Analysis of S-methylmethionine and S-adenosylmethionine in plant tissue by a dansylation, dual-isotope method

A method is presented for determining the levels of S-methylmethionine (MeMet) and S-adenosylmethionine (AdoMet) in the same plant tissue sample, utilizing readily available equipment. The bottom limit of sensitivity, ca. 100 pmol, can be lowered if required. A trichloracetic acid homogenate of the tissue is supplemented with [carboxyl-14C]MeMet and [carboxyl-14C]AdoMet. After separation of MeMet and AdoMet from each other and from endogenous homoserine on a phosphocellulose column, the two fractions are heat treated at appropriate pH values to liberate [14C]homoserine. Quantitation is via the 3H/14C ratio of [3H]dansyl-[14C]homoserine isolated by thin-layer chromatography. The method is validated with pea cotyledon, corn root, and cauliflower leaf.     

Inhibition of adenosylmethionine decarboxylase and perturbation of polyamine metabolism by 3-deaza-(+/-)aristeromycin

3-Deaza-(+/-)aristeromycin, previously known mainly as a potent inhibitor of adenosylhomocysteine hydrolase, can also inhibit the activity of adenosylmethionine decarboxylase. The release of [14C]CO2 from HeLa cells labeled with [carboxyl-14C]methionine was inhibited by more than 70% after 4 hours in the presence of 4 microM 3-deaza-(+/-)aristeromycin. Concomitant with this inhibition, there was a significant increase in the amount of putrescine in the HeLa cells. Adenosylmethionine decarboxylase isolated from HeLa cells could also be inhibited by 3-deaza-(+/-)aristeromycin and 3-deazaadenosine, 3-deazaadenosylhomocysteine, and 3-deaza-(+/-)aristeromycinylhomocysteine.     

Carotenoid biosynthesis in Rhodopseudomonas spheroides. S-Adenosylmethionine as the methylating agent in the biosynthesis of spheroidene and spheroidenone

[methyl-(14)C]Methionine and S-adenosyl[methyl-(14)C]methionine were incorporated into the methoxycarotenoids spheroidene and spheroidenone by Rhodopseudomonas spheroides. The incorporation was greatly enhanced in the presence of lysozyme. On degradation of labelled spheroidene by hydriodic acid, the (14)C label was recovered in methyl iodide. Degradation of spheroidenone by reduction and allylic dehydration and demethylation of the reduction product gave a mixture of unlabelled carotenoid hydrocarbons, including 3,4-didehydrolycopene and 3,4-didehydro-7',8'-dihydrolycopene. The label from [methyl-(14)C]methionine and S-adenosyl[methyl-(14)C]methionine was located specifically in the methoxy group of spheroidene and spheroidenone. The biosynthesis of methoxycarotenoids in Rps. spheroides involves methylation of the tertiary hydroxyl groups of intermediates with S-adenosylmethionine.     

Activation of lysine 2,3-aminomutase by S-adenosylmethionine

Lysine 2,3-aminomutase, which catalyzes the interconversion of L-lysine and L-beta-lysine, is S-adenosyl-methionine-dependent, and the adenosyl-C-5' methylene group of this coenzyme mediates the transfer of hydrogen from C-3 of lysine to C-2 of beta-lysine. We here report experiments that address the mechanism by which S-adenosylmethionine activates lysine 2,3-aminomutase. We also describe an updated and improved purification procedure that produces enzyme with a specific activity substantially higher than that previously reported. Activation of the enzyme by less than 1 mol of S-adenosyl[1-14C]methionine/mol of subunits in the presence of lysine leads to the production of [14C] methionine in a kinetically biphasic process. After 1.8 min at 30 degrees C, 10% of the 14C is reisolated as [14C] methionine, and the cleavage increases to 19% after 10 min and to 51% after 40 min. Similar experiments with S-[8-14C]adenosylmethionine produce 5'-deoxy[14C]adenosine in amounts similar to the formation of methionine. The major radioactive products isolated in each case are [14C]methionine or 5'-deoxy[14C]adenosine, respectively, and unchanged 14C-labeled S-adenosylmethionine. These experiments support the hypothesis that activation of lysine 2,3-aminomutase involves a transfer of the 5'-deoxyadenosyl moiety from S-adenosylmethionine to another species associated with the enzyme, presumably another cofactor, to form an adenosyl cofactor that functions as the proximal, hydrogen abstracting species in the mechanism.     

Rapid and convenient method for the assay of aminopropyltransferases.

A new method for the assay of aminopropyltransferase activity is described. The method measures the formation of [methyl-14C]methylthioadenosine from decarboxylated S-adenosyl[methyl-14C]methionine in the presence of an amine acceptor. When used with extracts from rat ventral prostate, kidney, liver or brain, and with putrescine or spermidine as amines, the method gave results in excellent agreement with those obtained by the much more time-consuming conventional method. It was found that 1,3-diamino-propane and 1,8-diamino-octane were not acceptors for the prostatic enzyme fraction, but 1,5-diaminopentane (cadaverine) was active and 1,9-diaminononane and 1,12-diaminododecane also lead to the production of [methyl-14C]methylthioadenosine.     

Regulation of polyamine biosynthesis in tobacco. Effects of inhibitors and exogenous polyamines on arginine decarboxylase, ornithine decarboxylase, and S-adenosylmethionine decarboxylase

Treatment of tobacco liquid suspension cultures with methylglyoxal bis(guanylhydrazone) (MGBG) an inhibitor of S-adenosylmethionine decarboxylase, resulted in a dramatic overproduction of a 35-kDa peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Malmberg, R.L., and McIndoo, J. (1983) Nature 305, 623-625). MGBG treatment also resulted in a 20-fold increase in the activity of S-adenosylmethionine decarboxylase. Purification of S-adenosylmethionine decarboxylase from MGBG-treated cultures revealed that the overproduced 35-kDa peptide and S-adenosylmethionine decarboxylase are identical. Precursor incorporation experiments using [3H] methionine and [35S]methionine revealed that MGBG does not induce any increased synthesis of S-adenosylmethionine decarboxylase but rather stabilizes the protein to proteolytic degradation. The half-life of the enzyme activity was increased when MGBG was present in the growth medium. In addition to stabilizing S-adenosylmethionine decarboxylase, MGBG also resulted in the rapid and specific loss of arginine decarboxylase activity with little effect ornithine decarboxylase. The kinetics of this effect suggest that arginine decarboxylase synthesis was rapidly inhibited by MGBG. Exogenously added polyamines had little effect on ornithine decarboxylase, whereas S-adenosylmethionine and arginine decarboxylase activities rapidly diminished with added spermidine or spermine. Finally, inhibition of ornithine decarboxylase was lethal to the cultures, whereas inhibition of arginine decarboxylase was only lethal during initiation of growth in suspension culture.     

S-adenosylmethionine decarboxylase from the thermophilic archaebacterium Sulfolobus solfataricus. Purification, molecular properties and studies on the covalently bound pyruvate.

S-Adenosylmethionine decarboxylase from Sulfolobus solfataricus, a thermoacidophilic archaebacterium optimally growing at 87 degrees C, has been purified to homogeneity. The specific activity of the homogeneous enzyme is 12 nmol CO2 formed min-1 (mg protein)-1 and the overall yield 8%. The enzyme is thermophilic with an optimum at 75 degrees C, is thermostable, and does not require divalent cations or putrescine for activity. It has a molecular mass of 32 kDa, and appears to be a monomeric protein. S-Adenosylmethionine decarboxylase from S. solfataricus contains covalently linked pyruvate as prosthetic group and is inactivated in a time-dependent process by NaCNBH3, in the presence of both the substrate and the product. Incubation with decarboxylated S-adenosyl[Me-3H]methionine and NaCNBH3 resulted in the labeling of the protein at the active site.     

Effects of 1-Aminocyclopropane-1-carboxylic Acid Production on the Changes in the Polyamine Levels in Hiproly Barley Callus after Auxin Withdrawal

Contents of polyamines and 1-aminocyclopropane-1-carboxylic acid (ACC) in Hiproly barley callus were examined under different culture conditions. After auxin withdrawal, the contents of free polyamines changed conversely to the contents of ACC. In the absence of auxin, incorporation of l-[3,4–¹⁴C]methionine into polyamines and the activity of S-adenosylmethionine decarboxylase (SAMDCase) in the callus increased, then remained stable, but incorporation of l-[3,4- ¹⁴C]methionine into ACC, precursor of ethylene and ACC synthase activity once declined and increased again.

Aminooxyacetic acid (AOA) affected the increase in the levels of polyamines in the callus. 1- Aminoisobutyric acid (AIB) had a slight effect on the polyamine production. The incorporation of l-[3,4–¹⁴C]methionine into ACC and ACC synthase activity were inhibited by AOA, but not by « 4 AIB. AOA stimulated the activity of SAMDCase, and also enhanced the incorporation of l-[3,4- ¹⁴C]methionine into polyamines in the callus. Methylglyoxal-bis(guanylhydrazone) (MGBG) greatly enhanced the ACC production. The rate of incorporation of l-[3,4–¹⁴C]methionine into ACC and ACC synthase activity in the callus were significantly enhanced by MGBG. MGBG strongly inhibited SAMDCase activity and the incorporation of l-[3,4–¹⁴C]methionine into polyamines. Moreover, the synthesis of polyamines was inhibited by MGBG.

These results suggested that in Hiproly barley callus ACC production has an important effect on changes in the polyamine levels, and that polyamine and ethylene biosynthetic pathways are regulated by competition against each other.     

Compensatory Aspects of the Biosynthesis of Spermidine in Tobacco Cells in Suspension Culture

The relationship between the biosynthesis of polyamines andethylene was examined in suspension cultures of Nicotiana tabacumL. cells. Aminooxyacetic acid (AOA), an inhibitor of 1-aminocyclopropane-1-carboxylicacid synthase, inhibited the production of ethylene and raisedlevels of spermidine by increasing the availability of S-adenosylmethionine(SAM) for the synthesis of polyamines. In contrast, methylglyoxalbis (guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethioninedecarboxylase (SAMDC), an enzyme involved in the biosynthesisof polyamines, caused a slight increase in the rate of biosynthesisof ethylene. However MGBG did not decrease the rate of biosynthesisof polyamines in 10-day-old senescing cells. Although MGBG inhibitedthe conversion of L-[U-^l4C]methionine into labeled spermidinevia SAM both in 4-day-and in 10-day-cultured cells, it stimulatedthe conversion of L-[U-^l4C]aspartic acid into labeled spermidinein 10-day-cultured cells. In actively dividing 4-day-culturedcells, L-[U-¹⁴C]homo-serine was also converted into polyamines.In senescing cells, which produce large amounts of ethylene,the biosynthesis of spermidine from aspartic acid coincidedwith that from methionine. In actively growing cells, whichproduce large amounts of polyamines, the biosynthesis of spermidinefrom homoserine coincided with that from methionine. These resultsindicate that homoserine and aspartic acid can be both usedas precursors in the biosynthesis of polyamines and help tomaintain appropriate titers of polyamines, when SAMDC is inhibitedand the level of decarboxylated SAM becomes limiting. (Received May 14, 1990; Accepted March 11, 1991)     

Homoserine kinase and threonine synthase in methionine-overproducing soybean tissue cultures

To gain understanding of the regulation of methionine level in plants, we assayed homoserine kinase and threonine synthase in extracts of wild type and several methionine-overproducing soybean [Glycine max (L.) Merr.] callus lines. The specific activity of homoserine kinase was depressed by 45–73%, and that of threonine synthase by 26–43% in the high methionine lines. Cysteine inhibited threonine synthase in wild type and variant lines. Threonine synthase in two variant lines showed significantly less inhibition by cysteine and in one line was inhibited by threonine. Depressed threonine synthase activity may increase the availability of homoserine phosphate to the competing methionine biosynthetic pathway.Abreviations MOPS morpholinopropane-sulfonate - EDTA ethylenediamine-tetraacetate - DTE dithioerythritol - AdoMet S-adenosylmethionine     

The Escherichia coli cysG gene encodes S-adenosylmethionine-dependent uroporphyrinogen III methylase.

The Escherichia coli cysG gene was successfully subcloned and over-expressed to produce a 52 kDa protein that was purified to homogeneity. This protein was shown to catalyse the S-adenosylmethionine-dependent methylation of uroporphyrinogen III to give a product identified as sirohydrochlorin on the basis of its absorption spectra, incorporation of 14C label from S-adenosyl[Me-14C]methionine and mass and 1H-n.m.r. spectra of its octamethyl ester. Further confirmation of the structure was obtained from a 14C-n.m.r. spectrum of the methyl ester produced by incubation of the methylase with uroporphyrinogen III, derived from [4.6-13C2]porphobilinogen, and S-adenosyl[Me-13C]methionine.     

Effects of Carboxylemethylation and Polyamine Synthesis Inhibitors on Methylation of Trypanosoma brucei Cellular Proteins and Lipids

ABSTRACT. The fate of methionine in eukaryotic cells is divided between protein synthesis and the branched pathway encompassing polyamine synthesis, methylation of proteins and lipids, and transsulphuration reactions. Aside from protein synthesis, the first step to all other uses of methionine is conversion to S-adenosylmethionine. Blockade of polyamine synthesis in African trypanosomes by the ornithine decarboxylase inhibitor DL-α-difluoromethylomithine (Ornidyl, DFMO) the AdoMet decarboxylase inhibitor 5′-{[(Z)-4-amino-2-butenyl]-methylamino}-5′-deoxyadenosine or the protein methylase inhibitor sinefungin induces dramatic increases in intracellular AdoMet. In a previous study, distribution and pool sizes of [¹⁵S] or [U-¹⁴C]methionine were followed in bloodform trypanosomes as incorporation into the total TCA precipitable fraction. In the present study, the effects of pretreatment with DFMO (1 mM), MDL 73811 (1 μM) and sinefugin (2 nM) on [³⁵S] and [U-¹⁴C]methionine incorporation were studied in blood forms. DFMO or MDL 73811 pretreatment increased protein methylation 1.5-fold through incorporation of [U¹⁴C]methionine, while sinefungin caused a 40% reduction of incorporation. The increases in incorporation of [U-¹⁴C]methionine due to DFMO and MDL 73811 were reduced 40% to 70% by including cold AdoMet (1 mM) in the incubation medium, an indication of AdoMet transport by bloodform trypanosomes and the utilization of [U-¹⁴C]methionine as AdoMet. Exogenous AdoMet had no effect on [³⁵S]methionine incorporation. The agents studied are curative for African trypnosomiasis infections, either clinically (DFMO) or in model infections (MDL 73811, sinefungin) and thus highlight interference with AdoMet metabolism and methylation reactions as biochemical consequences of these agents.     

The influence of nerve section on the metabolism of polyamines in rat diaphragm muscle.

Concentrations of spermidine, spermine and putrescine have been measured in rat diaphragm muscle after unilateral nerve section. The concentration of putrescine increased approx. 10-fold 2 days after nerve section, that of spermidine about 3-fold by day 3, whereas an increase in the concentration of spermine was only observed after 7-10 days. It was not possible to show enhanced uptake of either exogenous putrescine or spermidine by the isolated tissue during the hypertrophy. Consistent with the accumulation of putrescine, activity of ornithine decarboxylase increased within 1 day of nerve section, was maximally elevated by the second day and then declined. Synthesis of spermidine from [14C]putrescine and either methionine or S-adenosylmethionine bt diaphragm cytosol rose within 1 day of nerve section, but by day 3 had returned to normal or below normal values. Activity of adenosylmethionine decarboxylase similarly increased within 1 day of nerve section, but by day 3 had declined to below normal values. Activity of methionine adenosyltransferase was elevated throughout the period studied. The concentration of S-adenosylmethionine was likewise enhanced during hypertrophy. Administration of methylglyoxal bis(guanylhydrazone) produced a marked increase in adenosylmethionine decarboxylase activity and a large increase in putrescine concentration, but did not prevent the rise in spermidine concentration produced by denervation. Possible regulatory mechanisms of polyamine metabolism consistent with the observations are discussed.     

Polyamine Synthesis and Interconversion by the Microsporidian Encephalitozoon cuniculi

Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [1H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.     

Assay and regulation of S-adenosylmethionine synthetase in Saccharomyces cerevisiae and Candida utilis.

A simple and sensitive assay for S-adenosylmethionine (SAM) synthetase is described which depends on the quantitative separation of the product, [14CH3]S-adenosylmethionine, from the substrate, L-[14CH3]methionine, on a Bio-Rex 70 column. L-Methionine protects the enzyme during preparation of cell extracts by sonic treatment but causes repression of enzyme activity during growth of Candida utilis. The presence of 5 mM methionine in the growth medium repressed SAM synthetase specific activity threefold compared to the specific acitivity of the enzyme isolated from cells grown in unsupplemented medium. Conversely, the presence of methionine in the growth medium resulted in an 80-fold increase in the intracellular concentration of SAM as compared to the Sam accumulated intracellularly in unsupplemented cultures.     

Characterization of a long-chain 14C-labelled alcohol resulting from a plant enzyme-catalysed reaction with S-adenosyl[methyl-14C]methionine

A particulate enzyme from Phaseolus aureus seedlings catalyses the incorporation of radioactivity from S-adenosyl[Me-(14)C]methionine into several lipid acceptors present in the enzyme preparation. The structure of one of the radioactive lipids has been elucidated by using mass, n.m.r. and i.r. spectral data. This compound has a molecular formula C(28)H(57)OH, is a saturated primary alcohol and has a single branch of one methyl group in the interior of the hydrocarbon chain. The results of a Kuhn-Roth degradation demonstrate that the branch methyl group is the one derived from S-adenosylmethionine.     

Biogenesis of mitochondria. Phospholipid synthesis in vitro by yeast mitochondrial and microsomal fractions

The ability in vitro of yeast mitochondrial and microsomal fractions to synthesize lipid de novo was measured. The major phospholipids synthesized from sn-[2-(3)H]glycerol 3-phosphate by the two microsomal fractions were phosphatidylserine, phosphatidylinositol and phosphatidic acid. The mitochondrial fraction, which had a higher specific activity for total glycerolipid synthesis, synthesized phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidic acid, together with smaller amounts of neutral lipids and diphosphatidylglycerol. Phosphatidylcholine synthesis from both S-adenosyl[Me-(14)C]methionine and CDP-[Me-(14)C]choline appeared to be localized in the microsomal fraction.     

S-adenosylmethionine decarboxylase degradation by the 26 S proteasome is accelerated by substrate-mediated transamination

The short-lived enzyme S-adenosylmethionine decarboxylase uses a covalently bound pyruvoyl cofactor to catalyze the formation of decarboxylated S-adenosylmethionine, which then donates an aminopropyl group for polyamine biosynthesis. Here we demonstrate that S-adenosylmethionine decarboxylase is ubiquitinated and degraded by the 26 S proteasome in vivo, a process that is accelerated by inactivation of S-adenosylmethionine decarboxylase by substrate-mediated transamination of its pyruvoyl cofactor. Proteasome inhibition in COS-7 cells prevents the degradation of S-adenosylmethionine decarboxylase antigen; however, even brief inhibition of the 26 S proteasome caused substantial losses of S-adenosylmethionine decarboxylase activity despite accumulation of S-adenosylmethionine decarboxylase antigen. Levels of the enzyme's substrate (S-adenosylmethionine) increased rapidly after 26 S proteasome inhibition, and this increase in substrate level is consistent with the observed loss of activity arising from an increased rate of inactivation by substrate-mediated transamination. Evidence is also presented that this substrate-mediated transamination accelerates normal degradation of S-adenosylmethionine decarboxylase, as the rate of degradation of the enzyme was increased in the presence of AbeAdo (5'-([(Z)-4-amino-2-butenyl]methylamino]-5'-deoxyadenosine) (a substrate analogue that transaminates the enzyme); conversely, when the intracellular substrate level was reduced by methionine deprivation, the rate of degradation of the enzyme was decreased. Ubiquitination of S-adenosylmethionine decarboxylase is demonstrated by isolation of His-tagged AdoMetDC (S-adenosylmethionine decarboxylase) from COS-7 cells co-transfected with hemagglutinin-tagged ubiquitin and showing bands that were immunoreactive to both anti-AdoMetDC antibody and anti-hemagglutinin antibody. This is the first study to demonstrate that AdoMetDC is ubiquitinated and degraded by the 26 S proteasome, and substrate-mediated acceleration of degradation is a unique finding.     

Expression of an aromatic-dependent decarboxylase which provides growth-essential CO2 equivalents for the acetogenic (Wood) pathway of Clostridium thermoaceticum.

The acetogen Clostridium thermoaceticum generates growth-essential CO2 equivalents from carboxylated aromatic compounds (e.g., 4-hydroxybenzoate), and these CO2 equivalents are likely integrated into the acetogenic pathway (T. Hsu, S. L. Daniel, M. F. Lux, and H. L. Drake, J. Bacteriol. 172:212-217, 1990). By using 4-hydroxybenzoate as a model substrate, an assay was developed to study the expression and activity of the decarboxylase involved in the activation of aromatic carboxyl groups. The aromatic-dependent decarboxylase was induced by carboxylated aromatic compounds in the early stages of growth and was not repressed by glucose or other acetogenic substrates; nonutilizable carboxylated aromatic compounds did not induce the decarboxylase. The decarboxylase activity displayed saturation kinetics at both whole-cell and cell extract levels, was sensitive to oxidation, and was not affected by exogenous energy sources. However, at the whole-cell level, metabolic inhibitors decreased the decarboxylase activity. Supplemental biotin or avidin did not significantly affect decarboxylation. The aromatic-dependent decarboxylase was specific for benzoates with a hydroxyl group in the para position of the aromatic ring; the meta position could be occupied by various substituent groups (-H, -OH, -OCH3, -Cl, or -F). The carboxyl carbon from [carboxyl-14C] vanillate went primarily to 14CO2 in short-term decarboxylase assays. During growth, the aromatic carboxyl group went primarily to CO2 under CO2-enriched conditions. However, under CO2-limited conditions, the aromatic carboxyl carbon went nearly totally to acetate, with equal distribution between the carboxyl and methyl carbons, thus demonstrating that acetate could be totally synthesized from aromatic carboxyl groups. In contrast, when cocultivated (i.e., supplemented) with CO under CO2-limited conditions, the aromatic carboxyl group went primarily to the methyl carbon of acetate.     

The role of amino acids in the regulation of hydrogen sulfide production during ultradian respiratory oscillation of Saccharomyces cerevisiae

We previously demonstrated that periodic H2S production during aerobic continuous culture of Saccharomyces cerevisiae resulted in ultradian respiratory oscillation, and that H2S production was dependent on the activity of sulfate uptake and the level of sulfite. To investigate the mechanism of regulation of the sulfate assimilation pathway and of respiratory oscillation, several amino acids were pulse-injected into cultures during respiratory oscillation. Injection of sulfur amino acids or their derivatives perturbed respiratory oscillation, with changes in the H2S production profile. Four major regulators of H2S production in the sulfate assimilation pathway and respiratory oscillation were identified: (1) O-acetylhomoserine, not O-acetylserine, as a sulfide acceptor, (2) homoserine/threonine as a regulator of O-acetylhomoserine supply, (3) methionine/S-adenosyl methionine as a negative regulator of sulfate assimilation, and (4) cysteine (or its derivatives) as an essential regulator. The results obtained after the addition of DL-propargylglycine (5 microM and 100 microM) and cystathionine (50 microM) suggested that the intracellular cysteine level and cystathionine gamma-lyase, rather than methionine/S-adenosylmethionine, play an essential role in the regulation of sulfate assimilation and respiratory oscillation. Based on these results and those of our previous reports, we propose that periodic depletion of cysteine (or its derivatives), which is involved in the detoxification of toxic materials originating from respiration, causes periodic H2S production.