Isolation of cardiac myosin light‐chain isotypes by chromatofocusing

Abstract

1. 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.2. Light chains were resolved in order of decreasing isoelectric point in the pH range 5.2-4.5. Gradients of ApH = 0.004 -0.006/ml elution volume were achieved which were capable of resolving light chains with isoelectric point differences of only 0.03.3. Analytical isoelectric focusing of light chains in polyacrylamide gels could be used to predict the results of preparative chromatofocusing for method development.4. 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 foetal ventricular light chain 1 (FLC-1) from adult ventricular light chain 1 (VLC-1) present in foetal ventricles and the atrial light chain 1 (ALC-1) in adult atria.6. 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 three different proteases and amino acid compositions were similar with the exception of glycine content. The studies indicate that FLC-1 and ALC-1 are homologous, and possibly identical, light chains.

Atrial and ventricular myosins from human hearts. I. Isoenzyme distribution during development and in the adult

“…The existence of an embryonic form of myosin light chain (4,33) in the human embryonal, fetal, and neonatal cardiac muscle is confirmed by our present results and is probably a common feature of the myosin ontogenesis in the heart, just as it is in the human skeletal muscle (2). With regard to the light chain transitions of the myosin isoenzymes from fetal to neonatal and adult life our present results show that in the adult ventricle only VLC-1 and VLC-2 are expressed.…”

“…Developing human hearts possess a myosin distinguished from that of normal adults by the light chain composition. During fetal life and the first months after birth this isoenzyme contains a fetal light chain, which may be identical to the adult atrial one (4,23,33). Aging, as well as mechanical heart overloading, modifies the cardiac myosins and new forms are synthesized.…”