ABSTRACT
The effect of divalent cations--Co2+, Cu2+, Mn2+ and Ni2+ (5 mM) on the activity of actomyosin complex ATPase and ATPase of subfragment-1 (S1,head) of myosin from smooth muscle of the uterus was studied. It has been shown that Co2+, Mn2+ and Ni2+ inhibited, while Cu2+ activates the enzyme activity of both actomyosin and myosin S1. Mg and Mn ions had practically no effect on the emission intensity of eosin Y associated with actomyosin, while one could observe the most marked suppression of emission of related fluorescent probe in the presence of Cu cations and less pronounced suppression in the presence of Co2+. In the presence of Mn, Co and Ni cations the average hydrodynamic diameter (HD) of actomyosin complex and of subfragment-1 of the smooth muscle of the uterus is virtually identical to the HD in the presence of Mg2+. In the presence of Cu cations there is a considerable (ten-fold) increase in the size of the protein particles that may be a result of their aggregation. The results obtained evidence for the significant changes in the structure and function of the actomyosin complex of the myometrium in the presence of heavy metals and allow us to assume that the target of the effect of these metals on the contractile proteins is a subfragment-1 of myosin, where the active site of ATPase and actin-binding sites are localized.
Subject(s)
Actomyosin/metabolism , Adenosine Triphosphatases/metabolism , Cations, Divalent/pharmacology , Cytoskeleton/enzymology , Metals, Heavy/pharmacology , Myometrium/enzymology , Myosin Subfragments/metabolism , Actomyosin/chemistry , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Animals , Cations, Divalent/adverse effects , Cytoskeleton/drug effects , Eosine Yellowish-(YS)/analysis , Female , Metals, Heavy/adverse effects , Myometrium/drug effects , Myosin Subfragments/chemistry , Protein Binding , Spectrometry, Fluorescence , Spectrum Analysis , SwineABSTRACT
Influence of dielectric permeability and incubation medium temperature on sensitivity of ATPase of contractile protein complex to eosin Y was studied on preparations of uterine smooth muscle actomyosin. It was shown, that a decrease of dielectric permeability from 74.1 to 68.8 is accompanied by inhibition of activity of actomyosin ATPase by 30%. Administration of eosin Y to the incubation medium intensifies the inhibition of activity of actomyosin ATPase, that correlates with a decrease of dielectric permeability and increase of eosin Y concentration. Catalytic titration of actomyosin ATPase by eosin Y (from 10(-7) to 10(-5) M) on the background of a change of dielectric permeability (from 741 to 68.8) shows, that curve of dependence of ATPase activity on the inhibitor concentration is characterized by a lower value of enzymatic activity and its quicker decay at D 68.8 than at D 74.1. Under a decrease of dielectric permeability from 74.1 to 68.8 the observed decrease of the value of inhibition coefficient from 0.74 +/- 0.07 to 0.10 +/- 0.01 MKM, that can evidence for the essential role of electrostatic interactions in the binding of eosin Y with smooth muscle actomyosin. Studies of energy aspects of the mechanism of an eosin Y action on ATP-hydrolase activity of smooth muscle contractive proteins (determination of temperature dependence and activation energy E(o)) show that the inhibitory effect of eosin Y on myometrium actomyosin ATPase activity is connected with an increase of the reaction energy barrier. That is, the effect of eosin Y on actomyosin ATPase activity is shown not only on catalytic but also on thermodynamic levels.
