The metabolism of fluoroacetate in lettuce

1. Whole lettuce plants were incubated with (1) [1-(14)C]acetate, (2) fluoroacetate followed by [1-(14)C]acetate, (3) fluoro[1-(14)C]acetate, (4) fluoro[2-(14)C]acetate or (5) S-carboxy[(14)C]methylglutathione. 2. Fluoroacetate did not affect the expiration of (14)CO(2) from [1-(14)C]acetate and only a small amount of (14)CO(2) was produced from either fluoro[1-(14)C]-acetate or fluoro[2-(14)C]acetate in 43h. 3. Fluoroacetate at 50mg/kg wet wt. doubled the plant citrate concentration after 43h incubation, and depending on the age and size of the plant 50-100% of the compound was metabolized. 4. With both fluoro[1-(14)C]acetate and fluoro[2-(14)C]acetate all the radioactivity except that in the CO(2) was found in the water-soluble acid fraction. About 2% was in fluorocitrate and the remainder, apart from unchanged fluoroacetate, was in a number of compounds devoid of fluorine but containing nitrogen and sulphur. These were peptide-like and could be separated by chromatography on an amino acid analyser. 5. Identical compounds were obtained from the spontaneous reaction between iodo[2-(14)C]acetate and glutathione, the major product being S-carboxymethylglutathione. 6. S-Carboxymethylcysteine was also isolated and its mass spectrum compared with a commercial sample. 7. Reaction rates of all the monohaloacetates with glutathione were studied at pH7 at 25 degrees C. No reaction was observed with fluoroacetate. 8. The metabolism of fluoroacetate by lettuce is discussed in relation to that of aliphatic and aromatic halogen compounds, including fluoroacetate, by mammalian liver and to the metabolism of fluoroacetate by different plants reported by other workers.     

The role of biotin and sodium in the decarboxylation of oxaloacetate by the membrane-bound oxaloacetate decarboxylase from Klebsiella aerogenes

The biotin-containing oxaloacetate decarboxylase from Klebsiella aerogenes catalyzed the Na+-dependent decarboxylation of oxaloacetate to pyruvate and bicarbonate (or CO2) but not the reversal of this reaction, not even in the presence of an oxaloacetate trapping system. The enzyme catalyzed an avidin-sensitive isotopic exchange between [1-14C]pyruvate and oxaloacetate, which indicated the intermediate formation of a carboxybiotin enzyme. Sodium ions were not required for this partial reaction, but promoted the second partial reaction, the decarboxylation of the carboxybiotin enzyme, thus accounting for the Na+ requirement of the overall reaction. Therefore, the 14CO2-enzyme which was formed upon incubation of the decarboxylase with [4-15C]oxaloacetate, could only be isolated if Na+ ions were excluded. Preincubation of the decarboxylase with avidin also prevented its labelling with 14CO2. The isolated 14CO2-labelled oxaloacetate decarboxylase revealed the following properties. It was slowly decarboxylated at neutral pH and rapidly upon acidification. The 14CO2 residues of the 14CO2-enzyme could be transferred to pyruvate yielding [4-14C]oxaloacetate. In the presence of Na+ this 14CO2 transfer was repressed by the simultaneous decarboxylation of the 14CO2-enzyme. However, Na+ alone was insufficient as a cofactor for the decarboxylation of the isolated 14CO2-enzyme, since this required pyruvate in addition to Na+. It is therefore concluded that the decarboxylation of oxaloacetate proceeds over a CO2-enzyme--pyruvate complex and that free CO2-enzyme is an abortive reaction intermediate. The activation energy of the enzymic decarboxylation of oxaloacetate changed with temperature and was about 113 kJ below 11 degrees C, 60 kJ between 11 degrees C and 31 degrees C and 36 kJ between 31--45 degrees C.     

Studies on lung tumours. III. Oxidative metabolism of dimethylnitrosamine by rodent and human lung tissue.

The oxidative metabolism of the carcinogen dimethylnitrosamine (DMN) was studied in mouse, rat, hamster and human respiratory tissue. [14C]DMN was purified by Dowex-1-bisulfite column chromatography to remove a contaminant (probably [14C]formaldehyde) interfering with the enzyme assay. Since formaldehyde and methyl carbonium ions - yielding methanol with water - are considered to be the primary products of DMN metabolism, tissue slices were assayed for the production of [14C]CO2 from 14C-labelled methanol, formaldehyde, formate, and DMN. Oxidation of formaldehyde to formate was not, but oxidation of formate to CO2 was very much rate-limiting. This rate-limiting step was circumvented by introducing quantitative chemical oxidation of formate to CO2 by mercury(II)chloride following the enzymic reaction. Since oxidation of methanol to CO2 proved to be insignificant, production of CO2 from DMN by lung tissue enzymes and HgCl2 may serve as a parameter for N-demethylating activity and the production of the suspected carcinogenically active methyl carbonium ions. The DMN-N-demethylating activities of lung tissue slices of two mouse strains with widely different susceptibilities to formation of lung adenomas by DMN differed significantly, but the difference seemed too small to explain the divergence in tumourigenic response. The enzymatic activities decreased in hamster bronchus, hamster trachea, hamster lung, GRS/A mouse lung, C3Hf/A mouse lung, human lung, Sprague-Dawley rat lung, in that order. The reported resistance of the hamster respiratory system to tumour induction by DMN may therefore not be due to poor DMN-N-demethylating capacity.     

Ribose. An oxidation product of glucose 6-phosphate in microsomal fraction

An oxidative metabolism of glucose 6-phosphate was studied in rat liver microsomal fraction. Although radioactive 14CO2 was formed from [1-14C]glucose 6-phosphate in the microsomal fraction (Hino, Y., and Minakami, S. (1982) J. Biochem. (Tokyo) 92, 547-557), the formation was negligible when [2-14C]glucose 6-phosphate was used as a starting substrate. These results indicated an inability of the microsomal fraction to rearrange [2-14C]glucose 6-phosphate to form [1-14C] glucose 6-phosphate, and it was expected that a certain compound derived from glucose 6-phosphate accumulated as an end-product of the reaction. We, therefore, have tried to identify the product by high performance liquid chromatography, and found that ribose accumulated as the end-product. The formation of ribose was inhibited in the same manner as that of 14CO2 by antibodies against rat liver microsomal hexose-6-phosphate dehydrogenase, and the ratios of ribose to 14CO2 formed in the reaction were 0.5-0.8 on a molar basis. The finding of ribose formation further suggested the involvement of ribose phosphate isomerase and phosphatase activities in the reaction.     

A HPLC-fluorescence detection method for determination of cardiac phospholipase D activity in vitro

A nonradioactive assay for the investigation of phospholipase D (PLD) activity in cardiac membranes has been developed. A fluorescent derivative of phosphatidylcholine [2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3proprionyl)amino) undecyl) sn-glycero-3-phosphocholine] was utilized as substrate in an in vitro PLD-catalyzed transphosphatidylation reaction utilizing ethanol as second substrate. Unreacted phosphatidylcholine and the products of phospholipase activity (PEtOH, phosphatidylethanol; PA, phosphatidic acid; DAG, diacylglycerol) were separated by a binary gradient HPLC system and detected by fluorometry. The detection limit of this assay is approximately 0.6 pmol PEtOH. The reaction proceeded at a linear rate for up to 45 min and increased linearly with increasing amounts of rat cardiac membrane protein in a range of 0.625 microg up to at least 25 microg. In the presence of potassium fluoride, formation of fluorescent PA increased at the expense of DAG generation, demonstrating the presence of PA phosphohydrolase activity in rat cardiac membranes. PEtOH formation was unchanged in the presence of the PA phosphohydrolase inhibitor, indicating that the phosphatidylalcohol is not subject to further metabolism by this enzyme. Activation of protein kinase C by phorbol ester significantly increased PLD activity in cardiac membranes. This assay proved to be sensitive for accurate and rapid assessment of PLD activity in cardiac membranes permitting further characterization of the regulation of PLD signal transduction in the heart.     

Partial purification and properties of D-desthiobiotin synthetase from Escherichia coli

d-Desthiobiotin synthetase, an enzyme that catalyzes the synthesis of d-desthiobiotin from dl-7,8-diaminopelargonic acid and HCO(3) (-), was purified 100-fold from cells of a biotin mutant strain of Escherichia coli. Adenosine triphosphate and Mg(2+) were shown, especially in purified extracts, to be obligatory for enzyme activity, although concentrations higher than 5 mm caused severe inhibition of the reaction with unpurified cell-free extracts. Adenosine diphosphate and adenosine monophosphate were shown to inhibit the reaction, but fluoride (up to 50 mm) had no detectable effect. The product of the enzyme reaction was identical to d-desthiobiotin on the basis of biological activity and chromatography. Furthermore, when H(14)CO(3) (-) was used as a substrate, the radioactive product was shown to be (14)C-desthiobiotin labeled exclusively in the ureido carbon.     

Tritium isotope effects in the reaction catalyzed by 4-hydroxyphenylpyruvate dioxygenase from pseudomonas sp. strain P.J. 874

Tritium isotope effects in the reaction catalyzed by 4-hydroxyphenylpyruvate dioxygenase (4-hydroxyphenyl-pyruvate:oxygen oxidoreductase (hydroxylating, decarboxylating), EC 1.13.11.27) from Pseudomonas sp. strain P.J. 874 were studied with 14C- and different 3H-labelled 4-hydroxyphenylpyruvate. Tritium of ring-2,6-3H2-labelled substrate was released into water in 1:2 stoichiometry to 14CO2 formation. The tritium release from ring-3,5-3H2- and side chain-3-3H1-labelled 4-hydroxyphenylpyruvate was low as compared with 14CO2 formation. The apparent tritium isotope effects were below two, as judged by comparison of 3H/14C ratios of 4-hydroxyphenylpyruvate and homogentisate. The ratios showed no dependence on oxygen concentrations between 1 and 21% in the gas phase. Thus, a tritium assay can be used to determine the activity of 4-hydroxyphenylpyruvate dioxygenase. Apparently, none of the substrate hydrogens is involved in any rate-limiting step up to the first irreversible step. enol-4-Hydroxyphenylpyruvate was excluded as the active substrate tautomer.     

A fluorescence-based assay for 2-oxoglutarate-dependent oxygenases

A widely used generic assay for 2-oxoglutarate-dependent oxygenases relies upon monitoring the release of 14CO2 from labeled [1-14C]-2-oxoglutarate. We report an alternative assay in which depletion of 2-oxoglutarate is monitored by its postincubation derivatization with o-phenylenediamine to form a product amenable to fluorescence analysis. The utility of the procedure is demonstrated by assays with hypoxia-inducible factor hydroxylases where it was shown to give results similar to those reported with the radioactive assay, but it is more efficient and readily adapted to a multiwell format. The process should be amenable to the assay of other 2-oxoglutarate-consuming enzymes and to the discovery of inhibitors.     

Quantification of clarithromycin,its 14-hydroxy and decladinose metabolites in rat plasma,gastric juice and gastric tissue using high-performance liquid chromatography with electrochemical detection

A rapid, selective and sensitive HPLC assay has been developed for the simultaneous analysis of clarithromycin, its 14-hydroxy-clarithromycin metabolite, and its decladinose acid degradation product, in small volumes of rat gastric juice aspirate, plasma and gastric tissue. Sample were extracted with n-hexane/2-butanol (4:1) and the internal standard was roxithromycin. A Kromasil ODS 5 micrometer(75x4.6 mm I.D.) column was used with a mobile phase consisting of acetonitrile/aqueous phosphate buffer (pH 7, 0.086 M) (45:55 v/v). The column temperature was 30 degrees C and coulometric detection was used at 850 mV using a screen voltage of 600 mV. The analysis time was less than 8 min. The limits of quantitation for clarithromycin, 14-OH clarithromycin and decladinose clarithromycin were 0.15 microgram ml(-1) or lower in plasma (0.05 ml); 0.16 microgram ml(-1) or lower in gastric juice (0.2 ml); and 0.51 microgram g(-1) or lower for gastric tissue (0.25 g). The method was linear up to at least 20.3, 15.4 and 12.5 microgram ml(-1) for clarithromycin, 14-OH-clarithromycin and decladinose, respectively, in gastric juice aspirate and plasma and up to 40.6, 30.9 and 25.0 microgram g(-1) in gastric tissue. The assay was applied to the measurement of clarithromycin, 14-OH-clarithromycin and, for the first time, decladinose clarithromycin in pharmacokinetic studies of gastric transfer of clarithromycin in individual rats.     

Development of a sensitive radiorespiration method for detecting microbial activity at subzero temperatures

We have developed a simple and sensitive method to detect microbial respiration at subzero temperatures. Microbial activity was detected by measuring (14)CO(2) evolved during the microbial-mediated mineralization of [1-(14)C] acetic acid or [2-(14)C] glucose in microcosm assays using modified (14)CO(2) traps. Various (14)CO(2) traps, designed to withstand freezing at subzero temperatures, were tested for their quench characteristics during liquid scintillation spectrometry and their ability to trap (14)CO(2). Solutions consisting of 1 M KOH supplemented with 20% or 30% v/v ethylene glycol did not freeze at temperatures above -20 degrees C and had a minor quenching effect on liquid scintillation spectrometry. Addition of ethylene glycol did have an effect on the efficiency of (14)CO(2) trapping, as the cumulative recovery of (14)CO(2) was reduced by 14% and 32% in the 1 M KOH+20% ethylene glycol and 1 M KOH+30% ethylene glycol solutions, respectively. Using the modified (14)CO(2) traps, microbial activity in representative Canadian high Arctic environmental samples was detected at temperatures as low as -15 degrees C. This simple method allows for sensitive, specific, and reliable detection of microbial activity occurring at subzero temperatures and is readily adaptable for studies in other cryoenvironments.     

Does the pentose cycle play a major role for NADPH supply in the heart?

It was attempted to determine the substrate flux through the pentose cycle in isolated rat hearts which performed pressure-volume work employing 14CO2 production from [1-14C]glucose (Kühn & Scholz (1982) Eur. J. Biochem. 124, 611-617). Even under conditions of increased NADPH requirements (infusion of tert-butylhydroperoxide) and a diminished 14CO2 production from glucose via the citrate cycle (in the presence of oleate as additional substrate) or enhanced activity of glucose-6-phosphate dehydrogenase (pretreatment with isoproterenol), a substrate flux through the pentose cycle was not detectable. The lower limit of detection is 0.01 mumol/(min X g). The increase in 14CO2 production from [1-14C]- and [6-14C]glucose and the acceleration in the washout when tert-butylhydroperoxide was present suggest an increase of substrate flux through the citrate cycle; therefore it is concluded that NADPH required for the removal of peroxides via the glutathione system is derived from the isocitrate dehydrogenase reaction.     

Assay of tryptophan decarboxylase from Catharanthus roseus plant cell cultures by high-performance liquid chromatography

An assay is described for the enzyme tryptophan decarboxylase from plant cell suspension cultures. It is based on the fluorometric detection of tryptamine by HPLC on a LiChrosorb RP-8 Select B column. Tryptophan decarboxylase from Catharanthus roseus was induced by transferring 14-day-old cells into an induction medium. Optimum activity was found 2 days after transfer, the increase being 5- to 10-fold. When kept at -15 degrees C the crude enzyme lost half its activity in about 7 days. The rate of the decarboxylation reaction was linear for at least 3 h at 35 degrees C.     

Quantification and stability of everolimus (SDZ RAD) in human blood by high-performance liquid chromatography-electrospray tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug, everolimus (SDZ RAD), using HPLC-tandem mass spectrometry. Whole blood samples (500 microl) were prepared by protein precipitation, followed by C(18) solid-phase extraction. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface operating in positive ionization mode. The assay was linear from 0.5 to 100 microg/l (r(2) > 0.996, n = 9). The analytical recovery and inter-day imprecision, determined using whole blood quality control samples (n = 5) at 0.5, 1.2, 20.0, and 75.0 microg/l, was 100.3 - 105.4% and < or = 7.6%, respectively. The assay had a mean relative recovery of 94.8 +/- 3.8%. Extracted samples were stable for up to 24 h. Fortified everolimus blood samples were stable at -80 degrees C for at least 8 months and everolimus was found to be stable in blood when taken through at least three freeze-thaw cycles. The reported method provides accurate, precise and specific measurement of everolimus in blood over a wide analytical range and is currently supporting phase II and III clinical trials.     

Synthesis of acetyl coenzyme A by carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila.

The carbon monoxide dehydrogenase (CODH) complex from Methanosarcina thermophila catalyzed the synthesis of acetyl coenzyme A (acetyl-CoA) from CH3I, CO, and coenzyme A (CoA) at a rate of 65 nmol/min/mg at 55 degrees C. The reaction ended after 5 min with the synthesis of 52 nmol of acetyl-CoA per nmol of CODH complex. The optimum temperature for acetyl-CoA synthesis in the assay was between 55 and 60 degrees C; the rate of synthesis at 55 degrees C was not significantly different between pHs 5.5 and 8.0. The rate of acetyl-CoA synthesis was independent of CoA concentrations between 20 microM and 1 mM; however, activity was inhibited 50% with 5 mM CoA. Methylcobalamin did not substitute for CH3I in acetyl-CoA synthesis; no acetyl-CoA or propionyl coenzyme A was detected when sodium acetate or CH3CH2I replaced CH3I in the assay mixture. CO could be replaced with CO2 and titanium(III) citrate. When CO2 and 14CO were present in the assay, the specific activity of the acetyl-CoA synthesized was 87% of the specific activity of 14CO, indicating that CO was preferentially incorporated into acetyl-CoA without prior oxidation to free CO2. Greater than 100 microM potassium cyanide was required to significantly inhibit acetyl-CoA synthesis, and 500 microM was required for 50% inhibition; in contrast, oxidation of CO by the CODH complex was inhibited 50% by approximately 10 microM potassium cyanide.     

Bicarbonate is a recycling substrate for cyanase

Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to ammonia and bicarbonate. Previous studies provided evidence that carbamate is an initial product and that the kinetic mechanism is rapid equilibrium random (bicarbonate serving as substrate as opposed to activator); the following mechanism was proposed (Anderson, P. M. (1980) Biochemistry 19, 2282-2888; Anderson, P. M., and Little, R. M. (1986) Biochemistry 25, 1621-1626). (formula; see text) Direct evidence for this mechanism was obtained in this study by 1) determining whether CO2 or HCO3- serves as substrate and is formed as product, 2) identifying the products formed from [14C]HCO3- and [14C] OCN-, 3) identifying the products formed from [13C] HCO3- and [12C]OCN- in the presence of [18O]H2O, and 4) determining whether 18O from [18O]HCO3- is incorporated into CO2 derived from OCN-. Bicarbonate (not CO2) is the substrate. Carbon dioxide (not HCO3-) is produced in stoichiometric amounts from both HCO3- and OCN-. 18O from [18O]H2O is not incorporated into CO2 formed from either HCO3- or OCN-. Oxygen-18 from [18O]HCO3- is incorporated into CO2 derived from OCN-. These results support the above mechanism, indicating that decomposition of cyanate catalyzed by cyanase is not a hydrolysis reaction and that bicarbonate functions as a recycling substrate.     

A binding assay for serine hydroxymethyltransferase

A sensitive assay for measuring serine hydroxymethyltransferase activity has been developed, based on the binding of N5,N10-[14C]methylene tetrahydrofolate (THF) to DEAE-cellulose paper. The complete assay requires THF, pyridoxal 5'-phosphate, [14C]serine, and enzyme. The reaction is stopped by streaking an aliquot of the reaction mixture onto a square of DEAE-cellulose paper, washing the paper with water to remove unreacted serine, drying the paper, and counting the bound N5,N10-[14C]methylene-THF. To determine that the labeled product was N5,N10-methylene-THF, unlabeled formaldehyde, which exchanges with the labeled methylene carbon, was added after the product had accumulated; 2 min after the addition of formaldehyde the amount of labeled product was reduced by 50%, and by 85% after 10 min. In addition, glycine, which reverses the reaction, and hydroxylamine, which reacts with the methylene carbon, reduced the number of counts bound to the paper. Binding of product to the filter is proportional to both enzyme concentration and assay time. No counts were retained on phosphocellulose filters. This assay represents a new and simple method for measuring serine hydroxymethyltransferase activity, which can be used to measure enzyme activity in tissue homogenates and for screening large numbers of samples.     

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.     

Hormonal regulation of the alpha-ketoglutarate dehydrogenase complex in the isolated perfused rat liver

The metabolic flux through the alpha-ketoglutarate dehydrogenase reaction in perfused livers was monitored by measuring the rate of 14CO2 production from [1-14C]alpha-ketoglutarate. The rates of 14CO2 production and glucose production from [1-14C]alpha-ketoglutarate were increased with increasing perfusate alpha-ketoglutarate concentrations. Vasopressin, angiotensin II, and the alpha 1-adrenergic agonist phenylephrine stimulated transiently by 2.5-fold the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction in the presence and absence of Ca2+ in the perfusion medium. High concentrations of glucagon (1 x 10(-8) M) and 8-p-chlorophenylthio-cAMP (100 microM) (data not shown) also stimulated transiently the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction. However, lower glucagon concentrations (1 x 10(-9) M) stimulated the rate of 14CO2 production from [1-14C]alpha-ketoglutarate only under conditions optimized to fix the cellular oxidation-reduction state at an intermediate level, when glucagon (1 x 10(-9) M)-mediated elevation of cAMP content was greater than that observed under highly oxidizing and reducing conditions. These data indicate that agonists which increase cytosolic free Ca2+ levels stimulate the metabolic flux through the alpha-ketoglutarate dehydrogenase complex. Furthermore, the data presented here demonstrate for the first time that physiological glucagon concentrations stimulate the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction only under conditions known to be optimal for glucagon-mediated Ca2+ mobilization in the isolated perfused rat liver.     

Reduction potential of iron in transferrin

The reduction potential of Fe3+ in transferrin was measured spectrophotometrically by equilibration with methyl viologen in the presence of sodium dithionite. For an ionic strength near 0.1 M at 25 degrees C and pH 7.3 under 0.048 atm. CO2, half of the iron is reduced at a potential near -0.40 V (vs. standard hydrogen electrode). At least one disulfide bond of the protein is partially reduced at a potential of -0.44 V, as evidenced by reaction with [14C]iodoacetate.     

The decarboxylation of S-adenosylmethionine by detergent-treated extracts of rat liver

1. The production of (14)CO(2) from S-adenosyl[carboxyl-(14)C]methionine by rat liver extracts was investigated. It was found that, in addition to the well-known cytosolic putrescine-activated S-adenosylmethionine decarboxylase, an activity carrying out the production of (14)CO(2) could be extracted from a latent, particulate or membrane-bound form by treatment with buffer containing 1% (v/v) Triton X-100 [confirming the report of Sturman (1976) Biochim. Biophys. Acta428, 56-69]. 2. The formation of (14)CO(2) by such detergent-solubilized extracts differed from that by cytosolic S-adenosylmethionine decarboxylase in a number of ways. The reaction by the solubilized extracts did not require putrescine and was not directly proportional to time of incubation or the amount of protein added. Instead, activity a showed a distinct lag period and was much greater when high concentrations of the extracts were used. The cytosolic S-adenosylmethionine decarboxylase was activated by putrescine, showed strict proportionality to protein added and the reaction proceeded at a constant rate. Cytosolic activity was not inhibited by homoserine or by S-adenosylhomocysteine, whereas the Triton-solubilized activity was strongly inhibited. 3. By using an acetone precipitate of Triton-treated homogenates as a source of the activity, it was found that decarboxylated S-adenosylmethionine was not present among the products of the reaction, although 5'-methylthioadenosine and 5-methylthioribose were found. Such extracts were able to produce (14)CO(2) when incubated with [U-(14)C]-homoserine, and (14)CO(2) production was greater when S-adenosyl[carboxyl-(14)C]methionine that had been degraded by heating at pH6 at 100 degrees C for 30min (a procedure known to produce mainly 5'-methylthioadenosine and homoserine lactone) was used as a substrate than when S-adenosyl[carboxyl-(14)C]methionine was used. 4. These results indicate that the Triton-solubilized activity is not a real S-adenosylmethionine decarboxylase, but that (14)CO(2) is produced via a series of reactions involving degradation of the S-adenosyl-[carboxyl-(14)C]methionine. It is probable that this degradation can occur via several pathways. Our results would suggest that part of the reaction occurs via the production of S-adenosylhomocysteine, which can then be converted into 2-oxobutyrate via the transsulphuration pathway, and that part occurs via the production of homoserine by an enzyme converting S-adenosylmethionine into 5'-methylthioadenosine and homoserine lactone.