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Methylation of key biological molecules and proteins plays important roles in numerous biological systems, including signal transduction, biosynthesis, protein repair, gene silencing and chromatin regulation.
The S-adenosylmethionine (SAM) dependent methyltransferases use SAM, the second most commonly used enzymatic cofactor after ATP. SAM, also known as AdoMet, acts as a donor of a methyl group that is required for the modification of proteins and DNA. Aberrant levels of SAM have been linked to many abnormalities, including Alzheimer’s, depression, Parkinson’s, multiple sclerosis, liver failure and cancer.
The fluorescent SAMfluoro: SAM Methyltransferase Assay is a continuous enzyme coupled assay that can continuously monitor SAM-dependent methyltransferases without the use of radioactive labels or endpoint measurements.
The removal of the methyl group from SAM generates S-adenosylhomocysteine (AdoHcy), is rapidly converted to S-ribosylhomocysteine and adenine by the included AdoHcy nucleosidase. This rapid conversion prevents the buildup of AdoHcy and its feedback inhibition on the methylation reaction. Finally, the adenine is converted to hypoxanthine, by adenine deaminase, which in turn is converted to urate and hydrogen peroxide (H2O2). The rate of production of hydrogen peroxide is measured with 10-acetyl-3,7,-dihydroxyphenoxazine (ADHP), which produces the highly fluorescent compound resorufin. Resorufin production can easily be measured with an excitation wavelength of 530-540nm and an emission wavelength of 585-595nm.
The resorufin standard curve is generated from the supplied standards. The assay of human lysine specific histone methyltransferase Set7/9 is assayed with 20μM TAF-10 as the acceptor substrate.
The kit is supplied with enough reagents for 100 microwell assays. The assay is supplied with AdoHcy as a positive control. The assay can be adapted to be used with any purified SAM dependent methyltransferase or a purified enzyme that produces 5-adenosylhomocysteine or 5’-methylthioadenosine, due to the specificity of AdoHcy nucleosidase.
Features
- Kinetic analysis of purified methlytransferases or screening methylation inhibitors.
- Continuous enzyme coupled assay for kinetic studies.
- Fluorescent, non radioactive assay.
- Supplied with all reagents, including positive control.
- Adaptable for enzymes that generate S-adenosylhomocysteine or 5'-methylthioadenosine.
Applications
- For the kinetic analysis of protein SAM methyltransferase enzymes.
- Ideal for screening of methyltransferase inhibitors.
