Intermolecular tritium transfer in the transcarboxylase reaction.

Transfer of tritium from [3-3H]pyruvate into propionyl-CoA is found during the reaction of transcarboxylase: Methylmalonyl-CoA + pyruvate leads to oxalacetate + propionyl-CoA. About 5% of the tritium counts that are labilized in the reaction are found in a position of the propionate that exchanges rapidly with water in the presence of transcarboxylase. Transfer from [2-3H]propionate of propionyl-CoA to pyruvate is real but only about one-tenth as great. The tritium transfers between reactants on two subunits are difficult to explain by a "carbanion" mechanism of --C--H bond cleavage and support the cyclic mechanism in which carboxybiotin itself is the base and the enol form of biotin is the proton-transferring agent.     

Epoxidation of aflatoxin B1 by Aspergillus flavus microsomes in vitro: interaction with DNA and formation of aflatoxin B1-glutathione conjugate

Metabolism of aflatoxin B1 (AFB1) by subcellular preparations of Aspergillus flavus is least understood. The results reported here have demonstrated for the first time the epoxidation of AFB1 and subsequent conjugation with glutathione (GSH). Microsomes prepared from toxigenic mycelia catalysed [3H]AFB1 to calf thymus DNA to a greater extent (approximately 2-fold) as compared to that of non-toxigenic. The binding of [3H]AFB1 to exogenous and A. flavus nuclear DNA catalyzed by A. flavus microsomes was found to be comparable with that of mammalian extrahepatic tissue such as lung. Addition of phenobarbitone to the growing cultures resulted in 1.5-fold increase in [3H]AFB1-DNA binding mediated by microsomes prepared from either of the two strains. Tolnaftate, an inhibitor of aflatoxin synthesis enhanced the epoxidation rate in a dose-related manner. The binding of [3H]AFB1 to DNA catalyzed by A. flavus microsomes was significantly reduced (50% of control) upon addition of hamster liver cytosol, thereby substantiating the formation of the carcinogen adduct with DNA as reported in mammalian tissues. The metabolite formed by subcellular preparation of A. flavus was found to be AFB1-GSH having Rf value (6.5) similar to that obtained for mammalian liver preparations.     

Some chemical properties of carboxymethyl derivatives of amino acids

Tritium-labeled carboxymethyl derivatives of various functional groups in proteins show ready exchange of the tritium label when exposed to standard protein hydrolysis conditions (6 n HCl, 21 h, 110 °C). While N_?-[³H]carboxymethyl lysine does not show any tritium exchange, the two N-[³H]carboxymethyl histidine derivatives lose their tritium with a half time of about 15 h, and S-[³H]carboxymethyl cysteine loses its tritium with a half time of about 1.5 h. The tritium exchange of S-[³H]carboxymethyl methionine was so fast that the derivative could not be prepared with any of the tritium label intact. The rate of exchange for this compound was consequently determined by following the disappearance of the methylene NMR signal when S-carboxymethyl methionine was dissolved in D₂O. The half time for the exchange was about 12 min. Mechanisms involving either a sulfonium ion or enolization of the protonated conjugate carboxylic acid appear to give a satisfactory explanation of the relative stability of the different derivatives. The practical use of the differential rate of hydrogen exchange as a means of establishing rapidly and with small quantities of material the site of carboxymethylation in unknown proteins is suggested.     

The consequences of the isotope effect on proline dehydrogenation rates estimated by the tritium loss method.

Loss of tritium from a substrate is often used to estimate the rate of dehydrogenation. However, loss of 3H may be much slower than loss of H because of the tritium isotope effect. In order to assess the impact of the tritium isotope effect, loss of 3H from the C-5 position of proline during dehydrogenation by rat liver mitochondria and bacteroids from soybean (Glycine max [L.] Merrill) nodules was compared with appearance of 14C in products of [14C]proline dehydrogenation. Incubations were carried out in the presence of o-aminobenzaldehyde (added to trap the initial product, delta 1-pyroline-5-carboxylate). The fraction of total 14C products trapped by o-aminobenzaldehyde varied from 0.07 to 0.75 depending upon experimental conditions. With rat liver mitochondria, dehydrogenation of [14C]proline was between 3.27 and 9.25 times faster than dehydrogenation of 3H proline, depending upon assay conditions. Soybean nodule bacteriods dehydrogenated [14C]proline about 5 times faster than [3H]proline. We conclude the following: (i) the rate of proline dehydrogenation may be greatly underestimated by the tritium assay because of the tritium isotope effect, and (ii) the 14C assay may underestimate the rate of proline dehydrogenation if it is assumed that o-aminobenzaldehyde quantitatively traps delta 1-pyrroline-5-carboxylate under all conditions. The simplicity of the tritium assay makes it attractive for routine use. However, its use requires determination of the tritium isotope effect, under the specific conditions of the assay, in order to correct the results. The considerations discussed here have broad applicability to any dehydrogenase assay employing tritium loss.     

Denervation Supersensitivity of the Rat Pineal to Norepinephrine-Stimulated [3H]Inositide Turnover Revealed by Lithium and a Convenient Procedure

A convenient procedure for the assay of myo-[2-3H(N)]inositol ([3H]inositol) metabolites in cells or small amounts of tissue was developed. The procedure is a composite of modifications of published methods. After preincubation with [3H]inositol, rat pineal glands were disrupted in an acidified organic solvent mixture. Lipids were separated from the hydrophilic products and precursor using Sephadex G-25 columns and further analyzed by TLC. Hydrophilic products were further analyzed by anion-exchange column chromatography using Dowex AG1-X8 (formate form). In the presence of lithium, increases in inositol phosphates consequent to stimulation of the glands by norepinephrine were apparent within 10 min. The response in denervated glands was considerably greater than in intact pineals.     

Revised mechanism for inactivation of mitochondrial monoamine oxidase by N-cyclopropylbenzylamine

A mechanism previously proposed for inactivation of monoamine oxidase (MAO) by N-cyclopropylbenzylamine (N-CBA) [Silverman, R. B., & Hoffman, S. J. (1980) J. Am. Chem. Soc. 102, 884-886] is revised. Inactivation of MAO by N-[1-3H]CBA results in incorporation of about 3 equiv of tritium into the enzyme and release of [3H]acrolein. Treatment of inactivated enzyme with benzylamine, a reactivator for N-CBA-inactivated MAO, releases only 1 equiv of tritium as [3H]acrolein concomitant with reactivation of the enzyme. Even after MAO is inactivated by N-[1-3H]CBA, the reaction continues. At pH 7.2, a linear release of [3H]acrolein is observed for 70 h, which produces 55 equiv of [3H]acrolein while 2.3 equiv of tritium is incorporated into the enzyme. At pH 9, only 3.5 equiv of [3H]acrolein is detected in solution after 96 h, but 40 equiv of tritium is incorporated into the enzyme, presumably as a result of greater ionization of protein nucleophiles at the higher pH. N-[1-3H]Cyclopropyl-alpha-methylbenzylamine (N-C alpha MBA) produces the same adduct as N-CBA but gives only 1-1.35 equiv of tritium bound after inactivation of the enzyme. Denaturation of labeled enzyme results in reoxidation of the flavin without release of tritium, indicating attachment is not to the flavin but rather to an amino acid residue. Enzyme inactivated with N-[1-3H]C alpha MBA is reactivated by benzylamine with the release of 1 equiv of [3H]acrolein, which must have come from an adduct attached to an active site amino acid residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water

N-Acetyl-D-[2-³H]glucosamine was synthesized from N-acetyl-D-mannosamineby alkaline 2-epimerization in pyridine containing ³H₂O andnickelous acetate. The reaction involves reversible formationof an enol intermediate and therefore also resulted in incorporationof tritium into N-acetylmannosamine. After completed reaction,the two N-acetylhexosamines were separated from other radioactiveproducts and Morgan-Elson chromogens by chromatography on acolumn of Sephadex G-10, which was eluted with 10% ethanol,and were then separated from each other by chromatography onSephadex G-15 in 0·27 M sodium borate (pH 7·8).The location of the incorporated tritium was established bytreatment of the N-acetylhexosamines with borate under the conditionsof the Morgan-Elson reaction, which converts the sugars to Kuhn'schromogen I with concomitant loss of the C-2 hydrogen. As expected,this treatment resulted in the formation of ³H₂O, indicatingthat the tritium was located at C-2. [2-³H]Glucosamine was preparedby acid hydrolysis of the labelled N-acetylglucosamine and wasconverted to [2-³H]glucosamine 6-phosphate by incubation withhexokinase and ATP. The sugar phosphate was used as a substratefor glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10 [EC] )in a simple ³H₂O release assay. N-acetyl[2-³H]glucosamine N-acetyl[2-³H]mannosamine [2-³H]glucosamine glucosamine 6-phosphate deaminase [2-³H]mannosamine     

Characterization of A-2 Receptors in Postmortem Human Pineal Gland

We have examined the binding of the adenosine agonist radioligands [3N]N6-cyclohexyladenosine ([3H]CHA) and [3H]5'-N-ethylcarboxamidoadenosine ([3H]NECA) to membranes prepared from postmortem human pineal glands. The results showed that the A-1-specific ligand CHA did not bind to membranes. By contrast, [3H]NECA, a nonselective A-1/A-2 ligand, gave 68% specific binding of the total binding. This specific binding was nearly insensitive to the N-ethyl-maleimide pretreatment method. To characterize this binding, we used cyclopentyladenosine (50 nM). Under those conditions [3H]NECA binding at 30 degrees C was rapid and reversible; the KD determined from the kinetic studies was 141 nM. In postmortem human pineal gland, the rank order of potency of adenosine analogues and drugs competing with [3H]NECA showed the specificity for an A-2 receptor: NECA greater than 2-chloroadenosine greater than L-N6(2-phenylisopropyl)adenosine greater than 8-phenyltheophylline greater than 3-isobutyl-1-methylxanthine greater than caffeine. Guanylylimidodiphosphate (100 microM) induced a decrease in the affinity of [3H]NECA, a result suggesting the involvement of a G protein mechanism in the coupling of the adenosine receptor to other components of the receptor complex. Scatchard analysis revealed one class of binding sites for [3H]NECA with KD and Bmax ranging from 175 to 268 nM and 11.0 to 14.1 pmol/mg protein, respectively. The binding of [3H]NECA was not affected by age, sex, or postmortem delay. [3H]NECA should be a useful tool to assess brain A-2 receptor density in a variety of neuropsychiatric disorders.     

In Vivo Labeling of Serotonin Uptake Sites with [3H]Paroxetine

Previous work has shown that [3H]paroxetine is a potent and selective in vitro label for serotonin uptake sites in the mammalian brain. In the present study, [3H]paroxetine was tested in mice as an in vivo label for serotonin uptake sites. Maximum tritium concentration in the whole brain (1.4% of the intravenous dose) was reached 1 h after injection into a tail vein. Distribution of the tracer at 3 h after injection followed the distribution of serotonin uptake sites known from previous in vitro binding studies (r = 0.85). The areas of highest [3H]paroxetine concentration, in decreasing order, were: hypothalamus greater than frontal cortex greater than olfactory tubercles greater than thalamus greater than upper colliculi greater than brainstem greater than hippocampus greater than striatum greater than cerebellum. Preinjection of carrier paroxetine (1 mg/kg) significantly decreased [3H]paroxetine concentration in all areas except in the cerebellum, which is known to contain a relatively low number of specific binding sites. Kinetic studies showed highest specific [3H]paroxetine binding (tissue minus cerebellum) at 2 h after injection and slow clearance of activity thereafter (half-time of dissociation from the hypothalamus, 215 min). The specificity of in vivo [3H]paroxetine binding was studied by preinjecting monoamine uptake blockers or receptor antagonists 5 min before administration of [3H]paroxetine. Serotonergic or muscarinic cholinergic receptor antagonists and dopamine or norepinephrine uptake blockers did not reduce the in vivo binding of [3H]paroxetine. In contrast, there was an excellent correlation (r = 0.99) between the in vivo inhibitory potencies of serotonin uptake blockers in this study and previously published in vitro data on inhibition of [3H] serotonin uptake in brain synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The vitamin K-dependent carboxylase generates γ-carboxylated glutamates by using CO2 to facilitate glutamate deprotonation in a concerted mechanism that drives catalysis

The γ-glutamyl carboxylase converts Glu to carboxylated Glu (Gla) to activate a large number of vitamin K-dependent proteins with diverse functions, and this broad physiological impact makes it critical to understand the mechanism of carboxylation. Gla formation is thought to occur in two independent steps (i.e. Glu deprotonation to form a carbanion that then reacts with CO(2)), based on previous studies showing unresponsiveness of Glu deprotonation to CO(2). However, our recent studies on the kinetic properties of a variant enzyme (H160A) showing impaired Glu deprotonation prompted a reevaluation of this model. Glu deprotonation monitored by tritium release from the glutamyl γ-carbon was dependent upon CO(2), and a proportional increase in both tritium release and Gla formation occurred over a range of CO(2) concentrations. This discrepancy with the earlier studies using microsomes is probably due to the known accessibility of microsomal carboxylase to water, which reprotonates the carbanion. In contrast, tritium incorporation experiments with purified carboxylase showed very little carbanion reprotonation and consequently revealed the dependence of Glu deprotonation on CO(2). Cyanide stimulated Glu deprotonation and carbanion reprotonation to the same extent in wild type enzyme but not in the H160A variant. Glu deprotonation that depends upon CO(2) but that also occurs when water or cyanide are present strongly suggests a concerted mechanism facilitated by His-160 in which an electrophile accepts the negative charge on the developing carbanion. This revised mechanism provides important insight into how the carboxylase catalyzes the reaction by avoiding the formation of a high energy discrete carbanion.     

Facile oxygen exchanges of phosphoenolpyruvate and preparation of [18O]phosphoenolpyruvate

Phosphoenolpyruvate when heated in acidic solution exchanges its phosphoryl and carboxyl oxygens rapidly and its enolic oxygen much more slowly with oxygens from water. The incorporation of 18O into phosphoenolpyruvate was measured by gas chromatography-mass spectrometry and phosphorus-31 nuclear magnetic resonance after heating in H218O at 98 degrees C. The rates of exchange of all six oxygens of phosphoenolpyruvate with water increase with increasing acidity, and the phosphoryl oxygens exchange more rapidly than the carboxyl oxygens. The rate of exchange of each oxygen of the phosphoryl group is 16-fold greater than the hydrolysis rate at 1 N HCl. This provides a simple and useful method for the synthesis of [18O]phosphoenolpyruvate highly enriched in its phosphoryl-group oxygens. An enrichment of 89% was obtained with a 50% yield. The [18O]-phosphoenolpyruvate showed a binomial distribution of 18O in the phosphoryl-group oxygens. The exchange may be explained by the reversible formation of a transient cyclic phosphate and, for exchange of the enolic oxygen, a transient acyl phosphate. Preparation of [18O]phosphoenolypyruvate from [18O]Pi by a chemical synthesis from beta-chlorolactate was not satisfactory because of drastic loss of 18O during the procedures used. Some loss of 18O also occurred during an enzymic synthesis with KCNO, [18O]Pi, carbamate kinase, and pyruvate kinase.     

Intermolecular hydrogen transfer catalyzed by a flavodehydrogenase, bakers' yeast flavocytochrome b2

Bakers' yeast flavocytochrome b2 is a flavin-dependent L-2-hydroxy acid dehydrogenase which also exhibits transhydrogenase activity. When a reaction takes place between [2-3H]lactate and a halogenopyruvate, tritium is found in water and at the halogenolactate C2 position. When the halogenopyruvate undergoes halide ion elimination, tritium is also found at the C3 position of the resulting pyruvate. The amount tau of this intermolecular tritium transfer depends on the initial keto acid-acceptor concentration. At infinite acceptor concentration, extrapolation yields a maximal transfer of 97 +/- 11%. This indicates that the hydroxy acid-derived hydrogen resides transiently on enzyme monoprotic heteroatoms and that exchange with bulk solvent occurs only at the level of free reduced enzyme. Using a minimal kinetic scheme, the rate constant for hydrogen exchange between Ered and solvent is calculated to be on the order of 10(2) M-1 S-1, which leads to an estimated pK approximately equal to 15 for the ionization of the substrate-derived proton while on the enzyme. It is suggested that this hydrogen could be shared between the active site base and Flred N5 anion. It is furthermore shown that some tritium is incorporated into the products when the transhydrogenation is carried out in tritiated water. Finally, with [2-2H]lactate-reduced enzyme, a deuterium isotope effect is observed on the rate of bromopyruvate disappearance. Extrapolation to infinite bromopyruvate concentration yields DV = 4.4. An apparent inverse isotope effect is determined for bromide ion elimination. These results strengthen the idea that oxidoreduction and elimination pathways involve a common carbanionic intermediate.     

Enzymic generation of chiral acetates. A quantitative evaluation of their configurational assay.

1. R-Acetate was generated enzymically from R-acetate in the sequence acetate leads to malate leads to oxaloacetate leads to acetate, and S-acetate likewise from S-acetate. It was concluded that the formation of malate on malate synthase involves the operation of a normal isotopic effect combined with inversion of configuration. The malate synthase kH/k2H was determined as 3.7 +/- 0.5 by a method which yields results independently of the stereochemical purity of the chiral acetates used initially. 2. R-Acetate was also generated from R-acetate in the sequence acetate leads to citrate leads to malate leads to oxaloacetate leads to acetate, and S-acetate likewise from S-acetate. The conclusion is the same as given above, but refers to the formation of citrate on the re-synthase. 3. 2S,3R-[2-2H1,3-2H1,3H1]Malate and 2S,3S-[2-2H1,3-2H1]malate were prepared from 2S-[2,3-2H3]malate by treatment with fumarase in tritiated water and normal water, respectively. It was assumed that these malate specimens were pure with respect to chirality as generated by isotopic labelling. 4. These two malate specimens were partially converted (about 9%) to acetates in conditions where no racemization at the level of transiently formed oxaloacetate occurred. That no racemization took place was demonstrated experimentally. Oxidative enzymic hydrolysis of 2S,3R-[2-2H1,3-2H1,3H1]malate in normal water and of 2S,3S-[2-2H1,3-2H1]malate in tritiated water produced S-[2H1,3H1]acetate and R-[2H1,3H1]acetate, respectively. 5. The isolated R-[2H1,3H1]acetate and S-[2H1,3H1]acetate on configurational analysis yielded malates which in the presence of fumarase retained 79.7 +/- 0.7% and 20.3 +/- 0.9%, respectively, of their total tritium content. The symmetric deviation from the 50% value found with [3H1]acetate strengthens the conclusion that stereochemically pure chiral acetates were analyzed. The malate synthase kH/k2H was determined from the data of this study as 3.9 +/- 0.2. 6. The average of the values given under paragraphs 1 and 5 for the isotopic discrimination on malate synthase corresponds to kH/k2H=3.8 +/- 0.1. It was concluded that the configurational analysis of stereochemically pure R-[2H1,3H1]acetate and S-[2H1,3H1]acetate yields malates which in the presence of fumarase retain 79 +/- 2% and 21 +/- 2%, respectively, of their total tritium content. Hence, a deviation of 29 +/- 2% from the 50% value represents the actual amplitude of the configurational assay. 7. Outlines are given for an enzymic generation of chiral acetates in preparative scale.     

Chinese medicinal herbs modulate mutagenesis, DNA binding and metabolism of aflatoxin B1.

Oldenlandia diffusa (OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors while Astragalus membranaceus (AM) and Ligustrum lucidum (LL) are often used as an adjunct in cancer therapy. In this study, we determined the effects of aqueous extracts of these four herbs on aflatoxin B1 (AFB1)-induced mutagenesis using Salmonella typhimurium TA100 as the bacterial tester strain and rat liver 9000 x g supernatant as the activation system. The effects of these herbs on [3H]AFB1 binding to calf-thymus DNA were assessed. Organosoluble and water-soluble metabolites of AFB1 were extracted and analyzed by high-performance liquid chromatography (HPLC). Mutagenesis assays revealed that all of these herbs produced a concentration-dependent inhibition of histidine-independent revertant (His+) colonies induced by AFB1. At a concentration of 1.5 mg/plate, SB and OD in combination exhibited an additive effect. The trend of inhibition of these four herbs on AFB1-induced mutagenesis was: SB greater than LL greater than AM. LL, OD and SB significantly inhibited AFB1 binding to DNA, reduced AFB1-DNA adduct formation, and also significantly decreased the formation of organosoluble metabolites of AFB1. Our data suggest that these Chinese medicinal herbs possess cancer chemopreventive properties.     

Kinetic mechanism of histidinol dehydrogenase: histidinol binding and exchange reactions

Salmonella typhimurium histidinol dehydrogenase produces histidine from the amino alcohol histidinol by two sequential NAD-linked oxidations which form and oxidize a stable enzyme-bound histidinaldehyde intermediate. The enzyme was found to catalyze the exchange of 3H between histidinol and [4(R)-3H]NADH and between NAD and [4(S)-3H]NADH. The latter reaction proceeded at rates greater than kcat for the net reaction and was about 3-fold faster than the former. Histidine did not support an NAD/NADH exchange, demonstrating kinetic irreversibility in the second half-reaction. Specific activity measurements on [3H]histidinol produced during the histidinol/NADH exchange reaction showed that only a single hydrogen was exchanged between the two reactants, demonstrating that under the conditions employed this exchange reaction arises only from the reversal of the alcohol dehydrogenase step and not the aldehyde dehydrogenase reaction. The kinetics of the NAD/NADH exchange reaction demonstrated a hyperbolic dependence on the concentration of NAD and NADH when the two were present in a 1:2 molar ratio. The histidinol/NADH exchange showed severe inhibition by high NAD and NADH under the same conditions, indicating that histidinol cannot dissociate directly from the ternary enzyme-NAD-histidinol complex; in other words, the binding of substrate is ordered with histidinol leading. Binding studies indicated that [3H]histidinol bound to 1.7 sites on the dimeric enzyme (0.85 site/monomer) with a KD of 10 microM. No binding of [3H]NAD or [3H]NADH was detected. The nucleotides could, however, displace histidinol dehydrogenase from Cibacron Blue-agarose.(ABSTRACT TRUNCATED AT 250 WORDS)     

A rapid and simplified assay method for tyrosine hydroxylase

Tyrosine hydroxylase can be measured by release of tritiated water from labeled tyrosine, and the assay method has now been modified to allow recovery of 3H2O from the reaction mixture in a much more rapid and less tedious manner than previously possible. In the new method, the tyrosine hydroxylase reaction is stopped with sodium carbonate, pH 11.6. At this pH the tritium in 3H2O, but not other 3H species, is extracted into an organic scintillant containing 25% isoamyl alcohol, toluene, 2,5-diphenyloxazole, and p-bis-[2-(5-phenyloxazolyl)]benzene. The selective extraction occurs by means of exchange of tritium in 3H2O with the hydroxyl proton of isoamyl alcohol. It is the [3H]isoamyl alcohol that is then extracted into the scintillant and quantified by liquid scintillation spectrometry. Although the organic extraction method is somewhat less sensitive than the more frequently used ion-exchange method for isolating the 3H2O formed in the tyrosine hydroxylase reaction, it is much more rapid, as well as cost effective, since the enzyme reaction, extraction, and counting are carried out within the same vial.     

Reaction mechanism of gramicidin S synthetase 1, phenylalanine racemase, of Bacillus brevis

We have demonstrated that gramicidin S synthetase 1 (GS 1), phenylalanine racemase [EC 5.1.1.11], of Bacillus brevis catalyzes the exchange between a proton in the medium and alpha-hydrogen of phenylalanine in the course of the racemase reaction by using tritiated water or L-phenyl[2,3-3H]alanine. GS 1 from some gramicidin S non-producing mutants of B. brevis lacking phenylalanine racemase activity did not catalyze the tritium exchange reaction. The proton exchange between phenylalanine bound as thioester on the GS 1-phenylalanine complex and water in the medium was detected, but 5,5'-dithiobis(2-nitrobenzoic acid)-modified complex lacked both the proton exchange and phenylalanine racemase activity. It is suggested that a base group, probably a sulfhydryl group, on the enzyme functions as proton donor and acceptor during the phenylalanine racemase reaction.     

The metabolism of N-triphenylmethylmorpholine in the dog and rat

1. Rats were given N-triphenyl[(14)C]methylmorpholine, triphenyl[(14)C]carbinol, N-triphenylmethyl[G-(3)H]morpholine or [G-(3)H]morpholine as single oral doses; the routes of excretion were examined. 2. Dogs were given single oral doses of N-triphenyl[(14)C]methylmorpholine. 3. (14)C-labelled metabolites were excreted mainly in the faeces in both rats and dogs; no (14)CO(2) was expired and less than 3% remained in the carcass and skin after 96hr. 4. (3)H-labelled metabolites were excreted rapidly in urine; part of the label was found in the expired gases and over 10% remained in the carcass and skin after 96hr. 5. Differences in excretion pattern between the sexes were noticed in rats but not in dogs. 6. N-Triphenylmethylmorpholine was rapidly hydrolysed to form triphenylcarbinol and morpholine in the stomach; morpholine was absorbed rapidly and excreted largely unchanged, though some was degraded, since some of the (3)H was found in water. 7. Triphenylcarbinol was absorbed only slowly and was oxidized to p-hydroxyphenyldiphenylcarbinol. 8. Both triphenylcarbinol and its p-hydroxy derivative were found in urine, bile and faeces in the free form and conjugated with glucuronic acid. The proportion of conjugates was higher in rat bile than in faeces. 9. Traces of o-hydroxyphenyldiphenylcarbinol and m-hydroxyphenyldiphenylcarbinol were detected as metabolites both free and conjugated.     

The role of S-adenosylmethionine in the lysine 2,3-aminomutase reaction

The interconversion of L-lysine and L-3,6-diamino-hexanoate (L-beta-lysine) catalyzed by lysine 2,3-aminomutase is known to be stimulated by added S-adenosylmethionine (Chirpich, T. P., Zappia, V., Costilow, R. N., and Barker, H. A. (1970) J. Biol. Chem. 245, 1778-1789). In this paper we show that enzyme activated by S-[2,8,5'-3H]adenosylmethionine catalyzes the conversion of L-lysine to the equilibrium mixture of L-lysine and L-beta-lysine with incorporation of high levels of tritium into both isomers. The tritium levels in the isomers reflect the equilibrium constant for their interconversion, 84% in the L-beta-lysine and 16% in L-lysine compared with Keq = 5.3 +/- 0.3 in the direction of the formation of L-beta-lysine at pH 7.7 and 30 degrees C. No significant tritium is incorporated into lysine from S-[2,8-3H]adenosylmethionine or S-adenosyl[methyl-3H] methionine under comparable conditions. Therefore, the tritium incorporated into lysine in the former reaction arises from the 5'-position of the 5'-deoxyadenosyl group in S-adenosylmethionine. These experiments implicate the 5'-deoxyadenosyl portion of S-adenosylmethionine in the hydrogen transfer mechanism of this reaction, perhaps in a role analogous to that played by the 5'-deoxyadenosyl moiety of deoxyadenosyl cobalamin in coenzyme B12-dependent rearrangements.