ACTIN-BINDING PEPTIDE FROM SMOOTH-MUSCLE MYOSIN LIGHT-CHAIN KINASE

The objective of this study was to localize the actin-binding site in the smooth muscle myosin light chain kinase. Limited proteolysis by thermolysin indicated that hydrolysis of the kinase at the N-terminal end of the molecule resulted in loss of actin-binding ability. Various methods 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. This procedure yielded an actin-binding peptide of approximate M(r) 17 000. The peptide was purified and shown to possess the actin-binding properties of the native myosin light chain kinase. The binding constant 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 comparison with the sequence of gizzard myosin light chain kinase, it was suggested that the actin-binding site is located within the N-terminal sequence 1-114. Comparison with other actin-binding proteins shows some similarities to gizzard alpha-actinin and caldesmon.