Actin-binding peptide from smooth muscle myosin light chain kinase

• Published in: Biochemistry (Easton) 1993-08, Vol.32 (34), p.8902-8907
• Main Authors: Kanoh, Satoshi, Ito, Masaaki, Niwa, Eiji, Kawano, Yasushi, Hartshorne, David J
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Amino Acids - analysis; Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biological and medical sciences; Chickens; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Goats; Hydrolysis; Muscle, Smooth - enzymology; Myosin-Light-Chain Kinase - metabolism; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Peptide Fragments - metabolism; Transferases; Turkeys
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00085a023

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.