Half-stoichiometric trinitrophenylation of myosin subfragment 1 in the presence of pyrophosphate or adenosine diphosphate.
A molecule of myosin subfragment 1 (S1) from skeletal muscle has one reactive lysine residue (RLR) that is rapidly and stoichiometrically modified by trinitrobenzene sulfonate (TNBS). Previous studies on the RLR modification with TNBS in the presence of inorganic pyrophosphate (PPi) by others suggested the non-identical nature of the two myosin heads which is still controversial. To resolve this issue, we studied the effects of PPi and ADP on the trinitrophenylation reaction over a wide range of ligands more systematically, by using S1 containing the alkali 1 light chain. We herein show that MgPPi or MgADP reduces the maximum number of trinitrophenylated RLRs down to about 0.5 mol/mol of S1 and that this half-stoichiometric modification of RLRs is not the result of heterogeneity in the primary structure of S1. Instead, our results suggest the existence of two almost equimolar, different conformations of S1.PPi and S1.ADP. Furthermore, we show evidence that the two different conformations are in a slow equilibrium in the presence of TNBS. We also studied the effects of the stoichiometric and half-stoichiometric RLR modification on the EDTA- and Mg-ATPase activities of S1 and found no evidence for the functional heterogeneity of the myosin active site.
Komatsu
H
H
Department of Molecular Biology, Graduate School of Medical Science, Faculty of Science, Kyushu University, Fukuoka, Japan.
Emoto
Y
Y
Tawada
K
K
D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D011756:Diphosphates / Q000378:metabolism / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011487:Protein Conformation; D011817:Rabbits; D013997:Time Factors; D014302:Trinitrobenzenesulfonic Acid / Q000378:metabolism*
D011756:Diphosphates; D015879:Myosin Subfragments; D010446:Peptide Fragments; D000244:Adenosine Diphosphate; D014302:Trinitrobenzenesulfonic Acid; D004492:Edetic Acid; D009218:Myosins; D008274:Magnesium