The objective of this study was to localize the actin-binding site in
the smooth muscle myosin light chain kinase. Limited proteolysis by th
ermolysin indicated that hydrolysis of the kinase at the N-terminal en
d of the molecule resulted in loss of actin-binding ability. Various m
ethods of cleavage were investigated for the generation of a discrete
actin-binding peptide. The method chosen was cleavage at the cysteine
residues by the 5,5'-dithiobis(2-nitrobenzoic acid)-cyanide complex. T
his procedure yielded an actin-binding peptide of approximate M(r) 17
000. The peptide was purified and shown to possess the actin-binding p
roperties of the native myosin light chain kinase. The binding constan
t of the isolated peptide and parent enzyme to actin was estimated as
7.5 X 10(4) M-1. From the amino acid composition of the peptide and co
mparison with the sequence of gizzard myosin light chain kinase, it wa
s suggested that the actin-binding site is located within the N-termin
al sequence 1-114. Comparison with other actin-binding proteins shows
some similarities to gizzard alpha-actinin and caldesmon.