Evidence for the existence of two equilibrium conformations of the ternary complex of myosin subfragment-1, ADP, and orthovanadate.
In order to investigate the flexibility of the ternary complex consisting of myosin subfragment-1 (S1), ADP, and orthovanadate (Vi), i.e., S1.ADP.Vi, the exchangeability of the bound ADP was examined. After isolation of the ternary complex of S1.ADP.Vi by gel filtration, 3'-O-(N-methylanthraniloyl)-ADP (Mant-ADP), a fluorescent analogue of ADP, was added at 0.5 degrees C. The added Mant-ADP was incorporated into the ternary complex very slowly by replacing the bound ADP. The nucleotide exchange occurred without regeneration of the ATPase activity of S1. Similarly, the ternary complex of S1.Mant-ADP.Vi prepared and isolated by gel filtration according to Hiratsuka (3, 4), was incubated with ADP (2.4 mM) at 4.5 degrees C. The nucleotide exchange of S1.Mant-ADP.Vi with ADP occurred in two phases with the apparent rates of 4.5 x 10(-4) s-1 (the fast phase) and 6.7 x 10(-6) s-1 (the slow phase). Biphasic exchange of the bound nucleotide was also observed with S1(A1) isozyme, indicating that the biphasic exchange did not correspond to two S1 isozymes. The apparent rates of the fast and the slow phases increased with the concentration of the added ADP, but they became saturated at an ADP concentration of the order of 2 mM, indicating that the nucleotide exchange reaction involves a step (or steps) which is insensitive to the concentration of free ADP in the solution. This step might be a reversible isomerization.
Mihashi
K
K
Department of Physics, Faculty of Science, Nagoya University, Aichi.
Ooi
A
A
Hiratsuka
T
T
D000244:Adenosine Diphosphate / Q000032:analysis*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000032:analysis; D005990:Glycerol / Q000032:analysis; D007527:Isoenzymes / Q000032:analysis; D007700:Kinetics; D009132:Muscles / Q000032:analysis; D015879:Myosin Subfragments / Q000032:analysis*; D009711:Nucleotides / Q000032:analysis; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014638:Vanadates / Q000032:analysis*
D007527:Isoenzymes; D015879:Myosin Subfragments; D009711:Nucleotides; D014638:Vanadates; D000244:Adenosine Diphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D005990:Glycerol