Cross-linking of actin to myosin subfragment 1: course of reaction and stoichiometry of products.
The cross-linking of actin to myosin subfragment 1 (S-1) with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide was reexamined by using two cross-linking procedures [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Nature (London) 292, 301-306; Sutoh, K. (1983) Biochemistry 22, 1579-1585] and two independent methods for quantitating the reaction products. In the first approach, the cross-linked acto-S-1 complexes were cleaved with elastase at the 25K/50K and 50K/22K junctions in S-1. This enabled direct measurements of the cross-linked and un-cross-linked fractions of the 50K and 22K fragments of S-1. We found that in all cases actin was preferentially cross-linked to the 22K fragment and that the overall stoichiometry of the main cross-linked products was that of a 1:1 complex of actin and S-1. In the second approach, actin was cross-linked to tryptically cleaved S-1, and the course of these reactions was monitored by measuring the decay of the free 50K and 20K fragments and the formation of cross-linked products. After selecting the optimal cross-linking procedure and conditions, we determined that the rate of actin cross-linking to the 20K fragment of S-1 was 3-fold faster than the reaction with the 50K peptide. The overall rate of cross-linking actin to S-1 corresponded to the sum of the individual reactions of the 50K and 20K fragments, indicating their mutually exclusive cross-linking to actin. Thus, the reactions with tryptically cleaved S-1 were consistent with the 1:1 stoichiometry of actin and S-1 in the main cross-linked products and verified the preferential cross-linking of actin to the 20K fragment of S-1. These results are discussed in the context of the binding of actin to S-1.
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002234:Carbodiimides / Q000378:metabolism*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010196:Pancreatic Elastase; D010446:Peptide Fragments / Q000378:metabolism*; D011817:Rabbits
D000199:Actins; D002234:Carbodiimides; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D010446:Peptide Fragments; D010196:Pancreatic Elastase; D009218:Myosins; D005022:Ethyldimethylaminopropyl Carbodiimide