Further Characterization of Tissue Distribution and Metabolism of [14C]Aflatoxin B1 in Chickens

The distribution and metabolism of [(14)C]aflatoxin B(1) in chicken tissues were further investigated. Previously dried and frozen ethyl acetate extracts of liver, heart, gizzard, breast, leg, blood, and fecal samples were obtained from either layer or broiler chickens fed subclinical levels of [(14)C]aflatoxin B(1). Treatment of these extracts with either carboxypeptidase A, leucine aminopeptidase, pepsin, or trypsin revealed that an average of 50% of the (14)C detected in the acetate extracts was a liberated peptide (or amino acid) conjugate of [(14)C]aflatoxin B(2a). When a prepared standard of B(2a) was made by incubation of B(1) with cold dilute aqueous HCl, the R(f) values and absorbance maxima were identical with those of the tissue extracts after enzymatic treatment.     

The comparative metabolism of 2,6-dichlorothiobenzamide (prefix) and 2,6-dichlorobenzonitrile in the dog and rat

1. A single oral dose of either [(14)C]Prefix or 2,6-dichlorobenzo[(14)C]nitrile to rats is almost entirely eliminated in 4 days: 84.8-100.5% of (14)C from [(14)C]Prefix is excreted, 67.3-79.7% in the urine, and 85.8-97.2% of (14)C from 2,6-dichlorobenzo-[(14)C]nitrile is excreted, 72.3-80.7% in the urine. Only 0.37+/-0.03% of the dose of [(14)C]Prefix and 0.25+/-0.03% of the dose of 2,6-dichlorobenzo[(14)C]nitrile are present in the carcass plus viscera after removal of the gut. Rats do not show sex differences in the pattern of elimination of the respective metabolites of the two herbicides. The rates of elimination of (14)C from the two compounds in the 24hr. and 48hr. urines are not significantly different (P >0.05) from one another. 2. After oral administration to dogs, 85.9-106.1% of (14)C from [(14)C]Prefix is excreted, 66.6-80.9% in the urine, and 86.8-92.5% of (14)C from 2,6-dichlorobenzo[(14)C]nitrile is excreted, 60.0-70.1% in the urine. Dogs do not show sex differences in the pattern of eliminating the metabolites of either Prefix or 2,6-dichlorobenzonitrile. 3. Dogs and rats do not show species differences in the patterns of elimination of the two herbicides. 4. Prefix and 2,6-dichlorobenzonitrile are completely metabolized; unchanged Prefix and 2,6-dichlorobenzonitrile are absent from the urine and faeces, and from the carcasses when elimination is complete. In the hydrolysed urine of rats dosed with either [(14)C]Prefix or 2,6-dichlorobenzo[(14)C]nitrile, 2,6-dichloro-3-hydroxybenzonitrile accounts for approx. 42% of the (14)C, a further 10-11% is accounted for by 2,6-dichlorobenzamide, 2,6-dichlorobenzoic acid, 2,6-dichloro-3- and -4-hydroxybenzoic acid and 2,6-dichloro-4-hydroxybenzonitrile collectively, and 25-30% by six polar constituents, of which two are sulphur-containing amino acids. 5. In the unhydrolysed urines of rats dosed with either [(14)C]Prefix or 2,6-dichlorobenzo[(14)C]nitrile, there are present free 2,6-dichloro-3- and -4-hydroxybenzonitrile, their glucuronide conjugates, ester glucuronides of the principal aromatic acids that are present in the hydrolysed urines, and two sulphur-containing metabolites analogous to mercapturic acids or premercapturic acids. 6. Prefix is thus extensively transformed into 2,6-dichlorobenzonitrile: R.CS.NH(2)-->R.CN+H(2)S, where R=C(6)H(3)Cl(2). However, the competitive reaction: R.CS.NH(2)+H(2)O-->R.CO.NH(2)+H(2)S takes place to a very limited extent.     

The bacterial oxidation of N-methylisonicotinate, a photolytic product of Paraquat

Two bacteria have been isolated that are capable of oxidizing N-methylisonicotinate, a photodegradation product of Paraquat (1.1'-dimethyl-4,4'-bipyridylium ion). N-Methylisonicotinate-grown cells of strain 4C1, a Gram-positive rod, oxidized 2-hydroxy-N-methylisonicotinate without lag. Cell-free extracts of these cells converted 2-hydroxyisonicotinate into 2,6-dihydroxyisonicotinate; the reaction did not require molecular oxygen. Maleamate was deamidated and maleate isomerized to fumarate by soluble enzyme systems. [(14)C]Formaldehyde was isolated as the dimedone derivative from the supernatant of a cell suspension oxidizing N-[(14)C]methylisonicotinate, and no [(14)C]-methylamine was detected. Whole cells incubated with N-methyl[carboxy-(14)C]isonicotinate released 95% of the radioactivity as (14)CO(2). The second bacterium, strain 4C2, a Gram-negative rod, did not oxidize any of the mono- or di-hydroxypyridines or their N-methyl derivatives that were available or could be synthesized; nor did cell-free extracts oxidize any of these compounds. Methylamine was oxidized by whole cells without lag; cell-free extracts converted methylamine into formaldehyde when a soluble enzyme system requiring an electron acceptor was used; formaldehyde was oxidized to formate and formate to CO(2) by enzyme systems requiring NAD(+).     

Water-soluble metabolites of p-nitrophenol and 1-naphthyl N-methylcarbamate in flies and grass grubs. Formation of glucose phosphate and phosphate conjugates

Metabolites isolated from houseflies dosed with 1-napththol or p-nitrophenol were identified as the phosphate and glucose phosphate conjugates of these phenols by titrations, hydrolysis, ionophoresis, i.r. spectra and mixed melting point. [(3)H]Carbaryl (1-naphthyl N-methylcarbamate) was metabolized by houseflies, blowflies and grass grubs to water-soluble metabolites which had chromatographic and ionophoretic behaviour similar to those of the conjugates of 1-naphthol with glucose, sulphate, phosphate and glucose 6-phosphate.     

Microbial growth on C1 compounds. Incorporation of C1 units into allulose phosphate by extracts of Pseudomonas methanica

1. Incubation of cell-free extracts of methane- or methanol-grown Pseudomonas methanica with [(14)C]formaldehyde and d-ribose 5-phosphate leads to incorporation of radioactivity into a non-volatile product, which has the chromatographic properties of a phosphorylated compound. 2. Treatment of this reaction product with a phosphatase, followed by chromatography, shows the presence of two compounds whose chromatographic properties are consistent with their being free sugars. 3. The minor component of the dephosphorylated products has been identified as fructose. The major component has been identified as allulose (psicose) on the basis of co-chromatography, co-crystallization of the derived phenylosazone and dinitrophenylosazone with authentic derivatives of allulose and behaviour towards oxidation with bromine water. 4. It is suggested that the bacterial extracts catalyse the condensation of a C(1) unit identical with, or derived from, formaldehyde with ribose 5-phosphate to give allulose 6-phosphate. 5. Testing of hexose phosphates and pentose phosphates as substrates has so far shown the reaction to be specific for ribose 5-phosphate. 6. The condensation reaction is not catalysed by extracts of methanol-grown Pseudomonas AM1. 7. A variant of the pentose phosphate cycle, involving this condensation reaction, is suggested as an explanation for the net synthesis of C(3) compounds from C(1) units by P. methanica.     

A direct pathway for the conversion of propionate into pyruvate in Moraxella lwoffi

1. The identity of the organism previously known as Vibrio O1 (N.C.I.B. 8250) with a species of Moraxella is established. 2. The ability of cells to oxidize propionate is present only in cells with an endogenous respiration and this ability is increased 80-fold when the organism is grown with propionate. 3. Isocitrate lyase activity in extracts from propionate-grown cells is the same as that in extracts from lactate-grown cells, about tenfold greater than that in extracts from succinate-grown cells and slightly greater than half the activity in extracts from acetate-grown cells. 4. With arsenite as an inhibitor conditions were found in which the organism would catalyse the quantitative oxidation of propionate to pyruvate. When propionate was completely utilized pyruvate was metabolized further to 2-oxoglutarate. 5. The oxidation of propionate by cells was incomplete both in a ;closed system' with alkali to trap respiratory carbon dioxide and in an ;open system' with an atmosphere of oxygen+carbon dioxide (95:5). Acetate accumulated. Under these conditions [2-(14)C]- and [3-(14)C]-propionate gave rise to [(14)C]acetate. The rate of conversion of [2-(14)C]propionate into (14)CO(2), although much less than the rate of conversion of [1-(14)C]propionate into (14)CO(2), was slightly greater than the rate of conversion of [3-(14)C]propionate into (14)CO(2). 6. The oxidation of propionate by cells was complete in an ;open system' with an atmosphere of either oxygen or air. Under these conditions very little [1-(14)C]propionate was converted into (14)C-labelled cell material. The conversion of [2-(14)C]- and [3-(14)C]-propionate into (14)C-labelled cell material occurred at an appreciable rate, the rate for the incorporation of [3-(14)C]propionate being slightly more rapid. In the absence of a utilizable nitrogen source part of the [(14)C]propionate was incorporated into some reserve material, which was oxidized when added substrate had been completely utilized. 7. [(14)C]-Pyruvate produced from [(14)C]propionate was chemically degraded. The C((1)) of propionate was found only in C((1)) of pyruvate. At least 86% of C((2)) of pyruvate was derived from C((2)) of propionate and at least 92% of C((3)) of pyruvate from C((3)) of propionate. 8. These results are incompatible with the operation of any of the previously described pathways for propionate metabolism except the direct one, perhaps via an activated acrylate.     

The metabolism of [14C]nicotine in the cat

The metabolism of [2'-(14)C]nicotine given as an intravenous injection in small doses to anaesthetized and unanaesthetized cats has been studied. A method is described for the quantitative determination of [(14)C]nicotine and [(14)C]cotinine in tissues and body fluids. Nanogram amounts of these compounds have been detected. After a single dose of 40mug. of [(14)C]nicotine/kg., 55% of the injected radioactivity was excreted in the urine within 24hr., but only 1% of this radioactivity was unchanged nicotine. [(14)C]Nicotine is metabolized extremely rapidly, [(14)C]cotinine appearing in the blood within 2.5min. of intravenous injection. [(14)C]Nicotine accumulates rapidly in the brain and 15min. after injection 90% of the radioactivity still represents [(14)C]nicotine. Metabolites of [(14)C]nicotine have been identified in liver and urine extracts. [(14)C]Nicotine-1'-oxide has been detected in both liver and urine.     

Evidence that 1-naphthol is not an obligate intermediate in the covalent binding and the pulmonary bronchiolar necrosis by naphthalene

Recent studies of a number of volatile aromatic hydrocarbons have suggested that the formation of covalently bound metabolites arises solely through the intermediate formation of phenols. This study further examines the involvement of 1-naphthol in the in vivo and in vitro formation of covalently bound metabolites and pulmonary bronchiolar necrosis by naphthalene. Marked differences were observed in the rate of 1-naphthol formation in lung and liver microsomal incubations without correspondingly large differences between the rates of formation of covalently bound metabolites from naphthalene and 1-naphthol. Glutathione decreased covalent binding in hepatic microsomal incubations containing 14[C]1-naphthol but did not result in the formation of any of the glutathione adducts isolated from identical incubations containing 14[C]naphthalene. Tissue levels of covalently bound radioactivity in mice treated with 14[C]1-naphthol or 14[C]naphthalene were similar; however, in contrast to studies with naphthalene, 1-naphthol administration did not deplete tissue glutathione nor result in detectable tissue injury. These studies indicate that 1-naphthol is not an obligate intermediate in the formation of covalently bound metabolites from naphthalene nor does it appear to be a more proximate lung toxic metabolite.     

The role of valine in the biosynthesis of penicillin N and cephalosporin C by a Cephalosporium sp

1. The production of penicillin N, but not that of cephalosporin C, was inhibited by the addition of d-valine to suspensions in water of washed mycelium of Cephalosporium sp. 8650. The production of cephalosporin C was selectively inhibited by gamma-hydroxyvaline. 2. l-[(14)C]Valine was taken up rapidly and virtually completely by suspensions of washed mycelium but d-[(14)C]valine and alpha-oxo[(14)C]-isovalerate were taken up relatively slowly. 3. Part of the l-valine was rapidly degraded in the mycelium and part was incorporated into protein. Turnover of the valine in the amino acid pool was estimated to occur in 10-17min. 4. No detectable amount of l-[(14)C]valine was converted into the d-isomer in the mycelium. alpha-Oxo[(14)C]isovalerate was rapidly converted into l-[(14)C]valine in mycelium and mycelial extracts. 5. d-[(14)C]Valine was partially converted into the l-isomer in the mycelium and (14)C from d-valine was incorporated into protein. 6. The labelling of penicillin N and cephalosporin C by (14)C from l-[(14)C]valine was consistent with the view that l-valine is a direct precursor of C(5) fragments of both antibiotics and that any intermediates involved are present in relatively small pools in rapid turnover. 7. Labelling of the antibiotics with (14)C from d-[1-(14)C]valine appeared to occur after the latter had been converted into the l-isomer. Unlabelled d-valine did not decrease the efficiency of incorporation of (14)C from l-[1-(14)C]valine. 8. Intracellular peptide material which contained, among others, residues of alpha-aminoadipic acid, cysteine and valine, was rapidly labelled by (14)C from l-[1-(14)C]valine in a manner consistent with it being an intermediate in the biosynthesis of one or both of the antibiotics. 9. Labelling of penicillin N from l-[1-(14)C]valine occurred more rapidly than that of cephalosporin C. However, the effects of d-valine and gamma-hydroxyvaline on antibiotic production and the course of labelling of the antibiotics from l-[(14)C]valine could not readily be explained on the assumption that penicillin N was a precursor of cephalosporin C.     

The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of insulin, adrenaline and some metabolites in vitro

1. Adipose tissues from rats fed a balanced diet were incubated in the presence of glucose (20mm) with the following additions: insulin, anti-insulin serum, insulin+acetate, insulin+pyruvate, insulin+lactate, insulin+phenazine methosulphate, insulin+oleate+albumin, insulin+adrenaline+albumin, insulin+6-N-2'-O-dibutyryl 3':5'-cyclic AMP+albumin. 2. Measurements were made of the whole tissue concentrations of adenine nucleotides, hexose phosphates, triose phosphates, glycerol 1-phosphate, 3 phosphoglycerate, 6-phosphogluconate, long-chain fatty acyl-CoA, acid-soluble CoA, citrate, isocitrate, malate and 2-oxoglutarate, and of the release into the incubation medium of lactate, pyruvate and glycerol after 1h of incubation. 3. Fluxes of [(14)C]glucose carbon through the major pathways of glucose metabolism were calculated from the yields of (14)C in various products after 2h of incubation. Fluxes of [(14)C]acetate, [(14)C]pyruvate or [(14)C]lactate carbon in the presence of glucose were also determined. 4. Measurements were also made of the whole-tissue concentrations of metabolites in tissues taken directly from Nembutal-anaesthetized rats. 5. Whole tissue mass-action ratios for phosphofructokinase, phosphoglucose isomerase and the combined (aldolasextriose phosphate isomerase) reaction were similar in vivo and in vitro. The reactants of phosphofructokinase appeared to be far from mass-action equilibrium. In vitro, the reactants of hexokinase also appeared to be far from mass-action equilibrium. 6. Correlation of observed changes in glycolytic flux with changes in fructose 6-phosphate concentration suggested that phosphofructokinase may show regulatory behaviour. The enzyme appeared to be activated in the presence of oleate or adrenaline and to be inhibited in the presence of lactate or pyruvate. 7. Evidence is presented that the reactants of lactate dehydrogenase and glycerol 1-phosphate dehydrogenase may be near to mass-action equilibrium in the cytoplasm. 8. No satisfactory correlations could be drawn between the whole-tissue concentrations of long-chain fatty acyl-CoA, citrate and glycerol 1-phosphate and the observed rates of triglyceride and fatty acid synthesis. Under the conditions employed, the concentration of glycerol 1-phosphate appeared to depend mainly on the cytoplasmic [NAD(+)]/[NADH] ratios. 9. Calculated hexose monophosphate pathway flux rates roughly correlated with fatty acid synthesis rates and with whole tissue [6-phosphogluconate]/[glucose 6-phosphate] ratios. The relative rates of production of NADPH for fatty acid synthesis by the hexose monophosphate pathway and by the ;malic enzyme' are discussed. It is suggested that all NADH produced in the cytoplasm may be used in that compartment for reductive synthesis of fatty acids, lactate or glycerol 1-phosphate.     

Biosynthesis of ethylene from methionine in aminoethoxyvinylglycine-resistant avocado tissue

This study was conducted to determine if aminoethoxyvinylglycine (AVG) insensitivity in avocado (Persea americana Mill., Lula, Haas, and Bacon) tissue was due to an alternate pathway of ethylene biosynthesis from methionine. AVG, at 0.1 millimolar, had little or no inhibitory effect on either total ethylene production or [(14)C] ethylene production from [(14)C]methionine in avocado tissue at various stages of ripening. However, aminoxyacetic acid (AOA), which inhibits 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (the AVG-sensitive enzyme of ethylene biosynthesis), inhibited ethylene production in avocado tissue. Total ethylene production was stimulated, and [(14)C]ethylene production from [(14)C]methionine was lowered by treating avocado tissue with 1 millimolar ACC. An inhibitor of methionine adenosyltransferase (EC 2.5.1.6), l-2-amino-4-hexynoic acid (AHA), at 1.5 millimolar, effectively inhibited [(14)C]ethylene production from [(14)C]methionine in avocado tissue but had no effect on total ethylene production during a 2-hour incubation. Rates of [(14)C]AVG uptake by avocado and apple (Malus domestica Borkh., Golden Delicious) tissues were similar, and [(14)C]AVG was the only radioactive compound in alcohol-soluble fractions of the tissues. Hence, AVG-insensitivity in avocado tissue does not appear to be due to lack of uptake or to metabolism of AVG by avocado tissue. ACC synthase activity in extracts of avocado tissue was strongly inhibited (about 60%) by 10 micromolar AVG. Insensitivity of ethylene production in avocado tissue to AVG may be due to inaccessibility of ACC synthase to AVG. AVG-resistance in the avocado system is, therefore, different from that of early climacteric apple tissue, in which AVG-insensitivity of total ethylene production appears to be due to a high level of endogenous ACC relative to its rate of conversion to ethylene. However, the sensitivity of the avocado system to AOA and AHA, dilution of labeled ethylene production by ACC, and stimulation of total ethylene production by ACC provide evidence for the methionine --> SAM --> ACC --> ethylene pathway in avocado and do not suggest the operation of an alternate pathway.     

Utilization of l-threonine by a species of Arthrobacter. A novel catabolic role for `aminoacetone synthase''

1. A species of Arthrobacter (designated Arthrobacter 9759) was isolated from soil by its ability to grow aerobically on l-threonine as sole source of carbon atoms, nitrogen atoms and energy; the organism also grew well on other sources of carbon atoms including glycine, but no growth was obtainable on aminoacetone or dl-1-aminopropan-2-ol. 2. During growth on threonine, (14)C from l-[U-(14)C]threonine was rapidly incorporated into glycine and citrate, and thereafter into serine, alanine, aspartate and glutamate. 3. With extracts of threonine-grown cells supplied with l-[U-(14)C]threonine, evidence was obtained of the NAD and CoA-dependent catabolism of l-threonine to produce acetyl-CoA plus glycine. Short-term incorporation studies in which [2-(14)C]acetate and [2-(14)C]glycine were supplied (a) to cultures growing on threonine, and (b) to extracts of threonine-grown cells, showed that the acetyl-CoA was metabolized via the tricarboxylic acid cycle and glyoxylate cycle whereas the glycine was converted into pyruvate via the folate-dependent ;serine pathway'. 4. The threonine-grown organism contained ;biosynthetic' threonine dehydratase and a potent NAD-linked l-threonine dehydrogenase but possessed no l-threonine aldolase activity. 5. Evidence was obtained that the acetyl-CoA and glycine produced from l-threonine had their immediate origin in the alpha-amino-beta-oxobutyrate formed by the threonine dehydrogenase; the CoA-dependent cleavage of this compound was catalysed by an alpha-amino-beta-oxobutyrate CoA-ligase, which was identified with ;aminoacetone synthase'. A continuous spectrophotometric assay of this enzyme was developed, and it was found to be inducibly synthesized only during growth on threonine and not during growth on acetate plus glycine. 6. By using a reconstituted mixture of separately purified l-threonine dehydrogenase and alpha-amino-beta-oxobutyrate CoA-ligase (i.e. ;aminoacetone synthase'), l-[U-(14)C]threonine was broken down to [(14)C]glycine plus [(14)C]acetyl-CoA (trapped as [(14)C]citrate). 7. There was no evidence of aminoacetone metabolism by Arthrobacter 9759 even though a small amount of this amino ketone appeared in the culture medium during growth on threonine.     

Incorporation of [14C]histidine into homocarnosine and carnosine of frog brain in vivo and in vitro

1. Homocarnosine, carnosine and histidine were determined in brain from several species and the results compared with values in the literature. 2. [(14)C]Homocarnosine and [(14)C]carnosine were isolated from frog brain after intracerebral injection of [(14)C]histidine in vivo. 3. Whole frog brain, incubated in vitro with l-[(14)C]histidine, formed labelled homocarnosine and carnosine. 4. A frog brain homogenate or supernatant fraction catalysed the incorporation of l-[(14)C]histidine into homocarnosine and carnosine. 5. The results indicate that brain tissue can synthesize homocarnosine and carnosine.     

Further studies on the biosynthesis of gramicidin S and proteins in a cell-free system from Bacillus brevis

1. An improved 11000g cell-free system for the incorporation of [(14)C]valine into gramicidin S has been obtained. The cell-free extract used was the supernatant obtained by treating Bacillus brevis with ultrasonics for 1min. followed by centrifugation at 11000g. The optimum pH for the incorporation was 8.2-8.4 and the optimum Mg(2+) concentration 0.05m. The presence of ammonium sulphate (0.1m) and K(+) (0.01m) increased the incorporation. 2. Cell-free extracts prepared from cells harvested in the early phase of growth (extinction value 0.1) incorporated negligible amounts of [(14)C]valine into gramicidin S compared with that incorporated by cell-free extracts prepared from cells harvested in the late phase of growth (extinction value 0.5). This was not due to the presence of inhibitors in the cell-free extracts prepared from cells harvested early, since there was no marked decrease in gramicidin S synthesis in a mixture of extracts prepared from cells harvested early and late in the growth phase. 3. The small incorporation of [(14)C]valine into protein, which took place in cell-free extracts from cells harvested in the late growth phase, was not inhibited by puromycin, chloramphenicol and ribonuclease. However, the substantial incorporation that took place in cell-free extracts prepared from cells harvested in the early phase of growth was completely inhibited by puromycin, chloramphenicol and ribonuclease. On mixing cell-free extracts prepared from cells harvested early and late in the growth phase, it appeared that the small incorporation that occurs in extracts from cells harvested in the late phase of growth was not due to cellular inhibitors.     

Purine ribonucleotide biosynthesis, interconversion and catabolism in mouse brain in vitro

The relative rates of the synthetic, interconversion and catabolic reactions of purine metabolism in chopped mouse cerebrum were studied. The rates of incorporation of [(14)C]adenine and [(14)C]hypoxanthine into purine ribonucleotides were much less than the potential activities of adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase, and the rates of incorporation were stimulated by the addition of guanosine to the incubation mixture. The availability of ribose phosphates may be a limiting factor for the formation of ribonucleotides from purine bases. The rate of incorporation of [(14)C]adenosine into purine ribonucleotides was at least seven- to eight-fold higher than that of adenine. The radioactivity in adenine ribonucleotides synthesized from adenine and hypoxanthine was about 100- and ten-fold respectively higher than that in the radioactive guanine ribonucleotides. The conversion of inosinate into guanine ribonucleotides was probably limited by the amount of inosinate available, and the conversion of adenine ribonucleotides into guanine ribonucleotides was probably limited by the activity of adenylate deaminase. The rate of catabolism of [(14)C]adenosine was low in comparison with its rate of utilization for ribonucleotide synthesis. A fraction of the [(14)C]hypoxanthine was catabolized to xanthine and urate. [(14)C]Guanine was completely converted into xanthine, mostly by the guanine deaminase that was released during incubation of chopped mouse cerebrum.     

Calcium-regulated proteolysis of eEF1A

Eukaryotic elongation factor 1alpha (eEF1A) can be post-translationally modified by the addition of phosphorylglycerylethanolamine (PGE). [(14)C]Ethanolamine was incorporated into the PGE modification, and with carrot (Daucus carota L.) suspension culture cells, eEF1A was the only protein that incorporated detectable quantities of [(14)C]ethanolamine (Ransom et al., 1998). When 1 mM CaCl(2) was added to microsomes containing [(14)C]ethanolamine-labeled eEF1A ([(14)C]et-eEF1A), there was a 60% decrease in the amount of [(14)C]et-eEF1A recovered after 10 min. The loss of endogenous [(14)C]et-eEF1A was prevented by adding EGTA. Recombinant eEF1A, which did not contain the PGE modification, also was degraded by microsomes in a Ca(2+)-regulated manner, indicating that PGE modification was not necessary for proteolysis; however, it enabled us to quantify enodgenous eEF1A. By monitoring [(14)C]et-eEF1A, we found that treatment with phospholipase D or C, but not phospholipase A(2), resulted in a decrease in [(14)C]et-eEF1A from carrot microsomes. The fact that there was no loss of [(14)C]et-eEF1A with phospholipase A(2) treatment even in the presence of 1 mM Ca(2+) suggested that the loss of membrane lipids was not essential for eEF1A proteolysis and that lysolipids or fatty acids decreased proteolysis. At micromolar Ca(2+) concentrations, proteolysis of eEF1A was pH sensitive. When 1 microM CaCl(2) was added at pH 7.2, 35% of [(14)C]et-eEF1A was lost; while at pH 6.8, 10 microM CaCl(2) was required to give a similar loss of protein. These data suggest that eEF1A may be an important downstream target for Ca(2+) and lipid-mediated signal transduction cascades.     

A study on the tricarboxylic acid cycle and the synthesis of acetylcholine in the lobster nerve

1. The biosynthetic origin of the amide substituent of N-(alpha-hydroxyethyl)lysergamide has been studied. 2. [1-(14)C]Acetate, [(14)C]formate, [2-(14)C]mevalonic acid lactone, [2-(14)C]indole, dl-[3-(14)C]tryptophan, dl-[3-(14)C]serine, dl-[2-(14)C]alanine and [2-(14)C]pyruvate were efficiently incorporated into the alkaloid, but not dl-[1-(14)C]alanine or [1-(14)C]pyruvate. 3. Only the dl-[2-(14)C]alanine- and [2-(14)C]pyruvate-derived alkaloid contained appreciable radioactivity in the amide substituent. 4. l-[(15)N]Alanine-derived alkaloid was shown to be specifically labelled in the amide nitrogen. However, l-[(14)C,(15)N]alanine was found to be incorporated into the methylcarbinolamide substituent with an appreciable increase in the (15)N/(14)C ratio, suggesting that alanine is not the direct precursor of this moiety.     

5-3H]glucose overestimates glycolytic flux in isolated working rat heart: role of the pentose phosphate pathway

We set out to study the pentose phosphate pathway (PPP) in isolated rat hearts perfused with [5-3H]glucose and [1-14C]glucose or [6-14C]glucose (crossover study with 1- then 6- or 6- then 1-14C-labeled glucose). To model a physiological state, hearts were perfused under working conditions with Krebs-Henseleit buffer containing 5 mM glucose, 40 microU/ml insulin, 0.5 mM lactate, 0.05 mM pyruvate, and 0.4 mM oleate/3% albumin. The steady-state C1/C6 ratio (i.e., the ratio from [1-14C]glucose to [6-14C]glucose) of metabolites released by the heart, an index of oxidative PPP, was not different from 1 (1.06 +/- 0.19 for 14CO2, and 1.00 +/- 0.01 for [14C]lactate + [14C]pyruvate, mean +/- SE, n = 8). Hearts exhibited contractile, metabolic, and 14C-isotopic steady state for glucose oxidation (14CO2 production). Net glycolytic flux (net release of lactate + pyruvate) and efflux of [14C]lactate + [14C]pyruvate were the same and also exhibited steady state. In contrast, flux based on 3H2O production from [5-3H]glucose increased progressively, reaching 260% of the other measures of glycolysis after 30 min. The 3H/14C ratio of glycogen (relative to extracellular glucose) and sugar phosphates (representing the glycogen precursor pool of hexose phosphates) was not different from each other and was <1 (0.36 +/- 0.01 and 0.43 +/- 0.05 respectively, n = 8, P < 0.05 vs. 1). We conclude that both transaldolase and the L-type PPP permit hexose detritiation in the absence of net glycolytic flux by allowing interconversion of glycolytic hexose and triose phosphates. Thus apparent glycolytic flux obtained by 3H2O production from [5-3H]glucose overestimates the true glycolytic flux in rat heart.     

The biochemistry of aromatic amines. 2-Formamido-1-naphthyl hydrogen sulphate, a metabolite of 2-naphthylamine

1. 2-Formamido-1-naphthyl hydrogen sulphate is excreted by dogs and rats dosed with 2-naphthylamine or with 2-amino-1-naphthyl hydrogen sulphate and was isolated from dog urine. 2. 2-Formamido-1-naphthol, naphtho[2,1-d]oxazole and N-formyl-2-naphthylhydroxylamine are excreted as (2-formamido-1-naphthyl glucosid)uronic acid. 2-Formamidonaphthalene is converted into conjugates of 2-amino-6-naphthol and 2-amino-8-naphthol, but 2-methylaminonaphthalene is excreted as 2-amino-1-naphthyl hydrogen sulphate and its N-methyl and N-formyl derivatives and (2-amino-1-naphthyl glucosid)uronic acid. 3. 2-Methylamino-1-naphthyl hydrogen sulphate is converted into 2-formamido-1-naphthyl hydrogen sulphate and 2-amino-1-naphthyl hydrogen sulphate on charcoal. 2-Amino-1-naphthyl hydrogen sulphate and formaldehyde react on charcoal to yield 2-methylamino- and 2-formamido-1-naphthyl hydrogen sulphate. 4. 2-Formamido-1-naphthol, 2-formamido-1-naphthyl hydrogen sulphate, N-formyl-2-naphthylhydroxylamine and N-formyl-2-naphthylhydroxylamine-O-sulphonic acid were synthesized.     

Glutathione s-transferase omega 1-1 is a target of cytokine release inhibitory drugs and may be responsible for their effect on interleukin-1beta posttranslational processing

Stimulus-induced posttranslational processing of human monocyte interleukin-1beta (IL-1beta) is accompanied by major changes to the intracellular ionic environment, activation of caspase-1, and cell death. Certain diarylsulfonylureas inhibit this response, and are designated cytokine release inhibitory drugs (CRIDs). CRIDs arrest activated monocytes so that caspase-1 remains inactive and plasma membrane latency is preserved. Affinity labeling with [(14)C]CRIDs and affinity chromatography on immobilized CRID were used in seeking potential protein targets of their action. Following treatment of intact human monocytes with an epoxide-bearing [(14)C]CRID, glutathione S-transferase (GST) Omega 1-1 was identified as a preferred target. Moreover, labeling of this polypeptide correlated with irreversible inhibition of ATP-induced IL-1beta posttranslational processing. When extracts of human monocytic cells were chromatographed on a CRID affinity column, GST Omega 1-1 bound selectively to the affinity matrix and was eluted by soluble CRID. Recombinant GST Omega 1-1 readily incorporated [(14)C]CRID epoxides, but labeling was negated by co-incubation with S-substituted glutathiones or by mutagenesis of the catalytic center Cys(32) to alanine. Peptide mapping by high performance liquid chromatography-mass spectrometry also demonstrated that Cys(32) was the site of modification. Although S-alkylglutathiones did not arrest ATP-induced IL-1beta posttranslational processing or inhibit [(14)C]CRID incorporation into cell-associated GST Omega 1-1, a glutathione-CRID adduct effectively demonstrated these attributes. Therefore, the ability of CRIDs to arrest stimulus-induced IL-1beta posttranslational processing may be attributable to their interaction with GST Omega 1-1.