Muscle actin cleaved by proteinase K: its polymerization and in vitro motility.
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 polymerized slowly into F-actin (proK-F-actin), 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 = 2.8 microM, while Vmax = 7.6 s-1, and Kapp = 13 microM by F-actin. Correspondingly, in vitro this proK-F-actin slid very slowly on HMM attached to a glass surface at an average velocity of 0.47 microns/s, or 1/12 of that of intact F-actin. The fraction of sliding filaments was less than 50%. Assuming that the nonmotile filaments attached to HMM were not involved in ATPase activation, the sliding velocity correlated with the ATPase activity activated by proK-F-actin.
Higashi-Fujime
S
S
Department of Molecular Biology, Faculty of Science, Nagoya University, Aichi.
Suzuki
M
M
Titani
K
K
Hozumi
T
T
D000199:Actins / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D019286:Endopeptidase K; D004789:Enzyme Activation; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009132:Muscles / Q000378:metabolism*; D015879:Myosin Subfragments / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010590:Phalloidine / Q000494:pharmacology; D011108:Polymers / Q000378:metabolism; D011817:Rabbits; D012697:Serine Endopeptidases / Q000378:metabolism*
D000199:Actins; D015879:Myosin Subfragments; D010446:Peptide Fragments; D011108:Polymers; D010590:Phalloidine; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D000251:Adenosine Triphosphatases