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(Circulation Research. 2001;89:480.)
© 2001 American Heart Association, Inc.

Molecular Medicine

Proteomic Analysis of Pharmacologically Preconditioned Cardiomyocytes Reveals Novel Phosphorylation of Myosin Light Chain 1

D. Kent Arrell, Irina Neverova, Heather Fraser, Eduardo Marbán, Jennifer E. Van Eyk

From the Departments of Physiology (D.K.A., I.N., J.E.V.E.) and Biochemistry (J.E.V.E.), Queen’s University, Kingston, Ontario, Canada, and Institute of Molecular Cardiobiology (H.F., E.M.), Johns Hopkins University, Baltimore, Md.

Correspondence to Dr Jennifer E. Van Eyk, Room 429 Botterell Hall, Department of Physiology, Queen’s University, Kingston, Ontario, Canada, K7L 3N6. E-mail jve1{at}post.queensu.ca

Abstract — Proteomic analysis of rabbit ventricular myocytes revealed a novel posttranslational modification to myosin light chain 1 (MLC1), consisting of phosphorylation at two sites. Subproteomic extraction to isolate myofilament-enriched fractions enabled determination of the extent of phosphorylation, which increased from 25.7±1.6% to 34.0±2.7% (mean±SE, n=4; P<0.05) after adenosine treatment at levels sufficient to pharmacologically precondition the myocytes (100 µmol/L). Mass spectrometry of MLC1 tryptic digests identified two peptide fragments modified by phosphorylation. These two phosphopeptides were characterized by peptide mass fingerprinting to determine the phosphorylation sites within rabbit ventricular MLC1, which correspond to Thr69 and Ser200 of rat MLC1, and to Thr64 and Ser194 or 195 of human MLC1. This proteomic analysis of preconditioned myocardium has revealed a previously unsuspected in vivo posttranslational modification to MLC1.

Key Words: proteomics • myosin light chain • phosphorylation • adenosine • preconditioning

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