Effect of adenosine metabolites on methyltransferase reactions in isolated rat livers

Adenosine is rapidly metabolized by isolated rat livers. The major products found in the perfusate were inosine and uric acid while hypoxanthine could also be detected. S-Adenosylhomocysteine was also excreted when the liver was perfused with both adenosine and L-homocysteine. A considerable portion of the added adenosine was salvaged via the adenosine kinase reaction. The specific radioactivity of the resultant AMP reached 75–80% of the added [8-¹⁴C]adenosine within 90 min. When the liver was perfused with adenosine alone, hydrolysis of S-adenosyllhomosysteine, via S-adenosylhomocysteine hydrolase, appeared to be blocked resulting in the accumulation of this compound. As the intracellular level of S-adenosylhomocysteine increased, the rates of various methyltransferase reactions were reduced, resulting in elevated levels of intracellular S-adenosylmethionine. When the liver was perfused with normal plasma levels of methionine the S-adenosylmethionine : S-adenosylhomocysteine ratio was 5.3 and the half-life of the methyl groups was 32 min. Upon further addition of adenosien the S-adenosylmethionine : S-adenosylhomocysteine ratio shifted to 1.7 and the half-life of the methyl groups to 103 min. In the presence of adenosine and L-homocysteine such inordinate amounts of S-adenosylhomocysteine accumulated in the cell that methylation reactions were completely inhibited. Although adenine has been found to be a product of the S-adenosylhomocysteine hydrolase only trace quantities of this compound were detectable in the tissue after perfusing the liver with high concentrations of adenosine for 90 min.     

Boron deprivation decreases liver S-adenosylmethionine and spermidine and increases plasma homocysteine and cysteine in rats

Two experiments were conducted with weanling Sprague–Dawley rats to determine whether changes in S-adenosylmethionine utilization or metabolism contribute to the diverse responses to boron deprivation. In both experiments, four treatment groups of 15 male rats were fed ground corn-casein based diets that contained an average of 0.05 mg (experiment 1) or 0.15 mg (experiment 2) boron/kg. In experiment 2, some ground corn was replaced by sucrose and fructose to increase oxidative stress. The dietary variables were supplemental 0 (boron-deprived) or 3 (boron-adequate) mg boron/kg and different fat sources (can affect the response to boron) of 75 g corn oil/kg or 65 g fish (menhaden) oil/kg plus 10 linoleic acid/kg. When euthanized at age 20 (experiment 1) and 18 (experiment 2) weeks, rats fed the low-boron diet were considered boron-deprived because they had decreased boron concentrations in femur and kidney. Boron deprivation regardless of dietary oil increased plasma cysteine and homocysteine and decreased liver S-adenosylmethionine, S-adenosylhomocysteine, and spermidine. Plasma concentration of 8-iso-prostaglandin F_2α (indicator of oxidative stress) was not affected by boron, but was decreased by feeding fish oil instead of corn oil. Fish oil instead of corn oil decreased S-adenosylmethionine, increased spermidine, and did not affect S-adenosylhomocysteine concentrations in liver. Additionally, fish oil versus corn oil did not affect plasma homocysteine in experiment 1, and slightly increased it in experiment 2. The findings suggest that boron is bioactive through affecting the formation or utilization of S-adenosylmethionine. Dietary fatty acid composition also affects S-adenosylmethionine formation or utilization, but apparently through a mechanism different from that of boron.     

Simultaneous determination of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in biological samples using solid-phase extraction and high-performance liquid chromatography

A sensitive and rapid method for measuring simultaneously adenosine, S-adenosylhomocysteine and S-adenosylmethionine in renal tissue, and for the analysis of adenosine and S-adenosylhomocysteine concentrations in the urine is presented. Separation and quantification of the nucleosides are performed following solid-phase extraction by reversed-phase ion-pair high-performance liquid chromatography with a binary gradient system. N⁶-Methyladenosine is used as the internal standard. This method is characterized by an absolute recovery of over 90% of the nucleosides plus the following limits of quantification: 0.25–1.0 nmol/g wet weight for renal tissue and 0.25–0.5 μM for urine. The relative recovery (corrected for internal standard) of the three nucleosides ranges between 98.1±2.6% and 102.5±4.0% for renal tissue and urine, respectively (mean±S.D., n=3). Since the adenosine content in kidney tissue increases instantly after the onset of ischemia, a stop freezing technique is mandatory to observe the tissue levels of the nucleosides under normoxic conditions. The resulting tissue contents of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in normoxic rat kidney are 5.64±2.2, 0.67±0.18 and 46.2±1.9 nmol/g wet weight, respectively (mean±S.D., n=6). Urine concentrations of adenosine and S-adenosylhomocysteine of man and rat are in the low μM range and are negatively correlated with urine flow-rate.     

Induction and Repression in the S-Adenosylmethionine and Methionine Biosynthetic Systems of Saccharomyces cerevisiae

Two methionine biosynthetic enzymes and the methionine adenosyltransferase are repressed in Saccharomyces cerevisiae when grown under conditions where the intracellular levels of S-adenosylmethionine are high. The nature of the co-repressor molecule of this repression was investigated by following the intracellular levels of methionine, S-adenosylmethionine, and S-adenosylhomocysteine, as well as enzyme activities, after growth under various conditions. Under all of the conditions found to repress these enzymes, there is an accompanying induction of the S-adenosylmethionine-homocysteine methyltransferase which suggests that this enzyme may play a key role in the regulation of S-adenosylmethionine and methionine balance and synthesis. S-methylmethionine also induces the methyltransferase, but unlike S-adenosylmethionine, it does not repress the methionine adenosyltransferase or other methionine biosynthetic enzymes tested.     

Regulation of Rat Pineal Hydroxyindole-O-Methyltransferase: Evidence of S-Adenosylmethionine-Mediated Glucocorticoid Control

: Rat pineal hydroxyindole-O-methyltransferase is controlled similarly to adrenal medullary phenylethanolamine N-methyltransferase. S-adenosylmethionine (SAM), the in vivo cofactor utilized by the enzyme to convert N-acetylserotonin to melatonin, protects this methyltransferase against tryptic proteolysis in vitro. Furthermore, in vivo studies suggest that the nucleoside itself is controlled by glucocorticoids. Hypophysectomy decreases hydroxyindole-O-methyltransferase levels as compared with control animals, while dexamethasone and SAM administration restore enzyme levels toward control values. In vitro proteolytic studies further demonstrate that, although N-acetylserotonin does not stabilize the enzyme against trypsinization, this substrate acts synergistically with SAM to confer greater stabilization than observed with SAM alone.     

Purification and partial characterization of rat kidney histamine-N-methyltransferase

Histamine-N-methyltransferase (EC 2.1.1.8) was purified 1700-fold with a yield of 9% from rat kidney. Purification included ammonium sulfate precipitation, linear gradient DEAE-cellulose chromotography and S-adenosylhomocysteine affinity chromotography. The purified enzyme preparation showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 35 000. The isoelectric point of the enzyme was at pH 5.2. The purified enzyme preparation did not contain detectable amounts of histamine. The purified enzyme was totally inhibited in 100 μM parahydroxymercuric benzoate and in 10 μM iodoacetamide, and it was found to be stabilized with dithiothreitol (1 mM), suggesting that the enzyme has an SH-group in the active center. The K_m values for histamine and S-adenosylmethionine were 6.0 and 7.1 μM, respectively. 50% inhibition of histamine-N-methyltransferase was obtained at 28 μM S-adenosylhomocysteine and 100 μM methylhistamine. The purified enzyme was slightly inhibited in 1 mM methylthioadenosine. Histamine in concentrations higher than 25 μM caused substrate inhibition.     

The rate of transmethylation in mouse liver as measured by trapping S-adenosylhomocysteine

Periodate-oxidized adenosine has previously been shown to be a potent inhibitor in vitro of S-adenosylhomocysteine hydrolase (E.C. 3.3.1.1). This paper describes the inhibition of this enzyme in liver following injection of mice with periodate-oxidized adenosine. A maximally effective dose of 100 nmol/g of this compound causes liver S-adenosylhomocysteine to increase from 12 to 600 nmol/g within 30 min. This accumulation of S-adenosylhomocysteine provides an estimate of the rates of transmethylation, as well as adenosine and homocysteine production, as being at least 20 nmol/min/g liver. A doubling of S-adenosylmethionine in the liver of mice treated with periodate-oxidized adenosine suggests that the high levels of S-adenosylhomocysteine inhibit some transmethylation reactions.     

The S-n-propyl analogue of S-adenosylmethionine

A special strain of Saccharomyces cerevisiae responded to a supplement of S-n-propyl-l-homocysteine in the culture medium by synthesizing S-adenosyl-(S-n-propyl)l-homycysteine, the S-n-propyl analogue of S-adenosylmethionine. S-n-Butyl-l-homocysteine reacted sparingly with this strain, but S-isopropyl-l-homocysteine failed to form detectable quantities of the corresponding S-adenosylsulfonium were compound. The S-n-propyl compound was isolated by extraction of the cells, followed by ion-exchange chromatography, which separated it from endogenous S-adenosylmethionine. The structure was determined by hydrolytic procedures leading to overlapping fragments of known structure, 5′-n-propylthioadenosine and S-n-propyl-l-homocysteine. The new sulfonium compound was examined for its activity as n-propyl donor by substituting it for S-adenosylmethionine in methyltransferase systems. Enzymatic transpropylation was observed with S-adenosylmethionine: l-homocysteine S-methyltransferase (EC 2.1.1.10). Its rate was low in the S-adenosylmethionine: N-acetylserotonin O-methyltransferase system (EC 2.1.1.4), and below recognition with S-adenosylmethionine: guanidonoacetate methyltransferase (EC 21.1.2) and S-adnosylmethionine: histame N-methyltransferase (EC 2.1.1.8).     

Adenosine metabolism: Modification by S-adenosylhomocysteine and 5′-methylthioadenosine

To elucidate potential toxic properties of S-adenosylhomocysteine and 5′-methylthioadenosine, we have examined the inhibitory properties of these compounds upon enzymes involved with adenosine metabolism. S-Adenosylhomocysteine, but not S-adenosylmethionine, was a noncompetitive inhibitor of adenosine kinase with K_i values ranging from 100 to 400 μm. Methylthioadenosine competitively inhibited adenosine kinase with variable adenosine below 1 μm with a K_i of 120 μm, increased adenosine kinase activity when the adenosine concentration exceeded 2 μm, and did not appear to be a substrate for adenosine kinase. Methylthioadenosine inactivated S-adenosylhomocysteine hydrolase from erythrocytes, B-lymphoblasts, and T-lymphoblasts with K_i values ranging from 65 to 117 μm and “k₂” from 0.30 to 0.55 min^?1. Adenosine deaminase was not inhibited by 5′-methylthioadenosine up to 1000 μm. To clarify how 5′-methylthioadenosine might accumulate, 5′-methylthioadenosine phosphorylase was evaluated. This enzyme was not blocked by up to 500 μm adenosine, deoxyadenosine, S-adenosylhomocysteine, or S-adenosylmethionine and was not decreased in erythrocytes from patients with adenosine deaminase deficiency, purine nucleoside phosphorylase deficiency, or hypogammaglobulinemia. These observations suggest that the inhibitory properties of 5′-methylthioadenosine upon adenosine kinase and S-adenosylhomocysteine hydrolase may contribute to the toxicity of the exogenously added compound. The toxicity resulting from S-adenosylhomocysteine accumulation intracellularly may be related to adenosine kinase inhibition in addition to disruption of transmethylation reactions.     

Modulation by the ratio of cyclic AMP-dependent phosphorylation of the 50 kDa protein of rat liver phospholipid methyltransferase

The present results show that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates the 50 kDa protein of rat liver phospholipid methyltransferase at one single site on a serine residue. Phosphorylation of this site is stimulated 2- to 3-fold by S-adenosylmethionine. S-adenosylmethionine-dependent protein phosphorylation is time- and dose-dependent and occurs at physiological concentrations. S-adenosylhomocysteine has no effect on protein phosphorylation but inhibits S-adenosylmethionine-dependent protein phosphorylation. ratios varying from 0 to 5 produce a dose-dependent stimulation of the phosphorylation of the 50 kDa protein. In conclusion, these results show, for the first time, that the ratio can modulate phosphorylation of a specific protein.     

Selenite toxicity,depletion of liver S-adenosylmethionine,and inactivation of methionine adenosyltransferase

The possibility that dimethyl selenide production depletes liver S-adenosylmethionine was explored as a biochemical basis for selenite toxicity. Toxic doses of selenite (25 nmol/ g body weight) were found to rapidly decrease mouse liver S-adenosylmethionine and increase S-adenosylhomocysteine, indicative of an increased rate of transmethylation. However, S-adenosylmethionine levels remained depressed beyond the time when dimethyl selenide synthesis ceased, suggesting that selenite inactivated methionine adenosyltransferase. This was found to be the case in vivo by measuring the effect of graded doses of selenite on the conversion of the methionine analog, ethionine, to S-adenosylethionine. In vitro studies also indicated inactivation of this enzyme by selenite. Liver homogenates from mice injected with 25 nmol of selenite/g, as above, were found to have less than 50% of the methionine adenosyltransferase activity of saline-injected controls.     

Product inhibition studies and the reaction sequence of rabbit adrenal norepinephrine N-methyl transferase isozymes

The effects of reaction products on the steady-state kinetic properties of the five charge isozymes of rabbit adrenal norepinephrine N-methyl transferase have been investigated. Qualitative and quantitative differences were observed for the isozymes. The only characteristic that was common to all isozymes was the competition between S-adenosylmethionine and S-adenosylhomocysteine for the binding site. In most instances, the product inhibition constants were sufficiently low to suggest that product inhibition may be an important factor in regulating the activities of the isozymes. A reaction model is proposed for rabbit adrenal norepinephrine N-methyl transferase which is consistent with results observed in investigations of the steady-state kinetic properties of the five charge isozymes. The proposed model is that of an ordered sequential reaction sequence in which the active center contains a binding site for S-adenosylmethionine and S-adenosylhomocysteine, and a binding site for norepinephrine and epinephrine. The proposed model includes the formation of a number of abortive complexes between enzyme and substrate and product, but not all of the abortive complexes are significant kinetically in the case of some of the isozymes. The differences in the steady-state kinetic characteristics of the isozymes are attributed to differences in the magnitudes of the rate constants of some of the individual steps.     

1-Methylnicotinamide and NAD metabolism in normal and transformed normal rat kidney cells

NAD, 1-methylnicotinamide, S-adenosylmethionine, and S-adenosylhomocysteine levels were analyzed in different clones of untransformed normal rat kidney cells and in cells transformed by different viruses. No consistent changes in the levels of these metabolites were apparent as a result of malignant transformation, and also differences in the levels of metabolites did not correlate with growth rate in the various cell lines. 3-Deazaadenosine prevented synthesis of 1-methylnicotinamide but not of NAD. The S-denosylmethionine/S-adenosylhomocysteine ratio did not change in serum-starved, growth-arrested cells although 1-methylnicotinamide synthesis increased about twofold. These results were used to consider possible physiological roles for 1-methylnicotinamide. Its intracellular levels did not correlate with growth rate and were not altered by transformation. No evidence was obtained that its synthesis is involved with maintenance of nicotinamide of S-adenosylmethionine levels. Thus the biological function for 1-methylnicotinamide remains a mystery.     

S-Adenosylhomocysteine Hydrolase Deficiency in Cysteine Auxotrophs of Tetrahymena thermophila1

The biochemical lesion in two cysteine auxotrophs of Tetrahymena thermophila has been established as a defect in S-adenosylhomocysteine hydrolase, an enzyme of the transsulfuration pathway. As a result, these mutants require cysteine (or cystathionine or homocysteine) for growth in a denned medium. Cell-free extracts of the mutants contained < 5% of the level of the enzyme seen in the wild type. One of the mutant strains accumulated intracellular levels of S-adenosylhomocysteine as high as 1380 üM, a level 200 times normal. When both mutant strains were maintained in defined medium without cysteine, growth occurred after a long lag; this phenomenon was termed “adaptation.” Adaptation was a) reversed by passage through rich medium, b) was not a recovery of S-adenosylhomocysteine hydrolase, and c) was probably linked to induction of an alternate pathway for cysteine biosynthesis, involving a lysosomal S-adenosylhomocysteine nucleosidase activity.     

Putrescine N-methyltransferase – The start for alkaloids

Putrescine N-methyltransferase (PMT) catalyses S-adenosylmethionine (SAM) dependent methylation of the diamine putrescine. The product N-methylputrescine is the first specific metabolite on the route to nicotine, tropane, and nortropane alkaloids. PMT cDNA sequences were cloned from tobacco species and other Solanaceae, also from nortropane-forming Convolvulaceae and enzyme proteins were synthesised in Escherichia coli. PMT activity was measured by HPLC separation of polyamine derivatives and by an enzyme-coupled colorimetric assay using S-adenosylhomocysteine. PMT cDNA sequences resemble those of plant spermidine synthases (putrescine aminopropyltransferases) and display little similarity to other plant methyltransferases. PMT is likely to have evolved from the ubiquitous enzyme spermidine synthase. PMT and spermidine synthase proteins share the same overall protein structure; they bind the same substrate putrescine and similar co-substrates, SAM and decarboxylated S-adenosylmethionine. The active sites of both proteins, however, were shaped differentially in the course of evolution. Phylogenetic analysis of both enzyme groups from plants revealed a deep bifurcation and confirmed an early descent of PMT from spermidine synthase in the course of angiosperm development.     

S-adenosylmethionine synthetase in cultured normal and oncogenically-transformed human and rat cells

We have investigated the enzymatic formation of S-adenosylmethionine in extracts of a variety of normal and oncogenically-transformed human and rat cell lines which differ in their ability to grow in medium in which methionine is replaced by its immediate precursor homocysteine. We have localized the bulk of the S-adenosylmethionine synthetase activity to the post-mitochondrial supernatant. We show that in all cell lines a single kinetic species exists in a dialyzed extract with a K_m for methionine of about 3–12 μM. In selected lines we have demonstrated a requirement for Mg²⁺ in addition to that needed to form the Mg·ATP complex for enzyme activity and have shown that the enzyme can be regulated by product feedback inhibition. Because we detect no differences in the enzymatic ability of these cell extracts to utilize methionine for S-adenosylmethionine formation in vitro, we suggest that the failure of oncogenically-transformed cell lines to grow in homocysteine medium may result from the decreased methionine pools in these cells or from the loss of ability of these cells to properly metabolize homocysteine, adenosine, or their cellular product S-adenosylhomocysteine.     

An improved method for chemical alpha-oxidation

A nonisotopic assay for acetylserotonin methyltransferase (ASMT) was devised. Melatonin, the product of the enzyme reaction, is measured fluorometrically after its reaction with o-phthaldialdehyde (OPT). The reaction of melatonin with OPT is carried out in 1 n HCl to suppress the reaction of N-acetylserotonin, the substrate of ASMT, with OPT. The mixture is gassed with nitrogen just before incubation at 60°C for 60 min in order to secure the linear relationship between the concentration of melatonin and the fluorescence intensity. This method is much simpler than the isotopic assay and also has as much high sensitivity. Moreover, in this assay the enzyme can be well saturated with S-adenosylmethionine, whereas in the isotopic assay it cannot.     

A new structural class of S-adenosylhomocysteine hydrolase inhibitors

Effective inhibitors of S-adenosylhomocysteine hydrolase hold promise towards becoming useful therapeutic agents. Since most efforts have focused on the development of nucleoside analog inhibitors, issues regarding bioavailability and selectivity have been major challenges. Considering the marine sponge metabolite ilimaquinone was found to be a competitive inhibitor of S-adenosylhomocysteine hydrolase, new opportunities for developing selective new inhibitors of this enzyme have become available. Based on the activities of various hybrid analogs, SAR studies, pharmacophore modeling, and computer docking studies have lead to a predictive understanding of ilimaquinone’s S-adenosylhomocysteine hydrolase inhibitory activities. These studies have allowed for the design and preparation of simplified structural variants possessing new furanoside bioisosteres with 100-fold greater inhibitory activities than that of the natural product.     

The S-Methylmethionine Cycle in Lemna paucicostata

The metabolism of S-methylmethionine has been studied in cultures of plants of Lemna paucicostata and of cells of carrot (Daucus carota) and soybean (Glycine max). In each system, radiolabeled S-methylmethionine was rapidly formed from labeled l-methionine, consistent with the action of S-adenosyl-l-methionine:methionine S-methyltransferase, an enzyme which was demonstrated during these studies in Lemna homogenates. In Lemna plants and carrot cells radiolabel disappeared rapidly from S-methylmethionine during chase incubations in nonradioactive media. The results of pulse-chase experiments with Lemna strongly suggest that administered radiolabeled S-methylmethionine is metabolized initially to soluble methionine, then to the variety of compounds formed from soluble methionine. An enzyme catalyzing the transfer of a methyl group from S-methylmethionine to homocysteine to form methionine was demonstrated in homogenates of Lemna. The net result of these reactions, together with the hydrolysis of S-adenosylhomocysteine to homocysteine and adenosine, is to convert S-adenosylmethionine to methionine and adenosine. A physiological advantage is postulated for this sequence in that it provides the plant with a means of sustaining the pool of soluble methionine even when overshoot occurs in the conversion of soluble methionine to S-adenosylmethionine. The facts that the pool of soluble methionine is normally very small relative to the flux into S-adenosylmethionine and that the demand for the latter compound may change very markedly under different growth conditions make it plausible that such overshoot may occur unless the rate of synthesis of S-adenosylmethionine is regulated with exquisite precision. The metabolic cost of this apparent safeguard is the consumption of ATP. This S-methylmethionine cycle may well function in plants other than Lemna, but further substantiating evidence is neeeded.