Actin-binding peptide from smooth muscle myosin light chain kinase

Abstract

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.

The Structure and Function of the Actin-binding Domain of Myosin Light Chain Kinase of Smooth Muscle

“…In the presence of 1 mM EGTA, the NTCB fragment showed actin binding activity, confirming the results of Kanoh et al (14). Scatchard plots of the data showed that the NTCB fragment bound to actin filaments maximally at 0.2 mol/mol actin with a single K a of 4.8 ϫ 10…”

“…The fragment consisted of the Met 1 -Lys 114 sequence (14,18) and is referred to as the NTCB fragment. Skeletal muscle actin was purified from an acetone powder of chicken breast muscle (19) and used as actin filaments after polymerization.…”

“…13 for a review). However, there has been little study of the structure-function relationship of the actin binding activity except for the purification of an actinbinding fragment from MLCK (14).…”

“…Our approach has been to cleave MLCK to prepare native fragments containing the actin binding activity (14), to design MLCK cDNA to express the recombinant fragments of actin binding activity in Escherichia coli, and then to analyze them biochemically. A few peptides have been synthesized to confirm these analyses.…”

Properties of Long Myosin Light Chain Kinase Binding to F-Actin in Vitro and in Vivo

“…Interestingly, MLCK binds to actin thin filaments whereas its substrate, myosin, is in thick filaments (14,15). The actinbinding properties of the short MLCK are precisely identified with three repeated motifs of DFRXXL residues at the N terminus (1,16).…”