S 5 adenosyl l methionine

Manufactured by Merck Group

Sourced in Czechia, Canada, United States

S-(5′-adenosyl)-l-methionine is a chemical compound commonly used in laboratory research. It functions as a methyl group donor, facilitating the transfer of methyl groups in various biological processes.

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7 protocols using s 5 adenosyl l methionine

Methyltransferase Assay Protocol

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pUC19 DNA was acquired from New England BioLabs
(NEB) and propagated in the Escherichia coli XL-1
Blue strain under ampicillin selection. Plasmids were purified using
a miniprep plasmid extraction kit (Qiagen). DNA CpG methyltransferase
(M.SssI) and the restriction enzymes used for DNA mapping were also
purchased from NEB. Methionine analogues were either purchased or
synthesized as described previously^{11 (link)} and
were used as freshly prepared stock solutions adjusted to neutral
pH. Catechol O-methyltransferase (COMTase) was purchased
from Calzyme Laboratories Inc., while (−)-epinephrine (+)-bitartrate, S-(5′-adenosyl)-l-methionine (AdoMet), and S-(5′-adenosyl)-l-homocysteine (AdoHcy)
were obtained from Sigma-Aldrich. All of the other reagents were of
the highest purity available.

Wijayasinghe Y.S., Blumenthal R.M, & Viola R.E. (2014). Producing Proficient Methyl Donors from Alternative Substrates of S-Adenosylmethionine Synthetase. Biochemistry, 53(9), 1521-1526.

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Transfection and Drug Treatment in HeLa Cells

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HeLa cells grown to 40% confluence on 150-mm² culture flasks at 37°C with 8% (v/v) CO₂ were transfected with different siRNAs using RNAiMAX (Invitrogen) or shRNAs using Lipofectamine 3000 (Invitrogen). siSET7/9-1 (5′-GGGCACCUGGACGAUGACGGA-3′), siSET7/9-2 (5′-GCCUUGUAGGAGAAGUAAA-3′), siMad2 (5′-UCCGUUCAGUGAUCAGACA-3′), siBubR1 (5′-GUCUCACAGAUUGCUGCCU-3′), and siControl (5′-UAAAUGUACUGCGCGUGGAGAGGAA-3′) were synthesized from Qiagen. PLK1 shRNA targeting the 3′-UTR (5′-CCGGAGCTGCATCATCCTTGCAGGTCTCGAGACCTGCAAGGATGATGCAGCTTTTTT-3′) of PLK1 gene was purchased from Sigma-Aldrich (TRCN0000011006).
Nocodazole (100 ng/ml, ≥99%), monastrol (50 μM, ≥98%), MG132 (10 μM, ≥90%), PFI-2 (10 μM, ≥97%), cyproheptadine (2 μM, ≥99%), and S-(5′-adenosyl)-L-methionine (SAM, 1 mM, ≥80%) were from Sigma.

Yu R., Wu H., Ismail H., Du S., Cao J., Wang J., Ward T., Yang F., Gui P., Ali M., Chu L., Mo F., Wang Q., Chu Y., Zang J., Zhao Y., Ye M., Fang G., Chen P.R., Dou Z., Gao X., Wang W., Liu X, & Yao X. (2019). Methylation of PLK1 by SET7/9 ensures accurate kinetochore–microtubule dynamics. Journal of Molecular Cell Biology, 12(6), 462-476.

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Molecular Biology Reagents Preparation

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Eagle’s minimum essential medium (EMEM), MEM non-essential amino acid solution, l-glutamine solution, l-(−)-norepinephrine, S-(5′-Adenosyl)-l-methionine (SAM), l-glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB), 3′-phosphoadenosine 5′-phosphate (PAP), 2-naphthol, menadione, and Polyphenon 60 (GTE), the defined form of GTE, were obtained from Sigma-Aldrich (Prague, Czech Republic). Fetal bovine serum and streptomycin sulfate were purchased from Invitrogen (Carlsbad, CA, USA). (−)-EGCG was supplied by Extrasynthese (Lyon, France). We bought ProtoScript^® II Reverse Transcriptase from NEB (Ipswich, UK), TriReagent from Biotech (Prague, Czech Republic), and the qPCR Core kit for SYBR Green I from Eurogentec (Seraing, Belgium). All other chemicals, HPLC or analytical grade, were obtained from Sigma-Aldrich.

Lněničková K., Procházková E., Skálová L., Matoušková P., Bártíková H., Souček P, & Szotáková B. (2016). Catechins Variously Affect Activities of Conjugation Enzymes in Proliferating and Differentiated Caco-2 Cells. Molecules, 21(9), 1186.

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High-throughput Evaluation of Metabolic Mutant Growth and Gene Expression

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For Figures 1B, 6A and 6F, the working stock of each mutant, T2 cells, were thawed and diluted 1:100 into fresh C+Y medium, or C+Y medium with doxycycline at different final concentrations, or with different concentrations of S-(5’-Adenosyl)-L-methionine (A7007, Sigma Aldrich), as the initial cell culture. For Figure 6D, the T2 cells were thawed and diluted 1:100 into fresh Blood Like Medium (BLM) without nucleobases solution, or supplemented with individual nucleobases component (adenine, adenosine, guanine, uracil, uridine and xanthine), or with all the components (Aprianto et al., 2018 (link)). Then 300 μl of the initial culture was aliquoted into each well of 96-well plates with 3 replicates. Cell density were monitored by measuring OD595 every 10 minutes with a Tecan Spark microtiter plate reader at 37°C. Luciferase assay (Figure 1B) was performed as previously described (Liu et al., 2017 (link)). Luciferin (D-Luciferin sodium salt, SYNCHEM OHG) was added into C+Y medium at final concentration of 450 μg/ml as substrate of the luciferase. Luminescence signals were measured every 10 minutes with a Tecan Spark microtiter plate. Growth and luciferase activity curves were plotted with Prism 8 as described previously (Sorg and Veening, 2015 (link)).

Liu X., Kimmey J.M., Matarazzo L., de Bakker V., Maele L.V., Sirard J.C., Nizet V, & Veening J.W. (2020). Exploration of bacterial bottlenecks and Streptococcus pneumoniae pathogenesis by CRISPRi-seq. Cell host & microbe, 29(1), 107-120.e6.

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Enzymatic catechin metabolism analysis

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Green tea catechins [(+)catechin (C), (−)epicatechin (EC), (−)catechin gallate (CG), (−)epicatechin gallate (ECG), (−)gallocatechin (GC), (−)epigallocatechin (EGC), (−)epigallocatechin gallate (EGCG), (−)gallocatechin gallate (GCG)], synthetic gallate esters [ethyl gallate (EG), propyl gallate (PG), butyl gallate (BG), octyl gallate (OG) and lauryl gallate (LG)], gallic acid (GA), reduced L-glutathione (GSH), S-(5′-adenosyl)-L-methionine (SAM), nicotinamide adenine dinucleotide phosphate (NADP⁺), glucose-6-phosphate, magnesium chloride and glucose-6-phosphate dehydrogenase, potassium (mono- and di-basic) phosphate, in addition to HPLC-grade acetonitrile (ACN), methanol, and acetic acid were purchased from Sigma-Aldrich (Oakville, ON, Canada). Human liver microsomes (HLM, pooled from 50 donors) were purchased from Corning (Corning, NY, USA). Ultrapure water was obtained from a Millipore Synergy UV system (Billerica, MA, USA).

Ousji O, & Sleno L. (2022). Structural Elucidation of Novel Stable and Reactive Metabolites of Green Tea Catechins and Alkyl Gallates by LC-MS/MS. Antioxidants, 11(9), 1635.

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Recombinant Protein Expression in Pichia pastoris

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The easy select expression kit for expression of recombinant proteins using pPICZα in P. pastoris and zeocin were obtained from Invitrogen (Carlsbad, CA, USA). Bis-(1,3-dibutylbarbituric acid) trimethine oxonol was acquired from Molecular Probes^® (Part of Life technologies; Carlsbad, CA, USA). Yeast nitrogen base (YNB), dithiothreitol, S-(5′-adenosyl)-l-methionine, epinephrine (bitartrate salt), deoxyribonuclease (DNase), protease inhibitor cocktail, dl-metanephrine hydrochloride, glass beads (500 µm) and propidium iodide were purchased from Sigma-Aldrich (St. Louis, MO, USA). All chemicals used were of analytical grade, commercially available, and used without further purification.
E. coli TOP10F’ was used for DNA manipulations. E. coli transformants were selected on low-salt Luria–Bertani plates with 25 µg/mL Zeocin. P. pastoris X-33 and KM71H was used for fusion gene expression. YPD and YPDS media [40 ] were used for routine manipulation of Pichia cells. P. pastoris transformants were selected on YPDS plates with 200 µg/mL Zeocin. Small-scale fermentations were carried out in BMGH and BMMH media [40 ]. P. pastoris bioreactor cultures were carried out in modified basal salts medium (BSM) [27 (link)] with 200 µg/mL zeocin and supplemented with trace metal solution (SMT) [27 (link)].

Pedro A.Q., Martins L.M., Dias J.M., Bonifácio M.J., Queiroz J.A, & Passarinha L.A. (2015). An artificial neural network for membrane-bound catechol-O-methyltransferase biosynthesis with Pichia pastoris methanol-induced cultures. Microbial Cell Factories, 14, 113.

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Phospholipid and Metabolite Standards for Lipidomics

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Phosphatidylcholine (PC) and
phosphatidylethanolamine (PE) standards (Supporting Information Document 1, Table S1) and SPLASH Lipidomix
were acquired from Avanti Polar Lipids. Acetaminophen, caffeine, carnosine, S-(5′adenosyl)-l-methionine (SAM), and poly-dl-alanine were purchased from Sigma-Aldrich. Adenosine 5′-monophosphate
(AMP) was purchased from Cayman Chemical. l-Histidine, sucrose,
ammonium formate, formic acid (FA), HPLC grade acetonitrile (ACN),
methanol (MeOH), 2-propanol (BuOH), and water were purchased from
ThermoFisher Scientific. MassPREP enolase digest and alcohol dehydrogenase
digest were purchased from Waters. NIST SRM 1950 was acquired from
the National Institute of Standards and Technology (NIST).

Hynds H.M, & Hines K.M. (2024). MOCCal: A Multiomic CCS Calibrator for Traveling Wave Ion Mobility Mass Spectrometry. Analytical Chemistry, 96(3), 1185-1194.

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