A rapidly metabolizing pool of phosphatidylglycerol as a precursor of phosphatidylethanolamine and diglyceride in Bacillus megaterium.

Pulse-chase experiments in Bacillus megaterium ATCC 14581 with [U-14C]palmitate, L-[U-14C]serine, and [U-14C]glycerol showed that a large pool of phosphatidylglycerol (PG) which exhibited rapid turnover in the phosphate moiety (PGt) underwent very rapid interconversion with the large diglyceride (DG) pool. Kinetics of DG labeling indicated that the fatty acyl and diacylated glycerol moieties of PGt were also utilized as precursors for net DG formation. The [U-14C]glycerol pulse-chase results also confirmed the presence of a second, metabolically stable pool of PG (PGs), which was deduced from [32P]phosphate studies. The other major phospholipid, phosphatidylethanolamine (PE), exhibited pronounced lags relative to PG and DG in 14C-fatty acid, [14C]glycerol, and [32P]phosphate incorporation, but not for incorporation of L-[U-14C]serine into the ethanolamine group of PE or into the serine moiety of the small phosphatidylserine (PS) pool. Furthermore, initial rates of L-[U-14C]serine incorporation into the serine and ethanolamine moieties of PS and PE were unaffected by cerulenin. The results provided compelling in vivo evidence that de novo PGt, PS, and PE synthesis in this organism proceed for the most part sequentially in the order PGt yields PS yields PE rather than via branching pathways from a common intermediate and that the phosphatidyl moiety in PS and PE is derived largely from the corresponding moiety in PGt, whereas the DG pool indirectly provides an additional source for this conversion by way of the facile PGt in equilibrium or formed from DG interconversion.     

Niacin Biosynthesis in Seedlings of Zea mays

Evidence obtained from incubation of corn (Zea mays cv. Golden Bantam) seedlings in dl-[benzene ring-U-(14)C]tryptophan, l-[5-(3)H]tryptophan, l-[U-(14)C]aspartate and [U-(14)C]glycerol indicates that niacin is synthesized in these plants via oxidative degradation of tryptophan. Aspartate and glycerol do not appear to be precursors of niacin in corn seedlings.     

Studies on antibiotic biosynthesis by protoplasts and resting cells of Streptomyces echinatus. Part II. Effect of chromophore precursors

Washed cell and protoplast suspensions from Streptomyces echinatus A8331, which produces the quinoxaline antibiotic echinomycin, have been used to study the effects of analogues of the natural chromophore upon antibiotic biosynthesis. Addition of quinoline-2-carboxylic acid caused a decrease in the labelling of echinomycin from L-[methyl-14C]methionine and an increase in labelled chloroform-extractable material. Quinoxaline-2-carboxylic acid increased the incorporation of radioactivity into both fractions. Thieno[3,2-b]pyridine-5-carboxylic acid, 6-methylquinoline-2-carboxylic acid, and quinoline-2-carboxylic acid (also to a lesser extent 7-chloroquinoxaline-2-carboxylic acid) increased markedly the incorporation of radioactivity into chloroform-extractable material and virtually abolished echinomycin synthesis. Autoradiographs of extracts from suspensions supplemented with the latter four analogues revealed bis-substituted metabolites not found in unsupplemented cultures. When protoplast suspensions were incubated with L-[U-14C]serine, L-[U-14C]valine, or DL-[benzene ring-U-14C]tryptophan, quinoline-2-carboxylic acid, thieno[3,2-b]pyridine-5-carboxylic acid, and 6-methylquinoline-2-carboxylic acid directed the synthesis of antibiotically active bis derivatives at the expense of echinomycin. When analogues of quinoxaline-2-carboxylic acid previously found unsuitable for incorporation by growing cultures were tested in protoplast suspensions, only isoquinoline-3-carboxylic acid caused a large increase in the incorporation of radioactivity from L-[methyl-14C]methionine into chloroform-extractable material. With DL-[benzene ring-U-14C]tryptophan as the radiolabel, benzotriazoline-2-acetic acid and 6-bromoquinoxaline-2-carboxylic acid as well as isoquinoline-3-carboxylic acid sharply reduced the labelling of echinomycin.     

The metabolism of L-tryptophan by isolated rat liver cells. Quantification of the relative importance of, and the effect of nutritional status on, the individual pathways of tryptophan metabolism.

1. The metabolism of L-tryptophan by liver cells prepared from fed and 48 h-starved rats was studied. Methods are described, with the use of L-[ring-2-(14)C], L-[carboxy-14C]-and L-[benzene-ring-U-14C]-tryptophan, for the simultaneous determination of tryptophan 2,3-dioxygenase and kynureninase activities and of the oxidation of tryptophan to CO2 and non-aromatic intermediates of the kynurenine-glutarate pathway. 2. At physiological concentrations (0.1 mM), tryptophan was oxidized by tryptophan 2,3-dioxygenase at comparable rates in liver cells from both fed and starved rats. Kynureninase activity of hepatocytes from starved rats was 50% greater than that of cells from fed rats. About 10% of the tryptophan metabolized by tryptophan 2,3-dioxygenase was degraded completely to CO2. 3. In the presence of 0.5 mM-L-tryptophan, tryptophan 2,3-dioxygenase and kynureninase activities increased 5--6-fold. Liver cells from starved rats oxidized tryptophan at about twice the rate of these from fed rats. Degradation of tryptophan to non-aromatic intermediates of the glutarate pathway and CO2 was increased only 3-fold, suggesting an accumulation of aromatic intermediates of the kynurenine pathway. 4. Rates of metabolism with 2.5 mM-L-tryptophan were not significantly different from those obtained with 0.5 mM-tryptophan. 5. Rates of synthesis of quinolinic acid from 0.5 mM-L-tryptophan, determined either by direct quantification or indirectly from rates of radioisotope release from L-[carboxy-(14)C]- and [benzene-ring-U-14C]tryptophan, were essentially similar. 6. At all three concentrations examined, tryptophan was degraded exclusively through kynurenine; there was no evidence of formation of either indol-3-ylacetic acid or 5-hydroxyindol-3-ylacetic acid.     

Oxalic acid biosynthesis and oxalacetate acetylhydrolase activity in Streptomyces cattleya

In addition to producing the antibiotic thienamycin, Streptomyces cattleya accumulates large amounts of oxalic acid during the course of a fermentation. Washed cell suspensions were utilized to determine the specific incorporation of carbon-14 into oxalate from a number of labeled organic and amino acids. L-[U-14C]aspartate proved to be the best precursor, whereas only a small percentage of label from [1,5-14C]citrate was found in oxalate. Cell-free extracts catalyzed the formation of [14C]oxalate and [14C]acetate from L-[U-14C]aspartate. When L-[4-14C]aspartate was the substrate only [14C]acetate was formed. The cell-free extracts were found to contain oxalacetate acetylhydrolase (EC 3.7.1.1), the enzyme that catalyzes the hydrolysis of oxalacetate to oxalate and acetate. The enzyme is constitutive and is analogous to enzymes in fungi that produce oxalate from oxalacetate. Properties of the crude enzyme were examined.     

Synthesis of fluorescent and radiolabeled analogues of phosphatidic acid

Procedures for the synthesis of fluorescent and radiolabeled analogues of phosphatidic acid are described. The fluorophore 7-nitrobenzo-2-oxa-1,3-diazole (NBD) was coupled to 6-amino-caproic acid and 12-aminododecanoic acid by reaction of NBD-chloride with the amino acids under mild alkaline conditions at room temperature. 1,2-Dioleoyl-sn-[U-14C]glycerol 3-phosphate was prepared by acylation of sn-[U-14C]glycerol 3-phosphate with oleic acid anhydride using dimethylaminopyridine as the catalyst. This compound was converted to 1-oleoyl-sn-[U-14C]glycerol 3-phosphate by hydrolysis with phospholipase A2. The lysophosphatidic acid was reacylated with NBD-aminocaproyl imidazole or NBD-aminododecanoyl imidazole to form the fluorescent, radiolabeled analogue of phosphatidic acid. Fluorescent, non-radiolabeled analogues of phosphatidic acid were prepared by phospholipase D hydrolysis of fluorescent phosphatidylcholine.     

De novo synthesis of diacylglycerol from glucose. A new pathway of signal transduction in human neutrophils stimulated during phagocytosis of beta-glucan particles.

The phagocytosis of beta-glucan particles by human neutrophils and the associated activation of NADPH O2- forming oxidase were accompanied by an increased hydrolysis of phosphoinositides by phospholipase C, hydrolysis of phosphatidylcholine by phospholipase D, accumulation of diglyceride (DG) mass, and [Ca2+]i rise. The reaction of phospholipid hydrolysis played a minor role in the formation of DG, which was mainly formed by de novo synthesis from glucose. The activation of this pathway was shown by the stimulation of the incorporation of [U-14C]glucose into DG, which occurred very rapidly after the challenge of neutrophils with beta-glucan particles. This DG derived from glucose was found almost completely as 1-acyl-2-acyl-glycerol (DAG). On the basis of the finding that phosphatidic acid was the precursor of DAG, an increase in the incorporation of [U-14C]acetate into DAG did not occur, and the [14C]radioactivity was in the glycerol backbone, the synthesis of DAG from [U-14C]glucose occurred very likely via dihydroxyacetone phosphate and glycerol 3-phosphate, stepwise acylation to phosphatidic acid, and dephosphorylation by phosphatidate phosphatase.     

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.     

Fatty acid and glycerol labeling of glycerolipids of leukocytes in response to ionophore A23187.

The effect of divalent cation ionophore, A23187, on the incorporation of [1-14C]palmitic acid, [1-14C]linoleic acid and [U-14C]glycerol into glycerolipids of polymorphonulcear leukocytes was examined. Ionophore A23187 stimulated the labeling of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, and diacylglycerol by both labeled fatty acids and glycerol. [1-14C]Palmitic acid and [1-14C]linoleic acid incorporation into phosphatidylcholine and triacylglycerol was reduced by the presence of the ionophore in the incubation medium, while [U-14C]glycerol labeling of these lipids was not significantly changed under identical conditions. These data reflect that the acylation of sn-glycerol 3-phosphate is activated, and the acylations of lysophosphatidyl-choline and endogenous diacylglycerol are inhibited in cells incubated with ionophore A23187. External calcium was not required for the ionophore effect on the incorporation of labeled fatty acids and glycerol. It is suggested that the ionophore alters the metabolism of the fatty acid and glycerol moieties of glycerolipids by changing the distribution of intracellular calcium of leukocytes.     

Comparative utilization of glycerol and alanine as liver gluconeogenic substrates in the fed late pregnant rat

The appearance of plasma [14C]glucose in the inferior cava vein after a pulse of 0.2 mmol of [U-14C]L-alanine or [U-14C]glycerol/200 g body wt given through the portal vein was studied in fed 21 day pregnant rats and virgin controls under pentobarbital anesthesia. In both groups values were much higher when [U-14C]glycerol was the administered tracer than when [U-14C]L-alanine, and they were augmented in pregnant versus virgin animals at 1 min when receiving [U-14C]glycerol and at 2 min when receiving [U-14C]L-alanine. 20 min after the tracers rats receiving [U-14C]glycerol showed much higher liver [14C]glycogen and [14C]glyceride glycerol than those receiving [U-14C]L-alanine. Radioactivity present in liver as [14C]glyceride glycerol was greater in pregnant than in virgin rats receiving [U-14C]glycerol whereas radioactivity corresponding to [14C]fatty acids was lower in the former group receiving either tracer. At 20 min after maternal treatments fetuses showed lower plasma [14C]glycerol than [14C]alanine values but plasma [14C]glucose and liver [14C]glycogen values were much greater in fetuses from mothers receiving [U-14C]glycerol than [U-14C]L-amine. Besides showing the higher gluconeogenic efficiency in pregnant than in virgin rats, results indicate that at late gestation glycerol is used as a preferential substrate for both glucose and glyceride glycerol synthesis in liver.     

Biosynthesis of an unusual amino acyl moiety contained in bestatin

The biosynthetic pathway of an unusual amino acyl [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl (AHP)] moiety which is contained in bestatin has been studied by testing the incorporation of potential precursors. L-[U-14C]-Phenylalanine, L-[U-14C]leucine, and [U-14C]acetic acid were efficiently incorporated into bestatin, but the radioactivity of L-[1-14C]phenylalanine, [1-14C]glyoxylic acid, and [14C]oxalic acid were not incorporated. Incorporation of acetic acid into 1- and 2-carbon of the AHP moiety was confirmed by incorporation of [13C]acetic acid. Thus, the AHP moiety was shown to be biosynthesized from L-phenylalanine and two carbon atoms of acetic acid, accompanied by decarboxylation of the phenylalanine.     

Metabolic and secretory responses of parotid cells to cationic amino acids. Oxidation of the amino acids and interference with the oxidation of D-glucose or endogenous nutrients.

Cationic amino acids were recently found to stimulate amylase release from rat parotid cells. The possible relevance of their oxidative catabolism to such a secretory stimulation was investigated. D-Glucose, which was efficiently metabolized in parotid cells and which augmented O2 uptake above basal value, failed to affect basal or stimulated amylase release. L-Arginine, L-lysine and L-histidine failed to stimulate the oxidation of either exogenous D-[6-14C]glucose or endogenous nutrients in cells pre-labelled with [U-14C]palmitate or L-[U-14C]glutamine. The oxidation of L-[U-14C]arginine, L-[U-14C]ornithine, L-[U-14C]lysine and L-[U-14C]histidine, all tested at a 10 mM concentration, was much lower than that of D-[U-14C]glucose (5.6 mM). These findings argue against the view that the stimulation of amylase release by cationic amino acids would be related to their role as a source of energy in the parotid cells.     

L-methionine as an ethylene precursor in Saccharomyces cerevisiae.

L-Methionine induced production of ethylene by Saccharomyces cerevisiae growing in lactate medium. The production induced by L-methionine was inhibited by pyruvate, and elevated by glucose. Labeled ethylene was produced when L-[U-14C]methionine, but not [U-14C]glucose, was fed to the yeast. The mutant S. cerevisiae G1332 (ade-, met-) did not produce significant amounts of ethylene unless L-methionine was added. Thus L-methionine acts as a precursor of ethylene in S. cerevisiae. The role of glucose appears to be other than as a precursor.     

The biochemical pathway for the breakdown of N4-ethyl-L-asparagine in the bacterium Pseudomonas stutzeri.

N4-Ethyl-L-[u-14C]asparagine and L-[U-14C]aspartate give identical metabolites, mainly intermediates of the tricarboxylic acid cycle and related amino acids, in whole cells of Pseudomonas stutzeri. The labelled asparagine derivative is converted into [14C]-aspartate by cell-free extracts, and this reaction, which has an optimum pH of 8.8 +/- 0.2, is neither inhibited by unlabelled asparagine nor enhanced by unlabelled 2-oxoglutarate. No labelled keto acid corresponding to N4-ethylasparagine was detected in either whole cells or cell-free extracts. Thus N4-ethyl-L-asparagine, like asparagine, must be broken down by hydrolysis, at least in this bacterium.     

Inhibition of gamma-glutamyl transpeptidase decreases amino acid uptake in human keratinocytes in culture

Acivicin inhibits gamma-glutamyl transpeptidase activity in human keratinocytes in culture. Treatment of these cells with acivicin produces a decrease in the uptake of L-[U-14C]alanine, 2-amino-[1-14C]-isobutyrate, L-[U-14C]leucine and 1-aminocyclopentane-1-[14C]carboxylate. D-[U-14C]glucose uptake is not affected by the presence of acivicin. These results support, for the first time in vitro, the hypothesis that the gamma-glutamyl cycle may be involved in amino acid uptake by human cells.     

Metabolism of L-(U-14C)valine, L-(U-14C)leucine, L-(U-14C)histidine and L-(U-14C) phenylalanine by the isolated perfused lactating guinea-pig mammary gland.

1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.     

Studies on lipogenesis in vivo. Effect of dietary fat or starvation on conversion of [14C]glucose into fat and turnover of newly synthesized fat

1. Lipogenesis was studied in vivo by giving mice 250mg. meals of [U-(14)C]glucose and measuring the disposition and incorporation of label. About 48% of the (14)C dose was eliminated as (14)CO(2) in the first 2hr. At 60min. after administration, 1.0, 1.9 and 11.9% of the dose was recovered as liver glycogen, liver fatty acid and carcass fatty acid respectively. Of the [(14)C]glucose converted into fat in the epididymal pads about 90% was present as glyceride fatty acid and 10% as glyceride glycerol. 2. Hepatic synthesis of fatty acid was depressed by dietary fat to a much greater extent than was synthesis outside the liver. Both feeding with fat and starvation decreased the proportion of the label taken up by adipose tissue present as fat (triglyceride) and increased the proportion of triglyceride label present as glyceride glycerol. These results are consistent with the hypothesis that the primary action of both these conditions in decreasing fat synthesis is to inhibit synthesis of fatty acids. 3. Turnover of body fat labelled in vivo from [U-(14)C]glucose was estimated from the decline in radioactivity measured over the first 24hr. of the experiment. The half-life of liver and extrahepatic fatty acids (excluding epididymal fat) was 16hr. and 3 days respectively. In contrast, no measurable decrease in radioactivity of the fatty acids of epididymal fat was observed for 7 days after administration of the [U-(14)C]glucose.     

Decreased incorporation of L-[U-14C]serine into phosphatidylserine by polymorphonuclear leukocytes during phagocytosis

A decreased rate of L-[U-14C]serine incoroporation into phosphatidylserine of polymorphonuclear leukocytes exposed to starch granules was observed. L-[U-14C]serine uptake was also depressed under identical conditions. The degree of reduction in specific radioactivity of phosphatidylserine was parallel to that of L-[U-14C]serine uptake. Both uptake and efflux of 45Ca2+ were enhanced in cells with starch granules, but no significant change in cellular calcium levels was detected. These results suggest that the reduced L-[U-14C]serine incorporation into phospholipids may be attributable to decreased availability of this amino acid. The involvement of Ca2+ fluxes in phosphatidylserine synthesis in intact leukocytes cannot, however, be excluded.     

Effects of modulation of glycerol kinase expression on lipid and carbohydrate metabolism in human muscle cells.

Glycerol is taken up by human muscle in vivo and incorporated into lipids, but little is known about regulation of glycerol metabolism in this tissue. In this study, we have analyzed the role of glycerol kinase (GlK) in the regulation of glycerol metabolism in primary cultured human muscle cells. Isolated human muscle cells exhibited lower GlK activity than fresh muscle explants, but the activity in cultured cells was increased by exposure to insulin. [U-(14)C]Glycerol was incorporated into cellular phospholipids and triacylglycerides (TAGs), but little or no increase in TAG content or lactate release was observed in response to changes in the medium glycerol concentration. Adenovirus-mediated delivery of the Escherichia coli GlK gene (AdCMV-GlK) into muscle cells caused a 30-fold increase in GlK activity, which was associated with a marked rise in the labeling of phospholipid or TAG from [U-(14)C]glycerol compared with controls. Moreover, GlK overexpression caused [U-(14)C]glycerol to be incorporated into glycogen, which was dependent on the activation of glycogen synthase. Co-incubation of AdCMV-GlK-treated muscle cells with glycerol and oleate resulted in a large accumulation of TAG and an increase in lactate production. We conclude that GlK is the limiting step in muscle cell glycerol metabolism. Glycerol 3-phosphate is readily used for TAG synthesis but can also be diverted to form glycolytic intermediates that are in turn converted to glycogen or lactate. Given the high levels of glycerol in muscle interstitial fluid, these finding suggest that changes in GlK activity in muscle can exert important influences on fuel deposition in this tissue.     

Determination of evolved 14CO2 in decarboxylase reactions with application to measurement of [14C]oxalic acid.

An assay is described for the determination of the radioactive purity of [14C]oxalic acid preparations and the quantity of [14C]oxalic acid in biological samples. In this method oxalate decarboxylase is used to convert oxalate to formate and CO2. The entire procedure is carried out in a scintillation vial. The 14CO2 released in the enzymic reaction is allowed to diffuse off in a fume hood following acidification. Scintillation fluid is added to reacted and unreacted vials and the radioactivity measured. The loss of radioactivity from the reacted versus the unreacted vials provides the quantity of evolved 14CO2. This value is equal to 50% of the [14C]-oxalate (dpm) present. The radioactive purity of four preparations of [U-14C]oxalic acid was 99.0% while a fifth batch had a purity of 88%. A single batch of [U-14C]oxalic acid had a radioactive purity of 99.0% following storage of an aqueous solution, at -20 degrees C for 7 years. Recovery of [14C]oxalic acid from rat fecal extracts was 101.3%. Eight replicate analyses of a [U-14C]oxalic acid preparation gave a coefficient of variation of 0.3%. Following subcutaneous infusion of [U-14C]oxalic acid to rats, 100.2 +/- 2.9%, mean +/- SD, of the 14C in fecal extracts was present as [14C]oxalic acid (n = 10). The procedure provides a rapid, sensitive, and specific method to determine [14C]oxalic acid. It avoids the time consuming and inconvenient procedure for trapping and counting the evolved 14CO2. The approach used to determine the evolved 14CO2 may find application in other radiochemical methods that require its measurement.