The SAM510™: SAM Methyltransferase Assay is a continuous kinetic enzyme coupled assay that can continuously monitor purified SAM-dependent methyltransferases without the use of radioactive labels or endpoint measurements. This is a sensitive enzyme-coupled assay for protein SAM memthyltransferases.
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.
Figure 1 outlines the general scheme of the assay. Basically, the removal of the methyl group from SAM generates S-adenosylhomocysteine, which is rapidly converted to S-ribosylhomocysteine and adenine by the included adenosylhomocysteine nucleosidase. This rapid conversion prevents the buildup of adenosylhomocysteine 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. The rate of production of hydrogen peroxide is measured with a colorimetric assay by an increase in absorbance at 510nm.
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 adenosylhomocysteine nucleosidase.
The kit is supplied with enough reagents for 100 x 115µl microwell assays.
A fluorescent SAM methyltransferase assay are also available.
- Kinetic analysis of purified methlytransferases or screening methylation inhibitors.
- Continuous enzyme coupled assay for kinetic studies.
- Colorimetric, non radioactive assay.
- Supplied with all reagents, including positive control.
- Adaptable for enzymes that generate S-adenosylhomocysteine or 5'-methylthioadenosine.
- For the kinetic analysis of protein SAM methyltransferase enzymes.
- Ideal for screening of methyltransferase inhibitors.