Isolation of cardiac myosin light‐chain isotypes by chromatofocusing

Publisher Website
Google Scholar
Abstract
Cardiac myosin light chain isotypes have been resolved using chromatofocusing, a new preparative column chromatographic technique. The method relies on production of narrow-range, shallow and stable pH gradients, using ion-exchange resins and buffers, with even buffering capacity over the required pH range. Light chains were resolved in order of decreasing isoelectric point in the pH range 5.2-4.5. Gradients of .DELTA.pH = 0.004-0.006/ml elution volume were achieved which were capable of resolving light chains with isoelectric point differences of only 0.03. Analytical isoelectric focusing of light chains in polyacrylamide gels could be used to predict the results of preparative chromatofocusing for method development. Chromatofocusing was capable of resolving human and bovine cardiac light chain 1 and 2 subunits, atrial (ALC) and ventricular (VLC) light chain isotypes and homologous VLC-2 and VLC-2 light chains. The technique was used to purify and resolve the human fetal ventricular light chain 1 (FLC-1) from adult ventricular light chain 1 (VLC-1) present in fetal ventricles and the atrial light chain 1 (ALC-1) in adult atria. Comparative peptide mapping studies and amino acid analyses were carried out on FLC-1 and ALC-1. No differences were detected between FLC-1 and ALC-1 using 3 different proteases, and amino acid compositions were similar, with the exception of glycine content. FLC-1 and ALC-1 may be homologous, and possibly identical, light chains. Comparison of human FLC-1/ALC-1 with VLC-1 suggested marked structural and chemical differences in these light chain isotypes, in particular in the contents of methionine, proline, lysine and alanine residues. Differences in the contents of these residues were also apparent in the corresponding bovine atrial and ventricular light chains [Wikman-Coffelt, et Srivastava, 1979]. The latter 3 residues are known to be rich in the N-termini of cardiac and skeletal light chain 1 isotypes, an area that was implicated in actin binding, suggesting that atrial and ventricular light chains may differ functionally in this region.