Abstract
S-Adenosylmethionine:protein-arginine N-methyltransferase (EC 2.1.1. 23; protein methylase I) transfers the methyl group of S-adenosyl-L-methionine to an arginine residue of a protein substrate. The homogeneous liver protein methylase I was subjected to tryptic digestion followed by reverse phase high performance liquid chromatography (HPLC) separation and either "on-line" mass spectrometric fragmentation or "off-line" Edman sequencing of selected fractions. Data base searching of both the mass spectrometric and Edman sequencing data from several peptides identified the protein methylase as 10-formyltetrahydrofolate dehydrogenase (EC 1.5.1.6; Cook, R. J., Lloyd, R. S., and Wagner, C. (1991) J. Biol. Chem. 266, 4965-4973; Swiss accession number). This identification was confirmed by comparative HPLC tryptic peptide mapping and affinity chromatography of the methylase on the 5-formyltetrahydrofolate-Sepharose affinity gel used to purify the dehydrogenase. The purified rat liver methylase had approximately 33% of the 10-formyltetrahydrofolate dehydrogenase and 36% of the aldehyde dehydrogenase activity as compared with the recombinant dehydrogenase, which also had protein methylase I activity. Polyclonal antibodies against recombinant dehydrogenase reacted with protein methylase I purified either by polyacrylamide gel electrophoresis or 5-formyltetrahydrofolate affinity chromatography. In each instance there was only a single immunoreactive band at a molecular weight of approximately 106,000. Together, these results confirm the co-identity of protein-arginine methyltransferase and 10-formyltetrahydrofolate dehydrogenase.
Highlights
S-Adenosylmethionine:protein-arginine N-methyltransferase1 (EC. 2.1.1.23; protein methylase I) catalyzes transfer of the methyl group from S-adenosyl-L-methionine (AdoMet)2 to
The present article establishes the co-identity of the rat liver protein-arginine methyltransferase and the rat liver formyltetrahydrofolate dehydrogenase (FDH)
The protein methylase I has been isolated as an ϳ450-kDa protein tetramer of identical ϳ100-kDa subunits [3] while the dehydrogenase, which had been isolated initially as the folate-binding protein [23, 24], was reported to exist as various multimers, which had approximate sizes of 410, 350, and 210 kDa and which were all composed of an ϳ100-kDa monomer [21, 24]
Summary
Materials—S-Adenosyl-L-[methyl-14C]methionine (specific activity, 50 mCi/mmol) and S-adenosyl-L-[methyl-3H]methionine (specific activity, 78.5 Ci/mmol) were obtained from NEN Life Science Products Inc. LC-MS/MS Protein Identification—Approximately 10% (5 pmol) of the in-gel trypsin digests of protein methylase I and of the recombinant and rat liver FDH that had been isolated by SDS-PAGE were subjected to reverse phase HPLC/on-line mass spectrometry as described [39]. These studies were carried out on a Applied Biosystems Division Model 140 B HPLC that was directly interfaced to a Finnigan Corporation LCQ quadrupole ion trap mass spectrometer. The blotted paper was washed and visualized with 3,3Ј-diaminobenzidine, phosphatebuffered saline, 1% CoCl2, and water as described [42]
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