Improved PRMT Substrate Detection
In "Analysis of Protein Arginine Methylation and Protein Arginine Methyltransferase Activity" by Mowen and David, the authors outline procedures for the purification and use of recombinant arginine methyltransferases from bacteria. The methyltransferase activity assay supplied here relies on the PRMT-dependent incorporation of tritiated S-adenosyl-L-methionine (3H-SAM) into a protein or peptide substrate. The labeled substrate is then visualized by fluorography following a sizing reaction on SDS-PAGE. According to the protocol the recombinant PRMT (1 microgram) is incubated in 80 microliters of phosphate-buffered saline (1x PBS) supplemented with 2 microliters of 3H-SAM. Under these conditions, the reaction is carried out at a pH of approximately 7.0.
The basic outline of this protocol is well-known in the literature where it has been used to assess PRMT methylation in a variety of contexts. Thus, when our laboratory began studying methyltransferase modifications in rabbit recticulocyte lysates (RRL) in vitro translation reactions we modeled our assay after this one by simply supplementing RRL (50 microliters) with 1 to 2 microliters of 3H-SAM. We discovered that RRL had methyltransferase activity, but it appeared rather low. Indeed, to obtain quantifiable results using 1/3 of our reaction volume, the fluorgrams had to be exposed for 3 to 6 weeks. However, these results were at odds with Western blotting data showing robust levels of PRMT1 in RRL compared to equivalent protein loads from a variety of cultured cells.
This discrepancy was resolved when we noticed in a review article by Cheng et al. that the optimal pH for PRMT1 was in the basic range (1). As RRL in vitro translation reactions normally occur at pH 7 to 7.2, and as the commercially available 3H-SAM is supplied in dilute acid, we reasoned that increasing the pH of the reaction would increase PRMT-dependent incorporation of 3H-SAM into its substrates. In fact, we directly demonstrated this by carrying out methyltransferase reactions (20 microliters) with RRL-produced substrate (5 microliters RRL-generated substrate ca. 10 ng) supplemented with 1 microliter of 3H-SAM in both 1x PBS and in a higher pH buffer (25 mM Tris-HCl pH 9, 1 mM DTT and 1 mM PMSF). In the former reaction the pH is 7; in the latter reaction the pH is 8.5. The results showed that there was a 15-fold increase in 3H-SAM incorporation into protein at the higher pH. Direct methylation of myelin basic protein (MBP) by CARM1 is similarly affected by the use of high pH. The new conditions allow us to routinely assay 1 microliter of RRL and detect 3H-SAM incorporation into protein in 10 min at 30C, making it possible to perform repetitive simple kinetic measurements (2). We speculate that such conditions could be similarly applied in the assessment of (a) recombinant PRMT activity, in which case one would expect to have to use much less than 1 microgram of the purified enzyme to adequately detect activity, (b) PRMT activities in cultured cell lysates, and (c) in quantifying the effects of inhibitors on PRMT activity.
1. X. Cheng, R.E. Collins, X. Zhang, in Annual Reviews of Biophysics Biomolecular Structure. (Annual Reviews, Palo Alto, CA, 2005), vol. 34, pp. 267-294. [Online Journal]
2. N. Dolzhanskaya, G. Merz, R.B. Denman, Alternative splicing modulates protein arginine methyltransferase-dependent methylation of fragile X syndrome mental retardation protein. Biochemistry 45, 10385-10393 (2006). [PubMed Abstract]
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