism of purine, pyrimidine and pyridine nucleotides in mangrove (Bruguiera sexangula) cells, we examined the uptake and overall metabolism of radiolabelled intermediates involved in the de novo pathways and substrates of salvage pathways for nucleotide biosynthesis in the presence and absence of 100 mM NaCl. Uptake by the cells of substrates for the salvage pathways was much faster than uptake of intermediates of the de novo pathways. The activity of the de novo pyrimidine biosynthesis estimated by [2‐¹⁴C]orotate metabolism was not significantly affected by the salt. About 20–30% of [2‐¹⁴C]uridine, [2‐¹⁴C]uracil and more than 50% of [2‐¹⁴C]cytidine were salvaged for pyrimidine nucleotide biosynthesis. However, substantial quantities of these compounds were degraded to ¹⁴CO₂ via β‐ureidopropionate (β‐UP), and degradation of β‐UP was increased by the salt. The activities of the de novo pathway, estimated by [2‐¹⁴C] 5‐aminoimidazole‐4‐carboxamide ribonucleoside, and the salvage pathways from [8‐¹⁴C]adenosine and [8‐¹⁴C]guanosine for the purine nucleotide biosynthesis were not influenced by the salt. Most [8‐¹⁴C]hypoxanthine was catabolised to ¹⁴CO₂, and other purine compounds are also catabolised via xanthine. Purine catabolism was stimulated by the salt. [³H]Quinolinate, [carbonyl‐¹⁴C]nicotinamide and [carboxyl‐¹⁴C]nicotinic acid were utilised for the biosynthesis of pyridine nucleotides. The salvage pathways for pyridine nucleotides were significantly stimulated by the salt. Trigonelline was synthesised from all pyridine precursors that were examined; its synthesis was also stimulated by the salt. We discuss the physiological role of the salt‐stimulated reactions of nucleotide metabolism.     

The regiochemistry and stereochemistry of the biosynthesis of vitamin B6 from triose units

13C and 2H NMR spectroscopy has been employed to probe the biosynthesis of vitamin B6 in Escherichia coli. The 13C NMR spectrum of a sample of pyridoxol derived biosynthetically from D-[1,2,3,4,5,6-13C6]glucose shows that the bonds, C(2)-C(3) and C(4)-C(5), of the pyridine nucleus are the only two carbon-carbon bonds of pyridoxol which are generated de novo in the course of its biosynthesis from glucose. It follows that the pyridoxol skeleton is generated from two intact triose units and a triose-derived two-carbon unit, all of which are supplied by glucose. From the 2H NMR spectra of samples of pyridoxol derived from (R)-[1,1-2H2]glycerol and (S)-[1,1-2H2]glycerol, respectively, it can be deduced that the rehydroxymethyl group of glycerol enters C-2', C-4', and C-5' of the pyridoxol skeleton. It follows that each of the three fragments is derived from glycerol in stereo-specific fashion. These results answer questions concerning the regiochemistry and the stereochemistry of pyridoxol biosynthesis.     

Studies on the enzymic N-acylation of amino sugars in the sheep colonic mucosa

1. d-[2-(14)C]Glucose, [2-(14)C]acetate, hydroxy[3-(14)C]pyruvate, [3-(14)C]pyruvate and [U-(14)C]glycine were incorporated by surviving scrapings of sheep colonic mucosal tissue into glycoprotein. 2. d-[2-(14)C]Glucose, [2-(14)C]acetate, incorporated hydroxy-[3-(14)C]pyruvate and [3-(14)C]pyruvate resulted in labelling of each of the monosaccharide residues of the glycoprotein, namely N-glycollylneuraminic acid, N-acetylneuraminic acid, galactose, fucose, glucosamine and galactosamine. [U-(14)C]Glycine was incorporated as glycyl and seryl residues of the glycoprotein. 3. Despite N-glycollylneuraminic acid being quantitatively the predominant sialic acid (N-glycollylneuraminic acid and N-acetylneuraminic acid were 8.5 and 5.2% by weight of the glycoprotein respectively) the corresponding ratio of the radio-active labelling from d-[2-(14)C]glucose in N-glycollylneuraminic acid to that in N-acetylneuraminic acid was 1.00:7.27 (expressed as percentages of the total radioactivity in the glycoprotein). Neutral sugar, hexosamine and N-acetylneuraminic acid residues of the mucoprotein were each labelled to a similar extent. 4. Similarly, the ratio of the radioactivity in N-glycollylneuraminic acid to that in N-acetylneuraminic acid in the mucoprotein from tissue incubations with [2-(14)C]-acetate was 1.0:4.0. 5. Both [2-(14)C]acetate and [2-(14)C]glucose with whole tissue led to labelling of the N-glycollyl substituent and of the main nonose skeleton of the N-glycollylneuraminic acid. In whole-tissue incubations, [3-(14)C]pyruvate was also a precursor of radioactive N-glycollylneuraminic acid. 6. Hydroxy[3-(14)C]-pyruvate and [U-(14)C]glycine caused labelling of the carbohydrate and peptide residues of the glycoprotein, but did not give rise to labelling in the N-glycollylneuraminic acid residues. 7. With a wide variety of possible N-glycollyl precursors (fructose 6-phosphate, hydroxypyruvate, glycollate and chemically synthesized glycollyl-CoA) biosynthesis of N-glycollylglucosamine was not observed in cell-free preparations.     

Average structural and motional properties of a diunsaturated acyl chain in a lipid bilayer: effects of two cis-unsaturated double bonds

Isolinoleic acid (18:2 delta 6,9) deuterated at 10 different positions was esterified to form 1-palmitoyl-2-isolinoleoyl-sn-glycero-3-phosphocholine (PiLPC), and the average structural and motional properties of the diunsaturated chain, in aqueous dispersions of PiLPC, were examined by 2H NMR spectroscopy. For each sample, 2H spectra were acquired over a temperature range of 1-40 degrees C and the quadrupolar splittings interpreted in terms of carbon-deuterium bond order parameters, SCD. Furthermore, definition of the average orientation of the C8 methylene unit with respect to the bilayer normal [Baenziger, J. E., Smith, I. C. P., Hill, R. J., & Jarrell, H. C. (1988) J. Am. Chem. Soc. 110, 8229-8231] provided sufficient information to calculate both the average orientations and the molecular order parameters, Smol (which reflects the amplitudes of motion), for the C6-C7 and the C9-C10 double bonds. The results indicate that both the motional freedom (reflected in the order profile) and the average structure (reflected in the orientation of carbon segments with respect to the bilayer normal) are strongly affected by the presence of two cis-unsaturated double bonds. The data were interpreted in terms of two possible models whereby, in each case, the chain adopts a conformation consistent with the low-energy conformation of 1,4-pentadiene [Applegate, K. R., & Glomset, J. A. (1986) J. Lipid Res. 27, 658-680] but undergoes a two-site jump between the conformations. The jump motion arises mainly from rotations about the C7-C8 and the C8-C9 single bonds that disorder the C8 and the C9-C10 segments (Smol = 0.15 and 0.08, respectively) but leave the C6-C7 double bond relatively immobile (Smol = 0.55; all at 40 degrees C). It is suggested that acyl chains containing three or more double bonds could not undergo a similar jump motion and therefore would be highly ordered and not "fluid" as is generally thought.