Chemical crosslinking of myosin subfragment-1 to F-actin in the presence of nucleotide.
F-Actin and myosin subfragment-1 (S-1) were covalently crosslinked in the absence and presence of nucleotides, adenyl-5'-yl imidodiphosphate (AMPPNP), ATP, and ADP, at various KCl concentrations (0-0.5 M), using a zero-length crosslinker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). The rate of production of the covalent acto-S-1 complex was almost proportional to the amount of the acto-S-1(-nucleotide) complex existing in the reaction medium. However, the Mg-ATPase activity of the covalent acto-S-1 complex thus produced was affected by the presence of nucleotides. When S-1 was crosslinked to F-actin in the absence of nucleotide at 0-0.5 M KCl, the Mg-ATPase activity of S-1 was enhanced from 0.1-0.3 to 12-15 s-1. The Mg-ATPase activity of covalent complex produced in the presence of AMPPNP at 0 M KCl was 13 s-1 which was equal to that produced in the absence of nucleotide. The activity decreased with increasing KCl concentration of the crosslinking medium, reaching 6 s-1 at 0.5 M KCl. Covalent acto-S-1 complex was also produced when acto-S-1-ADP-P was formed during ATP hydrolysis. The Mg-ATPase activity of the covalent acto-S-1 complex crosslinked during ATP hydrolysis at 0 M KCl was about 5 s-1 which was about half of that obtained in the absence of nucleotide, and it decreased to about 2 s-1 at 0.15 M KCl of crosslinking medium. In contrast, the acto-S-1-ADP complex gave a covalent complex which was indistinguishable in its extent and Mg-ATPase activity from that obtained in the absence of nucleotide. These results suggest that the structures of acto-S-1-AMPPNP and acto-S-1-ADP-P complexes are different from those of acto-S-1 and acto-S-1-ADP complexes.
D000199:Actins / Q000378:metabolism*; D000227:Adenine Nucleotides / Q000494:pharmacology*; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D002234:Carbodiimides / Q000494:pharmacology*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology*; D007700:Kinetics; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011817:Rabbits
D000199:Actins; D000227:Adenine Nucleotides; D002234:Carbodiimides; D015879:Myosin Subfragments; D010446:Peptide Fragments; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins; D005022:Ethyldimethylaminopropyl Carbodiimide