Kinetic analysis of hydrolysis of free ATP and MgATP by natural actomyosin]

Computer analysis of experimental data published in 1-3 allowed to establish the presence of two non-interacting inequivalent hydrolytic sites in actomyosin molecule, one of them being specific for binding and hydrolysis of free ATP, the other--for MgATP. Thus both species of ATP are the substrates of actomyosin ATPase. Actomyosin molecule seems to bind on more (in additon to two active sites) substrate molecule (MgATP) at some non-catalytic regulatory site. The formation of the enzyme-substrate complex having three ATP molecules (one molecule of free ATP and two--of MgATP) is accompanied by the loss of the activity. An approach to the research of kinetic equations for complex systems considerably decreasing a number of variations to consider is given in this work.     

Polyphasic character of ATP hydrolysis in actomyosin system.

The interaction of 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris) with the metal ions (M2+) Mg2+, Ca2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ was studied by potentiometry and spectrophotometry in aqueous solution (I = 0.1 or 1.0 M, KNO3, 25 degrees C). Stability constants of the M(Tris)2+ complexes were determined; those constants which were measured by both methods agreed well. Ternary complexes containing ATP4- as a second ligand were also investigated and it is shown that in the presence of Tris, mixed-ligand complexes of the type M(ATP)(Tris)2- are formed. The values for delta log KM, where delta log KM = log KM(ATP)M(ATP)Tris--log KMM(Tris), are all negative, thus indicating that the interaction of Tris with M(ATP)2- is somewhat less pronounced than with M2+. However, it should be noted that even in mixed-ligand systems complex formation with Tris may still be considerable, hence great reservations should be exercised in employing Tris as a buffer in systems which also contain metal ions. Distributions of the complex species in dependence on pH are shown for several systems, and the structures of the binary M(Tris)2- and the ternary M(ATP)(Tris)2- complexes are discussed. The participation of a Tris-hydroxo group in complex formation is, at least for the M(Tris)2- species, quite evident.     

The inhibitory action of the light meromyosin component on the myofibrillar and actomyosin atp-ase.

The protein component of light meromyosin [LMM-1] was shown earlier to relax glycerinated muscle fibres and actomyosin. Presently its influence on ATP-ase activity of myofibrils, actomyosin, myosin and heavy meromyosin has been studied. LMM-1 decreases Mg-ATP-ase activity of myofibrils and of reconstructed actomyosin by 25-- 30% and does not change [or slightly increases] Ca-ATP-ase activity of this protein and of myosin; besides LMM-1 is able to increase Mg-ATP-ase of HMM substantially. LMM-1 markedly inhibits [preliminary data] the activation of ATP-ase activity of HMM by actin. It is suggested that LMM-1 protein interacts with myosin and decreases the actin-myosin affinity, displacing actin out of the complex. It reacts only with one of the heads of myosin. Probably this suggestion can account for a relatively slight inhibition of ATP-ase activity of complex by LMM-1. LMM-1 represents a natural and specific inhibitor of Mg-AM-ATP-ase activity, included in the structure of myosin protofibrils and interacting with the myosin active site region.     

Structural studies of natural actomyosin from thermally acclimated frogs.

Natural actomyosin was isolated from skeletal muscle of frogs (Rana catesbeiana) acclimated at 25 degrees C and 5 degrees C. It was found that preparations isolated from warm-acclimated frogs may display considerable degradation of myosin heavy chains as compared with preparations isolated from cold-acclimated frogs. However, degradation may be minimized by inclusion of protease inhibitors during purification, indicating enhanced protease activity in preparations of natural actomyosin from warm-acclimated frogs. When purified in the presence of protease inhibitors, natural actomyosin from both warm-acclimated and cold-acclimated frogs exhibits comparable subunit composition of SDS-gel electrophoresis. The overall gel pattern is similar to that obtained from rabbit natural actomyosin except that in the frog, troponin-T and troponin-C appear to co-migrate with tropomyosin and myosin light chain 2, respectively.     

Phosphorylation of guinea pig cardiac natural actomyosin and its effect on ATPase activity.

Guinea pig cardiac natural actomyosin incubated with commercial protein kinase, Mg²⁺-ATP, and cyclic AMP produced little or no change in actomyosin ATPase activity. However, addition of sodium fluoride, a known phosphatase inhibitor resulted in a decreased actomyosin ATPase at all measured calcium concentrations. The presence of phosphatase activity in actomyosin and protein kinase was confirmed with ?-nitrophenyl phosphate. These results indicate the importance of inhibiting phosphatase activity, particularly when measuring biological or enzymatic activity as a function of phosphorylation.     

The radiomodifying action of ATP and ADP on membrane-bound enzymes

A modifying action of ATP and ADP on the activity of some key membrane-bound enzymes of the brain and heart microsomes of rats exposed to 7 Gy radiation has been investigated. The difference in the reactions of energy-dependent enzymes is attributed to the compensatory systems involved at the molecular level.     

Kinetics of ATP and inorganic phosphate release during hydrolysis of ATP by rabbit skeletal actomyosin subfragment 1. Oxygen exchange between water and ATP or phosphate

We have used the technique of phosphate: water oxygen exchange to measure the rate of ATP and Pi release and Pi binding to myosin subfragment 1 and actomyosin subfragment 1 from rabbit skeletal muscle. The oxygen exchange distributions for ATP and Pi release fit a simple kinetic model with a single set of rate constants for each step. For actomyosin subfragment 1 (20 degrees C, pH 7.0, I = 50 mM), the rate constant governing ATP release is approximately 8 s-1, Pi release is at approximately 60 s-1 and Pi rebinds to an ADP state at greater than 120 M-1 s-1. These rate constants are similar to those that may occur for undistorted cross-bridges within glycerinated rabbit psoas fibers (Bowater, R., Webb, M. R., and Ferenczi, M. A. (1989) J. Biol. Chem. 264, 7193-7201.