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1.
Peter H. Yu 《Analytical biochemistry》1978,84(2):615-621
A simple, sensitive, and specific method for analysis of S-adenosyl-l-homocysteine (SAH) in tissue is described. The assay is based on the measurement of the inhibitory activity of SAH on catechol-O-methyltransferase (COMT). As little as 1 mg of fresh liver tissue may be used. The precedure involves extraction of tissues, thin-layer chromatographic separation, followed by measurement of COMT. The concentration of SAH in rat liver assessed by this method is 71.2 ± 3.8 nmol/g of wet tissue and is principally concentrated in the particulate fractions. 相似文献
2.
Lindsey J. Brown Matthias Baranowski Yun Wang Anna K. Schrey Thomas Lenz Sean D. Taverna Philip A. Cole Michael Sefkow 《Analytical biochemistry》2014
S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH–CC with biotin used in conjunction with streptavidin–horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the Kd values for COMT, SAHH, and PRDM2 (24.1 ± 2.2 μM, 6.0 ± 2.9 μM, and 10.06 ± 2.87 μM, respectively) and found them to be close to previously established Kd values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs. 相似文献
3.
A rapid, efficient method is described for the enzymatic conversion of S-adenosyl-l-[2(n)-3H]methionine to S-adenosyl-l-[2(n)-3H]homocysteine. Partially purified glycine N-methyltransferase is used in the reaction which yields 98% conversion. The product is purified using high-pressure liquid chromatography and is concentrated by lyophilization. S-Adenosyl-l-[2(n)-3H]homocysteine synthesized by this method is an active substrate for S-adenosylhomocysteine (SAH) hydrolase. A novel assay procedure for SAH hydrolase is also described, in which unreacted S-adenosyl-l-[2(n)-3H]homocysteine is removed by adsorption to dextran-coated charcoal. 相似文献
4.
5.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2001,762(1):59-65
An improved reversed-phase high-performance liquid chromatography (HPLC) procedure with ultraviolet detection is described for the simultaneous determination of S-adenosyl-l-methionine (SAM) and S-adenosyl-l-homocysteine (SAH) in mouse tissue. The method provides rapid resolution of both compounds in a 25-μl perchloric acid extract of the tissue. The limits of detection in 25-μl injection volumes were 22 and 20 pmol for SAM and SAH, respectively. The limits of quantitation in 25-μl injection volumes were 55 and 50 pmol for SAM and SAH, respectively, with recovery consistently >98%. The assay was validated over linear ranges of 55–11 000 pmol for SAM and 50–10 000 pmol for SAH. The intra-day precision and accuracy were ≤6.4% relative standard deviation (RSD) and 99.9–100.0% for SAH and ≤6.7% RSD and 100.0–100.1% for SAM. The inter-day precision and accuracy were ≤5.9% RSD and 99.9–100.6% for SAH and ≤7.0% RSD and 99.5–100.1% for SAM. Compared to earlier procedures, the HPLC method demonstrated significantly better separation, detection limit and linear range for SAM and SAH determination. The assay demonstrated applicability to monitoring in mice the time-course of the effect of methionine on SAM and SAH levels in the liver. Administering methionine to mice increased by 10-fold the liver concentration of SAM and SAH within 2 h, which then rapidly decreased to the control levels by 8 h. This indicated that methionine was promptly converted to SAM and then rapidly catabolized into SAH. Thus, the metabolism of methionine to SAM should be considered in the supplementation of methionine to maintain SAM levels in the body. 相似文献
6.
Giovanni Paolella Gennaro Ciliberto Cinzia Traboni Filiberto Cimino Francesco Salvatore 《Archives of biochemistry and biophysics》1982,219(1):149-154
S-Adenosylhomocysteine (SAH), a potent inhibitor of methyltransferases, and several thioethers structurally related to SAH, have been tested as potential inhibitors of tRNA (guanine-7)-methyltransferase from Salmonella typhimurium. The tested compounds are l-, d-, dl-S-adenosylhomocysteine, S-adenosylcysteine, methylthioadenosine, butylthioadenosine, thioethanoladenosine, isobutylthioadenosine, S-inosylhomocysteine, and methylthioinosine. Among them the highest inhibitory activity has been shown by SAH (Ki = 8 μM), whereas butylthioadenosine, isobutylthioadenosine, and thioethanoladenosine are almost inactive as inhibitors. The other compounds inhibit the enzyme with Ki values ranging between 400 and 800 μm. From these data it is possible to evaluate the importance of the -NH2 and -COOH groups of the substrate in the binding to the enzyme molecule, as well as other features such as the chirality at the α-carbon atom and the length of the hydrocarbon chain connecting the -NH2 and -COOH groups to the aromatic ring of adenosine. The aminic group of the adenosine is also critical, because S-inosylhornocysteine and methylthioinosine are poorer inhibitors in comparison with SAH and methylthioadenosine. 相似文献
7.
The second committed step in chlorophyll biosynthesis is the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to magnesium protoporphyrin IX (MgP) forming MgP monomethylester (MgPME). This reaction is catalyzed by the enzyme MgP methyltransferase (ChlM). Previous investigation of this enzyme has involved the use of time-consuming techniques requiring separation of products from substrates. More recent methyltransferase studies use coupling enzymes to monitor changes in absorption/fluorescence for the measurement of activity. However, due to the spectral properties of porphyrins, many of these assays are unsuitable for analysis of the catalytic properties of ChlM. Here we report the successful development of a coupled, continuous spectrophotometric assay to measure the activity of ChlM. The product of the methyltransferase reaction, S-adenosyl-l-homocysteine (SAH), is converted into adenine and then hypoxanthine by the recombinant coupling enzymes SAH nucleosidase and adenine deaminase, respectively. The appearance of hypoxanthine results in a decrease in absorbance at 265 nm.The utility of this assay was shown by the characterization of ChlM from the cyanobacterium Synechocystis sp. PCC 6803. Kinetic parameters obtained support data acquired using the discontinuous HPLC-based assay and provide further evidence for the stimulation of ChlM by the H subunit of magnesium chelatase (ChlH). 相似文献
8.
Although S-adenosyl-l-methionine (SAM) dependent caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) is one of the key enzymes in lignin biosynthesis, the present work demonstrates that alfalfa COMT methylates benzaldehyde derivatives more efficiently than lignin pathway intermediates. 3,4-Dihydroxy, 5-methoxybenzaldehyde and protocatechuic aldehyde were the best in vitro substrates for OMT activity in extracts from developing alfalfa stems, and these compounds were preferred over lignin pathway intermediates for 3-O-methylation by recombinant alfalfa COMT expressed in Escherichia coli. OMT activity with benzaldehydes was strongly reduced in extracts from stems of transgenic alfalfa down-regulated in COMT. However, although COMT down-regulation drastically affects lignin composition, it does not appear to significantly impact metabolism of benzaldehyde derivatives in alfalfa. Structurally designed site-directed mutants of COMT showed altered relative substrate preferences for lignin precursors and benzaldehyde derivatives. Taken together, these results indicate that COMT may have more than one role in phenylpropanoid metabolism (but probably not in alfalfa), and that engineered COMT enzymes could be useful for metabolic engineering of both lignin and benzaldehyde-derived flavors and fragrances. 相似文献
9.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1988,971(1):38-45
The effect of the cell cycle and differentiation on S-adenosylmethionine (SAM) metabolism in HL-60 cells has been investigated. Synthesis and pool sizes of SAM and S-adenosylhomocysteine (SAH) were cell-cycle-independent (SAM, 315, μM; SAH, 4.6 μM). The SAM-synthase (ATP: l-methionine S-adenosyltransferase) of HL-60 cells has a Km for methionine of 12.8±2.0 μM and thus appears to be of the intermediate Km type found in other malignant tissues. The enzyme does not show cell-cycle regulation. Treatment of cells with DMSO resulted in a rapid and marked decrease of SAM and SAH levels without affecting pool turnover or the SAM/SAH ratio. A decrease in SAM concentration could also be observed in a variant cell line resistant to differentiation with DMSO. DMSO inhibited SAM-synthase in cell-free extracts. This inhibition was noncompetitive with respect to l-methionine. Inhibition of SAM-synthase by cycloleucine lowered SAM levels in intact cells, but resulted in differentiation of only a minor percentage of cells. These data indicate that changes in SAM and SAH levels in HL-60 cells seem to be a consequence rather than a cause of differentiation. 相似文献
10.
《Bioorganic & medicinal chemistry letters》2014,24(12):2737-2740
Elevated plasma homocysteine (Hcy) levels are an independent risk factor for the onset and progression of Alzheimer’s disease. Reduction of Hcy to normal levels therefore presents a new approach for disease modification. Hcy is produced by the cytosolic enzyme S-adenosylhomocysteine hydrolase (AHCY), which converts S-adenosylhomocysteine (SAH) to Hcy and adenosine. Herein we describe the design and characterization of novel, substrate-based S-adenosylhomocysteine hydrolase inhibitors with low nanomolar potency in vitro and robust activity in vivo. 相似文献
11.
Andrea L. Edwards Francis E. Reyes Annie Héroux Robert T. Batey 《RNA (New York, N.Y.)》2010,16(11):2144-2155
S-adenosyl-(L)-homocysteine (SAH) riboswitches are regulatory elements found in bacterial mRNAs that up-regulate genes involved in the S-adenosyl-(L)-methionine (SAM) regeneration cycle. To understand the structural basis of SAH-dependent regulation by RNA, we have solved the structure of its metabolite-binding domain in complex with SAH. This structure reveals an unusual pseudoknot topology that creates a shallow groove on the surface of the RNA that binds SAH primarily through interactions with the adenine ring and methionine main chain atoms and discriminates against SAM through a steric mechanism. Chemical probing and calorimetric analysis indicate that the unliganded RNA can access bound-like conformations that are significantly stabilized by SAH to direct folding of the downstream regulatory switch. Strikingly, we find that metabolites bearing an adenine ring, including ATP, bind this aptamer with sufficiently high affinity such that normal intracellular concentrations of these compounds may influence regulation of the riboswitch. 相似文献
12.
Rosa Milagros Corrales Jade Leiba Martin Cohen-Gonsaud Virginie Molle Laurent Kremer 《Biochemical and biophysical research communications》2013,430(2):858-864
S-Adenosylhomocysteine hydrolase (SahH) is known as an ubiquitous player in methylation-based process that maintains the intracellular S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) equilibrium. Given its crucial role in central metabolism in both eukaryotes and prokaryotes, it is assumed that SahH must be regulated, albeit little is known regarding molecular mechanisms governing its activity. We report here that SahH from Mycobacterium tuberculosis can be phosphorylated by mycobacterial Ser/Thr protein kinases and that phosphorylation negatively affects its enzymatic activity. Mass spectrometric analyses and site-directed mutagenesis identified Thr2 and Thr221 as the two phosphoacceptors. SahH_T2D, SahH_T221D and SahH_T2D/T221D, designed to mimic constitutive phosphorylation, exhibited markedly decreased activity compared to the wild-type enzyme. Both residues are fully conserved in other mycobacterial SahH orthologues, suggesting that SahH phosphorylation on Thr2 and Thr221 may represent a novel and presumably more general mechanism of regulation of the SAH/SAM balance in mycobacteria. 相似文献
13.
Karin Heurlier Agns Vendeville Nigel Halliday Andrew Green Klaus Winzer Christoph M. Tang Kim R. Hardie 《Journal of bacteriology》2009,191(4):1293-1302
The activated methyl cycle (AMC) is a central metabolic pathway used to generate (and recycle) several important metabolites and enable methylation. Pfs and LuxS are considered integral components of this pathway because they convert S-adenosylhomocysteine (SAH) to S-ribosylhomocysteine (SRH) and S-ribosylhomocysteine to homocysteine (HCY), respectively. The latter reaction has a second function since it also generates the precursor of the quorum-sensing molecule autoinducer 2 (AI-2). By demonstrating that there was a complete lack of AI-2 production in pfs mutants of the causative agent of meningitis and septicemia, Neisseria meningitidis, we showed that the Pfs reaction is the sole intracellular source of the AI-2 signal. Analysis of lacZ reporters and real-time PCR experiments indicated that pfs is expressed constitutively from a promoter immediately upstream, and careful study of the pfs mutants revealed a growth defect that could not be attributed to a lack of AI-2. Metabolite profiling of the wild type and of a pfs mutant under various growth conditions revealed changes in the concentrations of several AMC metabolites, particularly SRH and SAH and under some conditions also HCY. Similar studies established that an N. meningitidis luxS mutant also has metabolite pool changes and growth defects in line with the function of LuxS downstream of Pfs in the AMC. Thus, the observed growth defect of N. meningitidis pfs and luxS mutants is not due to quorum sensing but is probably due to metabolic imbalance and, in the case of pfs inactivation, is most likely due to toxic accumulation of SAH. 相似文献
14.
Peter H. Yu 《Analytical biochemistry》1978,86(2):498-504
A rapid, sensitive, and specific method for the analysis of S-adenosyl-l-methionine (SAMe) in tissues is described. The assay is based on the measurement of the conversion of SAMe and [3H]dopamine to 3-[3H]methoxytyramine in the presence of catechol-O-methyltransferase (COMT). The linearity of the quantitative measurement is greatly improved by including an internal standard of [14C]SAMe in each test. Using the described technique, nanogram quantities of SAMe can be measured in tissue. SAMe is distributed relatively evenly throughout the anatomical regions of the brain. Studies of its subcellular distribution indicate that while SAMe is located primarily in the soluble fraction, a considerable amount is also associated with the synaptosomal fraction. 相似文献
15.
S-Adenosyl-l-methionine (SAM) is the biological methyl-group donor for the enzymatic methylation of numerous substrates including proteins. SAM has been reported to activate smooth muscle derived ryanodine receptor calcium release channels. Therefore, we examined the effects of SAM on the cardiac isoform of the ryanodine receptor (RyR2). SAM increased cardiac sarcoplasmic reticulum [3H]ryanodine binding in a concentration-dependent manner by increasing the affinity of RyR2 for ryanodine. Activation occurred at physiologically relevant concentrations. SAM, which contains an adenosine moiety, enhanced ryanodine binding in the absence but not in the presence of an ATP analogue. S-Adenosyl-l-homocysteine (SAH) is the product of the loss of the methyl-group from SAM and inhibits methylation reactions. SAH did not activate RyR2 but did inhibit SAM-induced RyR2 activation. SAH did not alter adenine nucleotide activation of RyR2. These data suggest SAM activates RyR2 via a site that interacts with, but is distinct from, the adenine nucleotide binding site. 相似文献
16.
Eiichi Tsuji Kosuke Okazaki Masayuki Isaji Kei Takeda 《Journal of structural biology》2009,165(3):133-139
Catechol-O-methyltransferase (COMT, EC 2.1.1.6) is a monomeric enzyme that catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the phenolic oxygen of substituted catechols. Although the inhibitor recognition pattern and AdoMet site have already been studied crystallographically, structural information on the catalytic cycle of COMT has not yet been obtained. In this study, comparison of the co-factor and inhibitor-bound structures revealed that the Apo form of COMT shows a conformational change and there was no cleft corresponding to the AdoMet-binding site; the overall structure was partially open form and the substrate recognition site was not clearly defined. The Holo form of COMT was similar to the quaternary structure except for the β6–β7 and α2–α3 ligand recognition loops. These conformational changes provide a deeper insight into the structural events occurring in reactions catalyzed by AdoMet. 相似文献
17.
Ilka Haferkamp Thomas Penz Melanie Geier Michelle Ast Tanja Mushak Matthias Horn Stephan Schmitz-Esser 《Journal of bacteriology》2013,195(14):3183-3192
All organisms require S-adenosylmethionine (SAM) as a methyl group donor and cofactor for various biologically important processes. However, certain obligate intracellular parasitic bacteria and also the amoeba symbiont Amoebophilus asiaticus have lost the capacity to synthesize this cofactor and hence rely on its uptake from host cells. Genome analyses revealed that A. asiaticus encodes a putative SAM transporter. The corresponding protein was functionally characterized in Escherichia coli: import studies demonstrated that it is specific for SAM and S-adenosylhomocysteine (SAH), the end product of methylation. SAM transport activity was shown to be highly dependent on the presence of a membrane potential, and by targeted analyses, we obtained direct evidence for a proton-driven SAM/SAH antiport mechanism. Sequence analyses suggest that SAM carriers from Rickettsiales might operate in a similar way, in contrast to chlamydial SAM transporters. SAM/SAH antiport is of high physiological importance, as it allows for compensation for the missing methylation cycle. The identification of a SAM transporter in A. asiaticus belonging to the Bacteroidetes phylum demonstrates that SAM transport is more widely spread than previously assumed and occurs in bacteria belonging to three different phyla (Proteobacteria, Chlamydiae, and Bacteroidetes). 相似文献
18.
Eun Young Park Seung-Ick Oh Jeong Sheop Shin 《Biochemical and biophysical research communications》2009,381(4):619-624
5′-Methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) are important metabolites in all living organisms. Two similar nucleosidases for hydrolyzing MTA in Arabidopsis thaliana (AtMTAN1 and AtMTAN2) exist, but only AtMTAN2 shows markedly broad substrate specificity for hydrolysis of SAH. To examine the biochemical characteristics of AtMTAN2, it was over-expressed in Escherichia coli and purified to homogeneity. Spectroscopic assays confirm AtMTAN2 catalyzes MTA as well as SAH hydrolysis, compared to AtMTAN1 which only hydrolyzes MTA. In addition, crystal structure of the AtMTAN2 enzyme in complex with, adenine was determined at 2.9 Å resolution. Finally, a structural comparison of AtMTAN2 performed with previously determined structures of AtMTAN1 and an E. coli homolog provides clues for the substrate specificity of MTA nucleosidases in A. thaliana. 相似文献
19.
The SAM-I riboswitch is a cis-acting element of genetic control found in bacterial mRNAs that specifically binds S-adenosylmethionine (SAM). We previously determined the 2.9-Å X-ray crystal structure of the effector-binding domain of this RNA element, revealing details of RNA-ligand recognition. To improve this structure, variations were made to the RNA sequence to alter lattice contacts, resulting in a 0.5-Å improvement in crystallographic resolution and allowing for a more accurate refinement of the crystallographic model. The basis for SAM specificity was addressed by a structural analysis of the RNA complexed to S-adenosylhomocysteine (SAH) and sinefungin and by measuring the affinity of SAM and SAH for a series of mutants using isothermal titration calorimetry. These data illustrate the importance of two universally conserved base pairs in the RNA that form electrostatic interactions with the positively charged sulfonium group of SAM, thereby providing a basis for discrimination between SAM and SAH. 相似文献
20.
Epigenetic regulation through protein posttranslational modifications is essential in development and disease. Among the key chemical modifications is protein methylation carried out by protein methyltransferases (PMTs). Quantitative and sensitive PMT activity assays can provide valuable tools to investigate PMT functions. Here we developed an enzyme-coupled luminescence assay for S-adenosyl-l-methionine (AdoMet/SAM)-based PMTs. In this assay, S-adenosyl-l-homocystine (AdoHcy/SAH), the by-product of PMT-involved methylation, is sequentially converted to adenine, adenosine monophosphate, and then adenosine 5′-triphosphate (ATP) by 5′-methylthio-adenosine/AdoHcy nucleosidase (MTAN), adenine phosphoribosyl transferase (APRT), and pyruvate orthophosphate dikinase (PPDK), respectively. The resultant ATP can be readily quantified with a luciferin/luciferase kit. This assay is featured for its quantitative linear response to AdoHcy and the ultrasensitivity to 0.3 pmol of AdoHcy. With this assay, the kinetic parameters of SET7/9 methylation were characterized and unambiguously support an ordered mechanism with AdoMet binding as the initial step, followed by the substrate binding and the rate-limiting methylation. The luminescence assay is also expected to be generally applicable to many other AdoMet-dependent enzymes. In addition, the mix-and-measure 96-/384-well format of our assay makes it suitable for automation and high throughput. Our enzyme-coupled luminescence assay, therefore, represents a convenient and ultrasensitive approach to examine methyltransferase activities and identify methyltransferase inhibitors. 相似文献