Muscle Actin Cleaved by Proteinase K: Its Polymerization and In Vitro Motility

• Published in: Journal of biochemistry (Tokyo) Vol. 112; no. 4; pp. 568 - 572
• Main Authors: Higashi-Fujime, Sugie, Suzuki, Masami, Titani, Koiti, Hozumi, Tetsu
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 1992
• Subjects: actin; Actins - metabolism; Adenosine Triphosphatases - metabolism; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; cleavage; Contractile proteins; electron microscopy; Endopeptidase K; Enzyme Activation; Fundamental and applied biological sciences. Psychology; Holoproteins; Kinetics; Microscopy, Fluorescence; motility; muscles; Muscles - metabolism; Myosin Subfragments - metabolism; Peptide Fragments - metabolism; Phalloidine - pharmacology; polymerization; Polymers - metabolism; proteinase K; Proteins; Rabbits; Serine Endopeptidases - metabolism; Vertebrata; Mammalia; Motility; Molecular structure; Enzyme; Proteinase; Contractile protein; Actins; Enzymatic digestion; Rabbit; Polymerization; Lagomorpha
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a123940

Skeletal muscle actin was lightly digested by proteinase K, which cleaved the peptide bond between Met-47 and Gly-48, producing a C-terminal 35 kDa fragment. Proteinase K-cleaved actin (proK-actin) did not polymerize into F-actin upon addition of salt. In the presence of phalloidin, however, it polymerLzed slowly Into F-aetln (proK-F-actln), indicating that the cleaved actin did not dissociate into the individual cleaved fragments but retained the global structure of actin. Electron microscopy showed that proK-F-actin had the typical double-stranded structure of a normal actin filament and formed the arrowhead structure when decorated with HMM. Heavy meromyosin ATPase was weakly activated by proK-F actin: Vmax=0.24 s−1 and Kapp=208μM while Vmax=7.6 s−1 and Kapp=13μ by F-actln. Correspondingly, in vitro this proK-F-actin slid very slowly on HMM attached to a glass surface at an average velocity of 0.47 μm/a, or 1/12 of that of intact F-actin. The fraction of sliding filaments was less than 50%. Assuming that the nnonmotile filaments attached to HMM were not involved in ATPase activation, the sliding velocity correlated with the ATPase activity activated by proK-F-actin.