Asparaginyl deamidation-methylation of rat ventricular myosin light chains.
Spontaneous asparaginyl deamidation can produce damage to cytoskeletal proteins, and may lead to their targeting for subsequent rapid intracellular breakdown or repair. To test if myofibrillar proteins are subject to spontaneous deamidation damage in vitro, purified rat ventricular myosin light chain 1 (MLC1v) and phosphorylatable myosin light chain 2 (MPLC2v) were incubated (37 degrees C, 4 h, pH 2-11), and tested as substrates for human erythrocyte and rat cardiac protein carboxyl methyltransferase (PCMT). PCMT catalyzes the transfer of a methyl group from [3H-methyl] S-adenosyl methionine to deamidated asparaginyl residues and altered aspartyl residues on damaged proteins. MLC1v and MPLC2v underwent extensive incubation damage at neutral and alkaline pH. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography revealed 3H-incorporation into MLC1v, MPLC2v, and a Mr = 14,000 polypeptide. 3H-methylated, CNBr-cleavage fragments of PCMT-methylated light chains were then separated by reverse-phase high performance liquid chromatography, and sequenced by automated Edman degradation. The major 3H-labeled peptide of the Mr = 14,000 protein proved homologous to residues 84 to 104 of rat MPLC2v, with a proposed deamidation site at Asn99-Ala100. The major 3H-labeled peptide from MLC1v proved homologous to residues 73 to 111 of rat cardiac MLC1v, with a proposed deamidation site at Asn108-Ser109. These results indicate that both myofibrillar protein subunits undergo selective non-enzymatic degradation at neutral and alkaline pH, resulting in the formation of methyl acceptor sites for human erythrocyte and rat cardiac PCMT. PCMT-catalyzed methylation of ventricular myosin light chains may be important in the repair, or subsequent proteolysis of these long-lived structural proteins of the myofibril.
Cassidy
E M
EM
Department of Medicine, Loyola University Stritch School of Medicine, Maywood, IL 60153.
Wakim
B T
BT
Ferguson
A G
AG
Samarel
A M
AM
D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006352:Heart Ventricles / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008745:Methylation; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D011497:Protein O-Methyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D001216:Asparagine; D011497:Protein O-Methyltransferase; D009218:Myosins; D003488:Cyanogen Bromide