JBC, Vol. 252, Issue 5, 1681-1688, Mar, 1977
Reaction of cardiac myosin with a purine disulfide analog of adenosine triphosphate. II. Stoichiometry and subunit location of labeling
L. E. Greene and R. G. Yount
The reaction of bovine cardiac myosin with the site-specific purine
disulfide analog of ATP, 6,6'-dithiobis (inosinyl imidodiphosphate), was
studied to determine the stoichiometry of labeling and subunit location of
the reactive cysteines. The analog inactivates myosin by forming a mixed
disulfide between the thiopurine nucleotide and certain key cysteines. The
thiopurine nucleotide was displaced quantitatively by 14CN to form the more
stable thiocyanato-enzyme derivatives. In cardiac myosin, the reactive
cysteines could be categorized into three classes, nonessential, critical,
and noncritical. The modification of the critical cysteines (two per
myosin) inactivated the EDTA and Ca2+ ATPase activities, the latter to a
lesser extent. The nonessential cysteines (two to three per myosin) and the
noncritical cysteines (two per myosin), differentiated by their rates of
reaction, had no effect on the ATPase activities after modification.
Thiocyanato-modified myosin was analyzed by sodium dodecyl sulfate gel
electrophoresis to determine the distribution of 14CN in the subunits. The
critical cysteines were found on the 21,000-dalton light chain (LC1) and
the noncritical cysteines on the heavy chains. More specifically, the
critical cysteine modified in cardiac LC1 (determined from the products
after cyclization and chain cleavage at the thiocyanatoalanyl residues) was
shown to be the thiol residue whose surrounding amino acid sequence is
homologous to that of the single cysteine of the skeletal myosin alkali
light chains, confirming the likely similar structure and function of these
light chains in the two different muscle types.
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