Sites of biological methylation of proteins in cultured chick muscle cells

Abstract

The methylation of myosin and other proteins has been studied using primary cultures of 12-day-old embryonic chick leg muscle. The methyl group of [Me-3H] methionine is incorporated into basic amino acid residues with the formation of Nepsilon-monomethyllysine, Nepsilon-dimethyllysine, Nepsilon-trimethyllysine, 3-methylhistidine, NG-monomethylarginine, and NG-dimethylarginine which are isolated from acid hydrolysates of purified myosin, and of proteins from polysomes and from the cytosol of the cultured muscle cells. In the presence of 0.1 mM cycloheximide, incorporation of [Me-3H] methionine into the polysome-bound proteins was decreased to 16.3% of control levels with no change in the pattern of incorporation into the basic amino acid residues, although protein synthesis was inhibited 97.5%. When protein synthesis was allowed to resume in such cultures by the removal of cycloheximide, polypeptides containing labeled N-methylated residues were released from polysomes into the soluble fraction. Polypeptides containing N-methylated amino acids were also released from polysomes following treatment with 2 mM puromycin. Peptidyl-tRNA, isolated from ribosomes after exposure of cultures to [Me-3H] methionine, contained labeled N-methylated amino acids. When [Me-3H] methionine was incorporated in the presence of cycloheximide, the isolated peptidyl-tRNA still contained N-methylated amino acids although the amount of methylation was 22.4% of control levels. These experiments demonstrate that N-methylation of basic amino acid residues in proteins may occur while the polypeptide is still being synthesized on the ribosome. In addition, N-methylation can occur on the nascent polypeptides in the absence of protein synthesis.

3-Methylhistidine Excretion and the Urinary 3-Methylhistidine/Creatinine Ratio Are Poor Indicators of Skeletal Muscle Protein Breakdown

“…The turnover of 3-methylhistidine in skeletal muscle protein was 35% of the overall rate of mixed muscle protein synthesis irrespective of whether it was measured 1 h or 4-5 h after injection of tracer [23,24]. This confirms the results of others, who have shown no differences between rates of methylation and overall protein synthesis [26,27].…”

Carnitine biosynthesis in mammals

“…In mammals, certain proteins contain N'trimethyl-lysine (TML) residues [16]. N-methylation of these lysine residues occurs as a post-translational event in proteins such as calmodulin, myosin, actin, cytochrome c and histones [17,18]. This reaction is catalysed by specific methyltransferases, which use S-adenosylmethionine as a methyl donor [16].…”

Cardiovascular Tests: Use & Limits of Biochemical Markers - Therapeutic Measurements of ADMA Involved in Cardiovascular Disorders

“…This process of basic amino acid methylation became well documented [17] and it was recognised that arginine methylation occurred shortly after protein synthesis. Incorporation of radiolabelled methyl groups onto the arginine residues of myelin basic protein revealed that once methylated the reaction appeared to be irreversible and the arginine-methylated protein took weeks to degrade [18,19].…”