Objective: A comparative study of the kinetics of proteolysis of myosin S1 heavy chain was performed using dog ventricular and atrial S1 to distinguish between protease sensitive sites in S1 isotypes and to determine the binding sites on S1 heavy chain for LC1, LC2, and actin. Methods: Digestion of S1 as a function of actin was performed at 25°C at a trypsin to S1 ratio (w/w) of 1:1500. Myofibrils were digested (trypsin/myofibrils w/w ratio=l:300) in the presence of ATP, ADP, and under rigor conditions. Light chain and actin binding sites were identified by the gel overlay method. Results: Ventricular and atrial S1 were proteolysed at 0.13 min-1 and 0.04 min-1 respectively. Actin significantly reduced the cleavage rate of both S1 heavy chains by blocking hydrolysis at the 50/20 kD site. Myofibrillar myosin heavy chains from ventricles were also hydrolysed faster than those of the atria in the presence of 4 mM MgATP. The calculated rates were 0.42-0.50 and 0.17-0.19 min-1 for ventricular and atrial myofibrils respectively. MgADP 2 mM or absence of nucleotides reduced the cleavage rates to 0.04-0.07 (ventricular myofibrils) and 0.02-0.03 min-1 (atrial myofibrils) respectively. Gel overlay experiments showed that 125I labelled LC1 and LC2 bound to the 20 kD fragment and actin mainly to the 50 and 20 kD peptides. Conclusions: The 50/20 kD site in either ventricular or atrial S1 was blocked when actin was present, while proteolysis at the 25/50 site proceeded regardless of the presence of actin. However, the 25/50 site was less accessible to trypsin in the α myosin heavy chain, since the roughly threefold reduction in the rates of hydrolysis of atrial S1 heavy chain was also maintained in the myofibrils in rigor or in the presence of ADP. Although actin made contact with the 70 kD and the 25 kD fragments, the 50 kD and 20 kD fragments appeared to be the central "anchor" for binding of both light chains and actin.
- Cardiac myosin S1 isoforms
- Cardiac myosin S1: S1 domains
- Kinetics of S1 heavy chain proteolysis
- Light chain and actin binding sites
- Myosin light chains.
ASJC Scopus subject areas
- General Medicine