Separation of Low Molecular Weight Components of Pig Cardiac Myosin and Myosin Subfragment-1, and Ca2+ Binding to One of the Components (g2)1

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Abstract
Cardiac myosin was found to contain two small polypeptide components (g1 and g2) as well as the main polypeptide chain (f-chain). A mixture of g1 and g2 was separated from the main chain in 4 M urea or 5 M guanidine-HCl and then these two proteins were separated by pCMB-Sepharose 4B conjugate column chromatography. Cardiac myosin subfragment-1 contained g1 as the only small component. Small amounts of unidentified polypeptide fragments were detected in this subfragrnent-1 preparation by acrylamide gel electrophoresis in the presence of SDS. These polypeptide fragments were removed by gel filtration using Sephadex G-200 in the presence of 4 M urea. The UV absorption spectra and electrophoretic mobilities of small polypeptide components of myosin and subfragment-1 were compared after isolation. It was concluded that i) g1 of subfragrnent-1 was identical with that of myosin, ii)g2 has an absorption spectrum characteristic of Ca1+ binding proteins, and iii)subfragment-1 was readily degraded by chymotrypsin into another form of subfragment-1 in which the small component was smaller than g1. Ca2+ binding to g2 was demonstrated using murexide as a color indicator. The UV difference spectrum of g2 was observed with and without Ca2+ The dissociation constant was estimated to be 3.1 × 10−4 M. No evidence for Ca2+ binding to g1 was obtained.