The Pre-Steady State of the Myosin-Adenosine Triphosphate System
VIII. Intermediate Formation and Activation of Myosin by ATP
1969 Volume 65 Issue 2 Pages 285-301
Details
1969 Volume 65 Issue 2 Pages 285-301
1. A stable NTP-myosin compound, which was produced by incubating myosin ATPase [ATP: phosphohydrolase, EC 3. 6. 1. 3] with NTP in the presence of ATP and Mg++, was isolated by gel-filtration through a Sephadex column. The amount of initial burst of Pi-liberation from the myosin-ATP system decreased almost linearly with increase of bound NTP, and was reduced to zero on binding about I mole of NTP per 4×105g of myosin.
2. The maximum velocity, Vmax, and the pH-activity curve of myosin ATPase in the steady state were scarcely changed on the stable binding of l mole of NTP to 4×105g of myosin. However, the value of the Michaelis constant, Km, decreased on binding of NTP to myosin.
3. Analysis of the ultraviolet absorption spectrum of NTP bound to myosin indicated that NTP combines with a residue in the myosin molecule through its sulfhydryl group. To determine the amino acid residue with which NTP combines, NTP-myosin was digested by Nagarse [Subtilopeptidase A; EC 3. 4. 4. 16] and Pronase [EC class 3. 4. 4], and the NTP-peptide mixture was isolated from the digest by chromatography on Amberlite IRC-50 resin. The bond between NTP and the peptide of the NTP-peptide mixture thus obtained was found to be stable at acidic and neutral pH values, but unstable at alkaline pH's. NTP-glutamic acid was isolated almost quantitatively by column chromatography from the complete digest of the NTP-peptide mixture by Ieucine aminopeptidase [EC 3. 4. 1. 1] and prolidase [EC 3. 4. 3. 7].
4. The velocity of liberation of free ADP after addition of 1 mole of ATP to 3.4×105g of H-meromyosin was determined by measuring the velocity of pyruvate-liberation on addition of pyruvate kinase [ATP: pyruvate phosphotransferase, EC 2. 7. 1. 40] and PEP at appropriate times after the start of the reaction. The rate of ADP-liberation P from the myosin-phosphate-ADP complex, _??_, was estimated by analysis of the ADP results. It was much lower than the rate of formation of this complex and far higher than the rate of decomposition of this complex to E+ADP+Pi+H+.
5. The report by Kanazawa and Tonomura on the existence of an extra-burst of Pi liberation in the initial phase of the reaction of myosin and ATP at a low concen-tration of Mg++ and high concentration of KCl at room temperature was confirmed. The amount of the extra-burst of Pi-liberation increased with increase in Mg++ concen-tration from 0 to 10μM, and reached a maximum level of 10-20 moles Pi per 4×105g of myosin at 10μM Mg++. It decreased gradually with further increase in Mg++ concen-tration. In the presence of more than 1mM Mg++, the initial burst remained at a stoichiometric amount of 1 mole Pi per 4×105g of myosin. The effects of temperature, KCl concentration and treatment with PCMB-β-mercaptoethanol on the amount of the extra-burst of P liberation were also examined.
6. The time-courses of TCA labile Pi and hydrogen ion-liberation from the myosin-ATP system were measured in the presence of 10μM Mg++. The extra-burst of TCA-labile Pi-liberation amounted to 17 moles per 4×105g of myosin and that of hydrogen ion-liberation to 10.5 moles per 4×105g of myosin. The initial rapid liberation of ADP from the myosin-ATP system during the extra-burst of Pi-liberation was followed by measuring pyruvate-liberation in a reaction medium coupled with the pyruvate kinase system. The initial rapid ADP-liberation was about I mole less than the amount of the initial burst of P liberation per 4 ×105g of myosin. These results indicate that during the extra-burst
