title: Modification of myosin subfragment 1 by carbodiimide in the presence of a nucleophile. Effect on adenosinetriphosphatase activities.
abstract: The modification of myosin subfragment 1 by N-cyclohexyl-N'-[2-(4-morpholinyl)ethyl]carbodiimide methyl p-toluenesulfonate in the presence of the nucleophile nitrotyrosine ethyl ester was investigated. For elimination of interference of the thiol groups, the two most reactive thiols were protected by cyanylation with 2-nitro-5-(thiocyanato)benzoic acid. The ATPase activity of the cyanylated myosin subfragment 1 was not lost, but had changed. At pH 5.9, carbodiimide in the presence of the nucleophile rapidly inactivated the cyanylated enzyme. The inactivation followed first-order kinetics. The K+(EDTA)--, Ca2+--, and Mg2+--ATPase activities decreased at the same rate. Inactivation and incorporation of nucleophile occurred simultaneously. A full loss of activity resulted from the incorporation of 1 mol of nitrotyrosine per mol of myosin subfragment 1. Pyrophosphate, ITP, ADP, and ATP protected against inactivation, and the efficiency of the protection was parallel to the ligand binding strength. These results suggested that one carboxyl group was essential for the active conformation of myosin.
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D003028:CME-Carbodiimide / Q000031:analogs & derivatives / Q000494:pharmacology*; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002234:Carbodiimides / Q000494:pharmacology*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits
chemical_list: D002234:Carbodiimides; D015879:Myosin Subfragments; D010446:Peptide Fragments; D003028:CME-Carbodiimide; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; C026049:EDTA-ATPase; D009218:Myosins; D000252:Calcium-Transporting ATPases