Graphical evaluation of alkylation of myosin's SH1 and SH2: the N-phenylmaleimide reaction.
Previous assertions about the effect of alkylation of SH1 and SH2 on the myosin high-salt calcium and EDTA ATPases have been summarized, and a simple procedure for obtaining the fractional labeling of SH1 and SH2 after treatment of myosin with alkylating agents has been derived. A simple graphical procedure for illustrating the degree of preference of a particular alkylating agent for SH1 over SH2 has also been developed. The procedures we developed were validated by applying them to two previously studied compounds, 4-(2-iodoacetamido)-TEMPO and 2,4-dinitrofluorobenzine, and then were used to determine a procedure for maximizing the extent of labeling of SH1 alone by N-phenylmaleimide, a compound not previously studied in this manner. It was found that approximately 80% of the SH1 sites could be alkylated without significant alkylation of SH2.
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Laboratory of Physical Biology, National Institute of Arthritis, and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Li
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Barnett
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Schoenberg
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D000478:Alkylation; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D003497:Cyclic N-Oxides / Q000378:metabolism; D004139:Dinitrofluorobenzene / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D007700:Kinetics; D008301:Maleimides / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D016658:Psoas Muscles / Q000737:chemistry; D011817:Rabbits; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000378:metabolism
D003497:Cyclic N-Oxides; D008301:Maleimides; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C003028:N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide; D004492:Edetic Acid; C028525:N-phenylmaleimide; D004139:Dinitrofluorobenzene; D009218:Myosins; D000252:Calcium-Transporting ATPases