Conformational differences in myosin, IV.[1-3] Radioactive labeling of specific thiol groups as influenced by ligand binding.
Changes in the mono- and divalentcation-stimulated ATPase activities of myosin progressively labeled with N-ethyl-[2,3-14C2]-maleimide were used to classify the readily reacting thiol groups into 3 types. The results show that one thiol-1 and one thiol-2 group are associated with each of the 2 active sites of myosin. Concentrations of KCl higher than 0.4M and/or temperatures above 10 degrees C lead to exposure of a variable number of thiol groups of a third class not affecting the enzymic properties. Although modification of thiol groups itself results in changes in structure and function of the protein, the patterns of incorporation of N-ethyl-[14C2]-malemide under various conditions of temperature, ionic strength and ligands bound to the protein revealed 9 different conformations of intact myosin. These were distinguished on the basis of the relative reactivity of the 3 different classes of thiol groups. The sequence of blockage of thiol groups reveals that cooperativity between the 2 active sites is induced by binding of a magnesium nucleotide complex to the protein. In the conformation of the long-lived myosin-product intermediate occuring during hydrolysis of Mg-ATP at 25 degrees C, 4 thiol groups of the third class react as well as or even more readily than those of the first and second classes.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology; D009218:Myosins / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D013438:Sulfhydryl Compounds; D011189:Potassium Chloride; D004492:Edetic Acid; D000251:Adenosine Triphosphatases; D009218:Myosins; D008274:Magnesium; D005033:Ethylmaleimide; D011188:Potassium; D002118:Calcium