Myosin filament ATPase is enhanced by intramolecularly cross-linked actin.
Reaction of rabbit skeletal muscle F-actin with the lysine-directed photolabile cross-linker, N-5-azido-2-nitrobenzoyloxy succinimide was limited to Lysine-328 and Lysine-326, with Lysine-328 being labelled to a greater extent. Photolysis of the modified actin enhanced the actin-activated MgATPase activity of filamentous scallop myosin 3-4-fold more than unmodified actin, without affecting calcium sensitivity. Unphotolysed modified actin behaved as untreated actin, indicating that photolysis was essential for the effect. The actin-activated ATPase of filamentous rabbit myosin was similarly increased by photolysed N-5-azido-2-nitrobenzoyloxy succinimide-modified actin. After photolysis in either the monomeric (G-) or filamentous (F-) form, N-5-azido-2-nitrobenzoyloxy succinimide-modified actin moved as a monomeric (42 kDa) species on SDS gels, and depolymerized and polymerized readily, demonstrating that any cross-linking event produced by photolysis must be intramolecular. In contrast to the substantial increase in actin-activated ATPase activity observed when photolysed ANB-NOS-modified actin was added to filamentous myosin, the enhancement was not observed with the soluble HMM and S-1 fragments of myosin. Photolysed modified actin showed only poor movement on a rabbit HMM-coated surface in vitro motility assays. These results can be explained if the internally cross-linked G-actin subunits which comprise only a fraction of the actin population, either weaken the actin-actin contacts or have an increased affinity for myosin.
Kwon
H
H
Department of Biology, Brandeis University, Waltham, MA 02254.
Hardwicke
P M
PM
Collins
J H
JH
Zhao
X
X
Szent-Györgyi
A G
AG
D000199:Actins / Q000187:drug effects* / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000494:pharmacology* / Q000528:radiation effects; D003432:Cross-Linking Reagents / Q000494:pharmacology* / Q000528:radiation effects; D004789:Enzyme Activation / Q000187:drug effects; D008239:Lysine / Q000737:chemistry; D008974:Mollusca / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D010782:Photolysis; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D013388:Succinimides / Q000494:pharmacology* / Q000528:radiation effects; D014466:Ultraviolet Rays
D000199:Actins; D001386:Azides; D003432:Cross-Linking Reagents; D013388:Succinimides; C049915:N-hydroxysuccinimidyl-5-azido-2-nitrobenzoate; D009218:Myosins; D008239:Lysine