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 (1). 

 

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 (2).

 

The SAM265:SAM Methyltransferase Assay is a sensitive, UV-based, continuous enzyme coupled assay that can continuously monitor SAM-dependent methyltransferases (3) without the use of radioactive labels or endpoint measurements.

 

Figure 1 outlines the general scheme of the assay. Basically, the removal of the methyl group from SAM, by the SAM methyltransferase, 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, the second enzyme in the assay. The rate of production of hypoxanthine is measured by absorbance change.

 

The assay can be adapted to be used with any SAM dependent methyltransferase or an enzyme reaction that produces 5-adenosylhomocysteine or 5'-methylthioadenosine, due to the specificity of adenosylhomocysteine nucleosidase.

The kit is supplied with a positive control to determine the ideal assay conditions and with enough reagents for 100 microwell assays.