Transmethylation reactions regulate affinity and functional activity of chemotactic factor receptors on macrophages

Methylation mediated by S-adenosyl-l-methionine is required for the chemotaxis of mononuclear leukocytes. We investigated whether transmethylation reactions are required for normal functioning of chemotactic factor receptors. Three chemoattracrant-mediated functions in macrophages, chemotaxis, the stimulated release of arachidonic acid from membrane phospholipids and superoxide production, are markedly depressed by agents that inhibit cellular methylation reactions. Treatment of macrophages with methylation inhibitors decreased the affinity of the N-formylated chemoattractant receptor present on these cells by a factor of 4.5, but did not significantly alter the total receptor number. These results suggest that the N-formylated chemoattractant receptor on macrophages can exist in more than one affinity state and that an ongoing methylation reaction is required for the maintenance of the receptor in its higher affinity form. Inhibition of methylation lowers the affinity of the receptor and renders it no nfunctional or “uncoupled” in its ability to produce chemotaxis, superoxide and the release of arachidonic acid from leukocyte membranes.     

Transmethylation and control of pattern formation in hydrozoa

Abstract. The control of pattern formation, cell differentiation and cell proliferation in hydroids involves inhibitory signals. In an attempt to identify their chemical nature, compounds from coelenterates which interfere with metamorphosis and pattern-forming processes in Hydractinia and Eirene were isolated. The most strongly metamorphosis-inhibiting compounds were determined to be N -methylpicolinic acid (homarine), N -methylnicotinic acid (trigonelline) and N -trimethylglycine (betaine). The overal concentration of these compounds within tissues is in the range of several millimoles, but micromolar quantities were found to affect development. Thus, the substances must be mainly present in a stored or an inactivated form. The compounds appear to exert their influence by transfering methyl groups to as yet unknown targets. Chemically related compounds that are not able to function as methyl donors have no or only a much lower inhibitory influence, while potential methyl or ethyl donors such as methionine and ethionine have a strong inhibitory influence. Cycloleucine, a competitor with methionine in the production of S-adenosyl-methionine (SAM), and sinefungin, a competitor with SAM in transmethylation, interfere with the intrinsic morphogenetically active compounds identified. One of the spatial patterns controlled by inhibitory signals is the distance between polyps in colonies. In Eirene , the addition of N -methylpicolinic acid led to an increase in the interpolyp distance, while sinefungin produced a decrease in this distance. The addition of sinefungin also stimulated stolon branching. Thus, control of methylation appears to play a key role in the control of metamorphosis and pattern formation in hydrozoa.     

In vivo hydrolysis of S-adenosyl-L-methionine in Escherichia coli increases export of 5-methylthioribose

Escherichia coli can not synthesize methionine from 5-methylthioribose (MTR) but instead exports this sulfur-containing, energy-rich molecule into the surrounding medium. Transforming E. coli with plasmids that direct expression of the cloned coliphage T3 S-adenosyl-L-methionine (SAM) hydrolase (SAMase) induces the met regulon by cleaving the SAM co-repressor to form 5'-methylthioadenosine, which is then cleaved to produce MTR. To test the effect of in vivo SAMase activity on MTR production and its fate, cultures were incubated in the presence of [35S]methionine and [methyl-3H]methionine. Cells with SAMase activity produced significantly enhanced levels (up to 40-fold in some trials) of extracellular MTR -- the only radiolabeled compound released in significant amounts -- when compared with controls. SAM synthetase (metK) mutants transformed with SAMase expression vectors did not show this increase, verifying the path through SAM as the sole route to MTR production. SAMase expression had little or no effect on intracellular MTR pools, levels of radiolabeled macromolecules, or the transfer of methyl groups to compounds that could be precipitated by trichloroacetic acid. Thus, MTR appears to be a dead-end metabolite in E. coli, begging questions about how this has evolved, the mechanism of MTR export for the cell, and whether the release of MTR is important for some other activity.     

Transmethylation of Sterols in Aerobically Adapting Saccharomyces cerevisiae

The transmethylation of methyl-(14)C-methionine and methyl-(14)C-adenosylmethionine into the nonsaponifiable lipids of anaerobically grown yeast during adaptation to aerobic conditions was investigated. The rate and extent of methyl transfer increased with aeration time and was dependent upon the presence of a fermentable carbon source and O(2). Methionine and adenosylmethionine uptake rates increased in adaptation buffer but did not seem to be the rate-limiting factor for transmethylation under the conditions studied. Thinlayer chromatography of the nonsaponifiable fraction after exposure to label showed the labeled product to be ergosterol. Samples taken after short-term exposure to label were composed of two labeled steroidal products, one with kinetics of an ergosterol precursor.     

Condensed phosphate deposition,sulfur amino acid use,and unidirectional transsulfuration in Synechococcus leopoliensis

Cells of the cyanobacterium, Synechococcus leopoliensis, have previously been shown to exhibit diminished growth, increased condensed phosphate accumulation, enlarged polyphosphate bodies, and severe chlorosis when cultured under conditions of sulfur deficiency. These characteristics were used to identify which of several sulfur amino acids and a tripeptide served as a sole sulfur source for this unicellular microorganism. Completely serving sulfur compounds were l-cystine, dl-lanthionine, l-djenkolic acid, and glutathione. Sulfur amino acids serving poorly or not at all were l-cystathionine, dl-homocystine, l-methionine, l-cysteic acid, and taurine. This pattern of use suggests that the unidirectional transsulfuration pathway demonstrated in enteric bacteria and green plants, i.e. l-cysteine to l-homocysteine, operates as well in cyanobacteria of the Synechococcus type.     

The transsulfuration pathway in Tetrahymena pyriformis.

Four enzymes necessary for the metabolism of methionine by the trans-sulfuration pathway, methionine adenosyltransferase (EC 2.5.1.6), adenosylhomocysteinase (EC 3.3.1.1), cystathionine beta-synthase (EC 4.2.1.22) and cystathionine gamma-lyase (EC 4.4.1.1) were identified in Tetrahymean pyriformis. The ability of these cells to transfer 35S from E135S]methionine to form [35S] cysteine was also observed and taken as direct evidence for the functional existence of this pathway in Tetrahymena. An intermediate in the pathway and an active methyl donor, S-adenosylmethionine, was qualitatively identified in Tetrahymena and its concentration was found to be greater in late stationary phase cells than in early stationary phase cells.     

Adrenergic reactions of sheep rumen in vivo

Pharmacodynamical analysis of alpha-adrenergic and beta-adrenergic reactions of sheep rumen was performed in vivo after administration of agonists and antagonists of alpha-adrenergic and beta-adrenergic receptors. It was found that phenylephrine, and in a lower degree propranolol, stimulated the motor activity of sheep rumen, while adrenaline, noradrenaline, isoprenaline, phenoxybenzamine and regitine depressed this activity. Propranolol abolished atonia produced by catecholamines in sheep rumen, and previous blockade of beta-adrenergic receptors with propranolol reversed the relaxing action of catecholamines on the muscular elements in the rumen. The experiments in vivo confirmed the adrenergic effects on the motor activity of the rumen of sheep obtained in earlier investigations on isolated muscles of the rumen. It is suggested that noradrenaline exerts an ambireceptro effect (similar to that of adrenaline) on the motor activity in sheep rumen.     

Desulfuration of l-cysteine through transamination and transsulfuration in rat liver

Desulfuration of L-cysteine by rat liver via a pathway of transamination followed by transsulfuration was studied using cyanide as a sulfur acceptor. More than a 5-fold increase in formation of thiocyanate from L-cysteine and cyanide was observed in the presence of 2-oxoglutarate and pyruvate. L-Cysteine aminotransferase and 3-mercaptopyruvate sulfurtransferase activities in the same preparations were also determined. It was concluded that L-cysteine was desulfurated through transamination and transsulfuration of the resulting 3-mercaptopyruvate, and that the rate-limiting step appears to be the transamination reaction.     

Methionine transsulfuration is increased during sepsis in rats

Methionine transsulfuration in plasma and liver, and plasma methionine and cysteine kinetics were investigated in vivo during the acute phase of sepsis in rats. Rats were infected with an intravenous injection of live Escherichia coli, and control pair-fed rats were injected with saline. Two days after injection, the rats were infused for 6 h with [(35)S]methionine and [(15)N]cysteine. Transsulfuration was measured from the transfer rate of (35)S from methionine to cysteine. Liver cystathionase activity was also measured. Infection significantly increased (P < 0.05) the contribution of transsulfuration to cysteine flux in both plasma and liver (by 80%) and the contribution of transsulfuration to plasma methionine flux (by 133%). Transsulfuration measured in plasma was significantly (P < 0.05) higher in infected rats than in pair-fed rats (0.68 and 0.25 micromol. h(-1). 100 g(-1), respectively). However, liver cystathionase specific activity was decreased by 17% by infection (P < 0.05). Infection increased methionine flux (16%, P < 0.05) less than cysteine flux (38%, P < 0.05). Therefore, the plasma cysteine flux was higher than that predicted from estimates of protein turnover based on methionine data, probably because of enhanced glutathione turnover. Taken together, these results suggest an increased cysteine requirement in infection.     

Co-production of S-adenosyl-L-methionine and L-isoleucine in Corynebacterium glutamicum

In this study, production of S-adenosyl-L-methionine in Corynebacterium glutamicum was investigated by overexpressing genes metK and vgb. Compared with vector control, overexpression of metK alone in C. glutamicum ATCC13032 and IWJ001 increased SAM production 5.11 and 11.65 times, respectively; while overexpression of metK and vgb in C. glutamicum ATCC13032 and IWJ001 increased SAM production 5.83 and 14.95 times, respectively. Further studies on IWJ001/pDXW-8-metk-vgb showed that the limiting factor for SAM production is intracellular ATP supply. Since IWJ001 is an L-isoleucine production strain, IWJ001/pDXW-8-metk-vgb could produce both SAM and L-isoleucine. After 72 h fermentation, SAM and L-isoleucine in IWJ001/pDXW-8-metk-vgb reached 0.67 g/L and 13.8 g/L, respectively. The results demonstrate the potential application of C. glutamicum for co-production of SAM and amino acids.     

Radical reactions in vivo - an overview

Summary Generation of radicals in vivo depends on metabolic activities. The reactions are usually influenced by(i) the presence and concentration of oxygen;(ii) the availability of transition metals (effects of binding and compartimentalization);(iii) the level of reductants and antioxidants (e.g. nutritional effects). The effects of radicals are thought to be due to(i) membrane damage (affecting passive or active transport through altered fluidity/function interrelationships, intercellular messenging through modifications in the synthesis of prostaglandins and leukotrienes);(ii) protein damage (e.g. affecting membrane transporters, channel proteins, receptor or regulatory proteins, immunomodulators);(iii) damage to DNA. Defense mechanisms consist of(i) prevention of the spreading of primary damage by low molecular weight antioxidants (e.g. vitamin E, GSH, vitamin C,-carotene, uric acid);(ii) prevention or limitation of secondary damage by enzymes (e.g. GSH-peroxidase, catalase, superoxide dismutase, DT-diaphorase) and/or chelators;(iii) repair processes, e.g. lipid degradation/membrane repair enzymes (phospholipases, peroxidases, some transferases and reductases), protein disposal or repair enzymes (proteases, GSSG-reductase), DNA degradation repair enzymes (exonuclease III, endonucleases III and IV, glycosylases, polymerases). Recent hypotheses on a messenging function of the superoxide anion O ₂ ^– are discussed and possible implications of cross-reactions between O ₂ ^– and nitric oxide (endothelium-derived relaxing factor EDRF) are shortly mentioned.Paper given at the workshop Molecular Radiation Biology. German Section of the DNA Repair Network, München-Neuherberg, 21.–23.3.90     

The dark reactions of rye phytochrome in vivo and in vitro

The dark reactions of Secale cereale L. cv. Balbo phytochrome have been investigated in coleoptile tips and in extensively purified extracts of large molecular weight phytochrome. Destruction, but not reversion, was detected in vivo. The effects of various inhibitors of an in vitro phytochrome-degrading protease did not support a view of proteolytic attack as the basis of in vivo destruction. In vitro, rye phytochrome (about 240,000 molecular weight) reverted extremely rapidly, even at 5 C. The reversion curves were resolved into two first order components. The previously studied 60,000 molecular weight species, obtained by controlled proteolysis of large rye phytochrome, showed a similar two-component pattern, but a much slower over-all reversion rate. This reduction in rate was caused mainly by the reversion of a greater percentage of the small phytochrome as the slow component. Sodium dithionite markedly accelerated the reversion rate of both large and small forms, but oxidants, at concentrations low enough to avoid chromophore destruction, had no effect. Both large and small crude Avena sativa L. phytochrome showed two-component reversion kinetics.     

Oscillations in peroxidase-catalyzed reactions and their potential function in vivo

The peroxidase-oxidase reaction has become a model system for the study of oscillations and complex dynamics in biochemical systems. In the present paper we give an overview of previous experimental and theoretical studies of the peroxidase-oxidase reaction. Recent in vitro experiments have raised the question whether the reaction also exhibits oscillations and complex dynamics in vivo. To investigate this possibility further we have undertaken new experimental studies of the reaction, using horseradish extracts and phenols which are widely distributed in plants. The results are discussed in light of the occurrence and a possible functional role of oscillations and complex dynamics of the peroxidase-oxidase reaction in vivo.     

Transmethylation inhibitors decrease chemotactic sensitivity and delay cell aggregation in Dictyostelium discoideum

In Dictyostelium discoideum, extracellular cyclic AMP (cAMP) induces chemotaxis and cell aggregation. Suspensions of cAMP-sensitive cells respond to a cAMP pulse with a rapid, transient increase of protein carboxyl methylation. The transmethylation inhibitors cycloleucine, L-homocysteine thiolactone, and coformycin decrease chemotactic sensitivity and delay cell aggregation when administered in concentrations which do not influence cAMP binding to cell surface receptors or the activity of total phosphodiesterase. The ability of the drugs to inhibit chemotaxis could be correlated with their capacity to convert the initial transient positive response of carboxyl methylation to cAMP into a negative one. This suggests that both protein O-methyltransferase and protein methylesterase are activated after stimulation of aggregative cells with cAMP, the net effect being a transient, positive response of methylation. In the presence of a sufficiently large dose of inhibitor, methyltransferase is inhibited, whereas methylesterase activity is much less affected, so that a transient negative response of methylation to cAMP is observed. The slow, positive response of carboxyl methylation to cAMP which occurs ca. 2.5 to 5 min after stimulus administration is not affected by inhibitors of transmethylation. These results suggest that methylation reactions are involved in the chemotactic response of D. discoideum cells to cAMP.