Identical behavior of the two active sites of myosin with respect to trinitrophenylation.
Myosin and its active subfragments were trinitrophenylated under conditions in which mainly the active site(s) was modified. Proteins modified at the active site(s) could be separated by affinity chromatography on agarose-ATP columns. By two independent methods, ATPase activity measurements and analysis of elution patterns on agarose-ATP columns, it was shown that the introduction of two trinitrophenyl groups per myosin or one per heavy meromyosin subfragment 1 molecule is responsible for the remarkable change in the ATPase activities. Heavy meromyosin subfragment 1 prepared from trinitrophenylated myosin retained the original degree of trinitrophenylation per "active head." The kinetic constant of trinitrophenylation of the epsilon-amino group of lysine at the active site was found to be 2000 S-1-M-1, whereas a much smaller constant of 2.2 S-1-M-1 was obtained for the trinitrophenylation of the unessential lysyl residues of myosin. By using affinity chromatography, we could follow the formation of mono- and ditrinitrophenyl myosin. The amounts of these myosin derivatives at various extents of the reaction corresponded approximately to the calculated amounts, assuming a random and independent trinitrophenylation of the two myosin "heads." It is concluded that in each of the two heads of myosin there is one ATPase active site and these two sites behave in an identical manner with respect to trinitrophenylation.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments / Q000378:metabolism; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism; D009596:Nitrophenols; D010206:Papain; D011188:Potassium / Q000494:pharmacology; D011485:Protein Binding; D011817:Rabbits; D013997:Time Factors; D014357:Trypsin
D015879:Myosin Subfragments; D009596:Nitrophenols; D014357:Trypsin; D010206:Papain; D000251:Adenosine Triphosphatases; D009218:Myosins; D008274:Magnesium; D011188:Potassium