Synthesis of fluoroacetate from fluoride, glycerol, and beta-hydroxypyruvate by Streptomyces cattleya.

Streptomyces cattleya produces fluoroacetate and 4-fluorothreonine from inorganic fluoride added to the culture broth. We have shown by 19F nuclear magnetic resonance (NMR) spectrometry that fluoroacetate is accumulated first in the culture broth and that accumulation of 4-fluorothreonine is next. To show precursors of the carbon skeleton of fluoroacetate, we carried out tracer experiments with various 14C- and 13C-labeled compounds. Radioactivity of [U-14C]glucose, [U-14C]glycerol, [U-14C]serine, and [U-14C]beta-hydroxypyruvate was incorporated into fluoroacetate to an extent of 0.2 to 0.4%, whereas [3-14C]pyruvate, [2,3-14C]succinate, and [U-14C]aspartate were less efficiently incorporated (0.04 to 0.08%). The addition of [2-13C]glycerol to the mycelium suspension of Streptomyces cattleya caused exclusive enrichment of the carboxyl carbon of fluoroacetate with 13C; about 40% of carboxyl carbon of fluoroacetate was labeled with 13C. We studied the radioactivity incorporation of [3-14C]-, [U-14C]-, and [1-14C]beta-hydroxypyruvates to show that C-2 and C-3 of beta-hydroxypyruvate are exclusively converted to the carbon skeleton of fluoroacetate. These results suggest that the carbon skeleton of fluoroacetate derives from C-1 and C-2 of glycerol through beta-hydroxypyruvate, whose hydroxyl group is eventually replaced by fluoride.     

Metabolism of (3-(14)C)tryptophan and (2-(14)C)alanine in cattle tissues

In the experiments involving incubation of the liver, brain cortex, muscle and adipose tissues homogenates with [3-14C] tryptophan for an hour 43.2-89.3% of the label was found in proteins, 7.2-47.2%--in lipids, 2.6-9.4%--in CO2. Following incubation of the above-mentioned tissue homogenates with [2-14C] alanine, proteins, lipids and CO2 contain 28.8-49.3%; 22.6-31.9% and 21.6-49.3% of radioactive label, respectively. Radioactivity of lipids synthesized by the homogenates of the investigated tissues from [3-14C] tryptophan and [2-14C] alanine is 23.5-63.5 and 21.1-56.0%, respectively, the radioactivity of CO2 being 1.4-5.1 and 9.3-11.8% of the above-mentioned compounds synthesized from [1-14C] acetate. The results obtained testify to the considerable contribution of [3-14C] tryptophan and [2-14C] alanine to protein synthesis as well as to their involvement in the substrate supply of lipogenesis and energetic processes in various organs and tissues of cattle.     

Distribution, metabolism and excretion of [1-14C]dolichol injected intravenously into rats.

[1-14C]Dolichol mixed in vitro with rat serum and injected intravenously into rats was rapidly cleared from the circulation in a manner consistent with a two-compartment model. About 80% of the radioactivity recovered from animals killed after 1 day was in the liver, with smaller amounts being found in lung, carcass (internal organs removed), gastrointestinal tract and contents, and spleen. The kidneys, testes and heart contained little radioactivity, and the brain did not appear to take up any [1-14C]dolichol. The half-life for the turnover of radioactivity from [1-14C]dolichol in tissues varied considerably, being 2 days for the lung, 17 for liver and about 50 days for the carcass. After 1 day, and also after 4 and 21 days, most of the radioactivity in all tissues was as [1-14C]dolichol and as [1-14C]dolichyl fatty acyl ester, although a small amount of incorporation of [1-14C]dolichol radioactivity into phospholipids was also observed. Faeces collected over the first 4 days after injection contained 13% of the [1-14C]dolichol dose, but urine and expired air contained only small amounts of radioactivity. Radioactivity in faeces was nearly all as unchanged [1-14C]dolichol and as [1-14C]dolichyl fatty acyl ester. The [1-14C]dolichol remaining in liver after 21 days appeared to be in a pool (possibly lysosomes) where most of it was not subject to excretion.     

Asymmetric incorporation of 4-(2′-carboxyphenyl)-4-oxobutyrate into phylloquinone by Zea mays

Radioactivity from 4-(2′-carboxyphenyl)-4-oxobutyrate-[2-¹⁴C] and 4-(2′-carboxyphenyl)- 4-oxobutyrate-[3-¹⁴C] was incorporated into C-3 and C-2 respectively of phylloquinone in maize shoots. These results show that this substrate is incorporated in the same asymmetric manner into phylloquinone as it is into the bacterial menaquinones.     

Phosphonopyruvic acid: A probable precursor of phosphonic acids in cell-free preparation of Tetrahymena.

1. In cell-free preparations of Tetrahymena, doubly labelled [32P]phosphoenol-[3-14C]pyruvate gives rise to 2-aminoethylphosphonate and 2-amino-3-phosphonopropionate, labelled with the two isotopes in the same ratio as the starting compound. The result is consistent with an intra-molecular rearrangement of phosphoenolpyruvate in the biosynthetic sequence of carbon-phosphorus bond formation. 2. Incubation of [32P]phosphoenolpyruvate with the same preparation, followed by treatment with 2,4-dinitrophenylhydrazine, yielded labelled hydrazones. When these were subjected to hydrogenolysis, the radioactivity was recovered in 2-aminoethylphosphonate and 2-amino-3-phosphonopropionate, suggesting that 2-phosphonoacetaldehyde and 3-phosphonopyruvic acid were probable precursors of the aminoalkylphosphonic acids. 3. Radioactivity from 2-amino-3-phosphono-[3-14C]propionic acid was incorporated into 2-aminoethylphosphonic acid, but incorporation of the radioactivity into lipids was negligible.     

Measurement of sodium ion concentration in the unstirred layer of rat small intestine by polymer Na+-sensitive electrodes

[14C]Formate is incorporated into the C-2 of the pyrimidine moiety of thiamin by Escherichia coli and Salmonella typhimurium. In Saccharomyces cerevisiae, it is incorporated into C-4. Radioactive carbons of [1-14C]glycine and [2-14C]glycine are incorporated by S. typhimurium into the C-4 and C-6 of the pyrimidine, respectively, but not by S. cerevisiae. These facts suggest that procaryotes and eucaryotes have different biosynthetic pathways for pyrimidine. In this study, the procaryotes tested incorporated [14C]formate into the C-2 and the eucaryotes incorporated it into the C-4 of the pyrimidine.     

In vitro synthesis and O acetylation of peptidoglycan by permeabilized cells of Proteus mirabilis.

The synthesis and O acetylation in vitro of peptidoglycan by Proteus mirabilis was studied in microorganisms made permeable to specifically radiolabelled nucleotide precursors by treatment with either diethyl ether or toluene. Optimum synthesis occurred with cells permeabilized by 1% (vol/vol) toluene in 30 mM MgCl2 in in vitro experiments with 50 mM Tris-HCl buffer (pH 6.80). Acetate recovered by mild base hydrolysis from sodium dodecyl sulfate-insoluble peptidoglycan synthesized in the presence of UDP-[acetyl-1-14C]N-acetyl-D-glucosamine was found to be radioactive. Radioactivity was not retained by peptidoglycan synthesized when UDP-[acetyl-1-14C]N-acetyl-D-glucosamine was replaced with both unlabelled nucleotide and either [acetyl-3H]N-acetyl-D-glucosamine or [glucosamine-1,6-3H]N-acetyl-D-glucosamine. In addition, no radioactive acetate was detected in the mild base hydrolysates of peptidoglycan synthesized in vitro with UDP-[glucosamine-6-3H]N-acetyl-D-glucosamine as the radiolabel. Chasing UDP-[acetyl-1-14C]N-acetyl-D-glucosamine with unlabelled material served to increase the yield of O-linked [14C]acetate, whereas penicillin G blocked both peptidoglycan synthesis and [14C]acetate transfer. These results support the hypothesis that the base-labile O-linked acetate is derived directly from N-acetylglucosamine incorporated into insoluble peptidoglycan via N----O transacetylation and not from the catabolism of the supplemented peptidoglycan precursors followed by subsequent reactivation of acetate.     

Mechanism of pyruvate-uridine diphospho-N-acetylglucosamine transferase. Evidence for an enzyme-enolpyruvate intermediate.

The enzyme, phosphoenolpyruvate:uridine-5-diphospho-N-acetyl-2-amino-2-deoxyglucose-3-enolpyruvyltransferase, which catalyzes the transfer of enolpyruvate from phosphoenolpyruvate to uridine diphospho-N-acetylglucosamine with the liberation of Pi, was found to form a covalent intermediate with the enolpyruvate moiety. Radioactivity from [1-14-C]phosphoenolpyruvate in the forward reaction and from UDP-GlNAc-[1-14-C]enolpyruvate in the reverse reaction was incorporated into the enzyme and remained bound to the protein after precipitation with ammonium sulfate or treatment with sodium dodecyl sulfate and heat. This incorporation from UDP-GlcNAc-[1-14-C]enolpyruvate took place in the absence of Pi. When [32-P,1-14C]phosphoenolpyruvate was used, only 14-C appeared to be incorporated. In the forward reaction, the incorporation was contingent on the removal of UDP-GlcNAc from the transferase. Consistent with the formation of an enzyme-enolpyruvate intermediate, exchange of UDP-[6-3-H]GlcNAc with UDP-GlcNAc-enolpyruvate was observed in the absence of Pi. Nonstoichiometric incorporation of 3H from 3H2O into the product, UDP-GlcNAc-enolpyruvate, was observed and was shown to be due to a product isotope effect. Based on these observations, a mechanism of action for this enzyme is proposed which involves synchronous addition-elimination followed by a second addition-elimination step.     

Biosynthesis of riboflavin. Enzymatic formation of 6,7-dimethyl-8-ribityllumazine from pentose phosphates

The xylene ring of riboflavin originates by dismutation of the precursor, 6,7-dimethyl-8-ribityllumazine. The formation of the latter compound requires a 4-carbon unit as the precursor of carbon atoms 6 alpha, 6, 7, and 7 alpha of the pyrazine ring. The formation of riboflavin from GTP and ribose phosphate by cell extract from Candida guilliermondii has been observed by Logvinenko et al. (Logvinenko, E. M., Shavlovsky, G. M., Zakal'sky, A. E., and Zakhodylo, I. V. (1982) Biokhimiya 47, 931-936). We have studied this enzyme reaction in closer detail using carbohydrate phosphates as substrates and synthetic 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione or its 5'-phosphate as cosubstrates. Several pentose phosphates and pentulose phosphates can serve as substrate for the formation of riboflavin with similar efficiency. The reaction requires Mg2+. Various samples of ribulose phosphate labeled with 14C or 13C have been prepared and used as enzyme substrates. Radioactivity was efficiently incorporated into riboflavin from [1-14C]ribulose phosphate, [3,5-14C]ribulose phosphate, and [5-14C]ribulose phosphate, but not from [4-14C]ribulose phosphate. Label from [1-13C]ribose 5-phosphate was incorporated into C6 and C8 alpha of riboflavin. [2,3,5-13C]Ribose 5-phosphate yielded riboflavin containing two contiguously labeled segments of three carbon atoms, namely 5a, 9a, 9 and 8, 7, 7 alpha. 5-Amino-6-[1'-14C] ribitylamino-2,4 (1H,3H)-pyrimidinedione transferred radioactivity exclusively to the ribityl side chain of riboflavin in the enzymatic reaction. It follows that the 4-carbon unit used for the biosynthesis of 6,7-dimethyl-8-ribityllumazine consists of the pentose carbon atoms 1, 2, 3, and 5 in agreement with earlier in vivo studies.     

alpha-Ketoglutarate dehydrogenase complex of Escherichia coli. A hybrid complex containing pyruvate dehydrogenase subunits from pyruvate dehydrogenase complex

The alpha-ketoglutarate dehydrogenase complex of Escherichia coli utilizes pyruvate as a poor substrate, with an activity of 0.082 units/mg of protein compared with 22 units/mg of protein for alpha-ketoglutarate. Pyruvate fully reduces the FAD in the complex and both alpha-keto[5-14C]glutarate and [2-14C]pyruvate fully [14C] acylate the lipoyl groups with approximately 10 nmol of 14C/mg of protein, corresponding to 24 lipoyl groups. NADH-dependent succinylation by [4-14C]succinyl-CoA also labels the enzyme with approximately 10 nmol of 14C/mg of protein. Therefore, pyruvate is a true substrate. However, the pyruvate and alpha-ketoglutarate activities exhibit different thiamin pyrophosphate dependencies. Moreover, 3-fluoropyruvate inhibits the pyruvate activity of the complex without affecting the alpha-ketoglutarate activity, and 2-oxo-3-fluoroglutarate inhibits the alpha-ketoglutarate activity without affecting the pyruvate activity. 3-Fluoro[1,2-14C]pyruvate labels about 10% of the E1 components (alpha-ketoacid dehydrogenases). The dihydrolipoyl transsuccinylase-dihydrolipoyl dehydrogenase subcomplex (E2E3) is activated as a pyruvate dehydrogenase complex by addition of E. coli pyruvate dehydrogenase, the E1 component of the pyruvate dehydrogenase complex. All evidence indicates that the alpha-ketoglutarate dehydrogenase complex purified from E. coli is a hybrid complex containing pyruvate dehydrogenase (approximately 10%) and alpha-ketoglutarate dehydrogenase (approximately 90%) as its E1 components.     

Biosynthesis of a mycobacterial lipopolysaccharide. Incorporation of [14C]acyl groups by whole cells in vivo

1. Mycobacterium phlei (A.T.C.C. 356) cells were incubated with (14)C-labelled short-chain fatty acids and the 6-O-methylglucose-containing lipopolysaccharides that became esterified with radioactive acyl groups were isolated. The pattern of labelling of these lipopolysaccharides with the different acyl groups, the effects of different conditions on labelling patterns, and the kinetics of the turnover of (14)C-labelled acyl groups were studied. 2. The labelling patterns are summarized as follows. [1-(14)C]Acetate was incorporated into all of the acyl groups. [1-(14)C]Propionate led to labelling of propionate and succinate, while [1-(14)C]isobutyrate was incorporated mostly as such, along with a trace amount in iso-octanoate. 3. Under the conditions of the experiments, [1-(14)C]acetate was rapidly incorporated into succinyl (3-carboxypropionyl) and octanoyl groups, whereas the acetyl groups themselves were labelled more slowly. Radioactivity in propionyl and succinyl groups, originating from [1-(14)C]propionate, attained maximum values and then gradually decreased in both. Incorporation of [1-(14)C]isobutyrate proceeded slowly but reached a plateau and remained constant. While n-butyrate is not a normal constituent of methyl-glucose-containing lipopolysaccharides, it was incorporated as such when n-[1-(14)C]-butyrate was supplied in the medium. 4. [1-(14)C]Acetyl groups were readily displaced by unlabelled acetate. On the other hand, the specific radioactivity of the succinyl group continued to increase during a 3h incubation with unlabelled succinate. Propionyl and succinyl groups, labelled by [1-(14)C]propionate, were displaced slowly by unlabelled propionate or succcinate. The isobutyryl group of the lipopolysaccharides did not turn over, in contrast to the results obtained with the other acyl substituents.     

Escherichia coli pyruvate dehydrogenase complex. Thiamin pyrophosphate-dependent inactivation by 3-bromopyruvate

Inactivation of the pyruvate dehydrogenase complex by 3-bromopyruvate is thiamin pyrophosphate (TPP)-dependent. Inactivation with 2-14C- or 3-14C-labeled 3-bromopyruvate results in TPP-dependent covalent labeling of more than 60 sites in the complex, all of which are associated with the dihydrolipoyl transacetylase component. Inactivation by 3-bromo[1-14C]pyruvate labels up to 20 sites associated with dihydrolipoyl transacetylase, also with TPP dependence. Systemic chemical degradation of the complex inactivated by 3-bromo[2-14C]pyruvate under conditions that would convert lipoyl groups to S,S,-biscarboxymethyl dihydrolipoic acid produces S,S,-bis[14C]carboxymethyl dihydrolipoic acid. It is concluded that 3-bromopyruvate inactivates this complex by initially undergoing the first two steps of the usual catalytic pathway, TPP-dependent decarboxylation followed by reductive bromoacetylation of lipoyl moieties. The sulfhydryl groups of S-bromoacetyl dihydrolipoyl moieties generated by reductive bromoacetylation are then alkylated by 3-bromopyruvate as well as by bromoacetyl thioester groups associated with the complex.     

Incorporation of shikimate and 4-(2′-carboxyphenyl)-4-oxobutyrate into phylloquinone

The patterns of incorporation of d-[G-¹⁴C]shikimate and variously labelled ¹⁴C-4-(2′-carboxy-phenyl)-4-oxobutyrate into the naphthoquinone nucleus of phylloquinone by maize shoots have been investigated. The results show that (a) the alicyclic ring and C-7 of shikimate give rise to Ring A and either C-1 or C-4, and (b) the phenyl ring, 2′-carboxy and C-4, and C-2 and -3 of 4-(2′-carboxyphenyl)-4-oxobutyrate give rise to Ring A, C-1 and -4 and C-2 and -3. Radioactivity from α-[1-¹⁴C]naphthol, 1,4-[1,4-¹⁴C]naphthoquinone and [Me-¹⁴C]menadione is not incorporated into phylloquinone to any significant extent.     

Metabolism of methyl n-amyl ketone (2-heptanone) and its binding to DNA of rat liver in vivo and in vitro

Methyl n-amyl ketone (2-heptanone), a reported metabolite of 2-ethylhexanol which in turn is a primary metabolite of plasticizers such as di-(2-ethylhexyl) phthalate, is metabolized in male Fischer 344 rats to CO2, acetate and a variety of compounds that could be either anabolic or catabolic or a combination of the two. A significant percentage of the radioactivity given orally (gavage) as [2-14C]-2 heptanone, at least 10%, was not excreted from the body in 48 h. Radioactivity was incorporated into liver protein in the form of three unidentified products as well as [14C]arginine, and into DNA both as 14C-labeled normal nucleosides (50-75%) and as presently unidentified hydrophobic materials (25-50%). Urea and cholesterol were significantly labeled, indicative of anabolic reutilization of [2-14C]-2-heptanone breakdown products. The 2-heptanone also bound to DNA spontaneously in vitro, to the extent of 400 pmol/mg DNA.     

The origin of the sulfur atom of thiamin

The incorporation of the sulfur atom of 35S-labeled amino acids into thiamin in Escherichia coli and Saccharomyces cerevisiae was studied. The specific radioactivity of the S atoms was incorporated at similar levels into thiamin and cysteine residues in cell proteins. However, the specific radioactivity of the S atoms from [35S]methionine was not incorporated into thiamin but into methionine residues in cell proteins. Thus, the origin of the S atom of thiamin was established as being the S atom of cysteine. No activity from [U-14C]cysteine was recovered in thiamin, proving that the carbon skeleton of this amino acid was not utilized in synthesizing the thiazole moiety of thiamin.     

Metabolic fate of 14C-labelled nicotinamide and adenine in germinating propagules of the mangrove Bruguiera gymnorrhiza

We studied the metabolic fate of [carbonyl-14C]nicotinamide and [8-(14)C]adenine in segments taken from young and developing leaves, stem, hypocotyls, and roots of a shoot-root type emerging propagule of the mangrove plant Bruguiera gymnorrhiza. Thin-layer chromatography was used together with a bioimaging analyser system. During 4 h of incubation, incorporation of radioactivity from [carbonyl-14C]nicotinamide into NAD and trigonelline was found in all parts of the propagules; the highest incorporation rates into NAD and trigonelline were found in newly emerged stem and young leaves, respectively. Radioactivity from [8-(14)C]adenine was distributed mainly in the salvage products (adenine nucleotides and RNA), and incorporation was less in catabolites (allantoin, allantoic acid, and CO2). Adenine salvage activity was higher in young leaves and stem than in hypocotyls and roots. Over a short time, the effect of 500 mM NaCl on nicotinamide and adenine metabolism indicated that NaCl inhibits both salvage and degradation activities in roots.     

Amino acid sequence of the diazooxonorleucine binding site of Acinetobacter and Pseudomonas 7A glutaminase--asparaginase enzymes

Acinetobactor glutaminase-asparaginase was treated with [6-14C]diazo-5-oxonorleucine, reduced with sodium borohydride, and cleaved with cyanogen bromide. Radioactivity was present only in a 96-residue-N-terminal peptide which eluted as the second peptide peak on Sephadex G-50. Radioactivity was released with the threonine in position 12 during automatic sequencing of this peptide. The amino acid sequence of a 60-residue tn-terminal segment and a 16-residue C-terminal segment of this peptide was determined. Pseudomonas 7 A glutaminase-asparaginase was treated with [6-14C]diazo-5-oxonorleucine and reduced with sodium borohydride. Radioactivity was released with the threonine in residue 20 during automatic sequencing of the whole enzyme. Analysis of 26 N-terminal residues showed that an 8-residue segment containing the radioactive threonine was identical with that in Acinetobacter glutaminase-asparaginase and in Escherichia coli asparaginase. Additional identical residues were noted in the N-terminal regions of these enzymes.     

Glycine formation during growth of Pseudomonas AM1 on methanol and succinate

1. The mechanism of regeneration of glycine during the growth of Pseudomonas AM1 on C(1) compounds has been investigated by brief incubation of bacterial suspensions with [2,3-(14)C(2)]succinate and observing the incorporation of radioactivity into various metabolites. 2. With the wild-type organism growing on methanol, radioactivity appeared rapidly in glycine and tricarboxylic acid-cycle intermediates, but there was a relatively slow labelling of serine and phosphorylated compounds. Serine became labelled predominantly in the C-2 position. 3. The proportion of radioactivity incorporated into glycine at earliest times was greatly diminished when succinate-grown cells were used. 4. Radioactivity was also incorporated from [2,3-(14)C(2)]succinate into glycine and serine by methanol-grown mutant 20S, which lacks phosphoserine phosphohydrolase. Both the glycine and serine were labelled mainly in C-2. 5. The formation of predominantly [2-(14)C]serine from [2,3-(14)C(2)]succinate in wild-type Pseudomonas AM1, and of [2-(14)C]serine and [2-(14)C]glycine in the mutant lacking the phosphorylated pathway from succinate to serine, is taken as strong evidence for a mechanism of glycine regeneration involving cleavage of a C(4) skeleton between C-2 and C-3, rather than by a direct combination of two C(1) units derived from the growth substrate. 6. The cleavage mechanism is quantitatively more significant during growth on methanol than on succinate.     

Incorporation of [14C] arachidonic acid into ovine conceptus and endometrial lipids.

The purpose of this study was to quantitate conceptus and endometrial incorporation of [14C]arachidonic acid (AA) into individual neutral and polar lipids. Endometrium and conceptuses from pregnant ewes and endometrium from nonbred ewes were collected 14 and 16 d after onset of estrus (d 0). Tissues were incubated for 8 h at 37 degrees C in medium containing 1 microCi of [14C]AA. Thin-layer chromatographic procedures were used to separate 12 lipids. Radioactivity was measured in each lipid, and the amount (ng) of [14C]AA incorporated into each lipid was calculated. Conceptuses and endometrium incorporated more [14C]AA into triacylglycerols than into any other lipid. Day and tissue type affected differentially (i.e., day X tissue interaction) the incorporation of [14C]AA into several lipids; d-14 conceptuses incorporated [14C]AA more actively than did any other day-tissue combination. Results indicate that triacylglycerols may be an important reservoir for conceptus and endometrial AA. The remarkable ability of d-14 conceptuses to incorporate [14C]AA into various lipids may be important for their accelerated elongation and active prostaglandin synthetic system.     

Plasmodium knowlesi: In vitro biosynthesis of methionine

Methionine biosynthesis was studied in rhesus monkey erythrocytes infected with Plasmodium knowlesi malaria which were cultured in vitro with l-[3-¹⁴C]serine, methyl-[¹⁴C]tetrahydrofolic acid, and l-[³⁵S]homocysteine. Radioactivity derived from [3-¹⁴C]serine was detected in approximately equivalent amounts in methionine and thymidylic acid by thin-layer chromatography of acid-hydrolysates of washed erythrocytes. The results with methyl-[¹⁴C]tetrahydrofolic acid were inconclusive. Radioactivity from l-[³⁵S]homocysteine also appeared in methionine but the level of homocysteine required for maximal activity was tenfold that of serine. The results indicate that the serine: 5,10-methylenetetrahydrofolic acid: 5-methyl-tetrahydrofolic acid: methionine biosynthetic pathway is present in the P. knowlesi malaria parasite.