T. O. Veklich1, O. A. Shkrabak1, S. O. Kosterin1, R. V. Rodik2, S. O. Cherenok2, V. I. Boyko2, V. I. Kalchenko2
In the experiments carried out with the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution the authors investigated influence of the сalixarenes С-97 and С-107 (codes are shown) on ouabain effect on the Nа+,K+-АТРase activity. It was shown that calixarenes in concentration 100 ?M inhibited by 97–98% the enzymatic Na+,K+-АТРase activity, while they did not practically influence on the basal Mg2+-АТРase activity, and suppressed much more effective than ouabain the sodium pump enzymatic activity: in the case of the action of the calixarenes the value of the apparent constant of inhibition I0.5 was <0.1 ?M while for ouabain it was 15–25 ?M. The negative cooperative effect was typical of the inhibitory action of сalixarenes, as well as ouabain: the value of Hills factor nH?=?0.3–0.5 <1. The modelling compound М-3 (0.1 ?M?– 4??M) – a fragment of the calixarene С-107 – did not practically influence the enzymatic activities as Na+,K+-АТРase and basal Mg2+-АТРаse. Hence the influence of calixarene С-107 on the Na+, K+-АТРаse activity is caused by cooperative action of two fragments М-3 and effect of calixarene bowl, rather than by simple action of the fragment М-3. Calixarenes С-97 and С-107, used in concentration corresponding to values of I0.5 (40 and 60?nM, accordingly), essentially stimulated inhibiting action of ouabain on the specific Na+, K+-АТРаse activity in the memrane fraction. Under coaction of ouabain with calixarene С-97 or С-107 there was no additive effect of the action of these inhibitors on the Nа+,K+-АТРase activity. Calixarene С-97 brought in the incubation medium in concentration of 10 nM not only led to inhibition of the Na+,K+-АТРase activity relative to control, but also simultaneously increased the affinity of the enzyme for the cardiac glycoside: the magnitudes of the apparent constant of inhibition I0.5 were 21.0?± 5.2 ?M and 5.3 ± 0.7 ?M. It is concluded, that highly effective inhibitors of the Na+,K+-АТРase activity – сalixarenes С-97 and С-107 can enhance the effect of the sodium pump conventional inhibitor – ouabain, increasing the affinity of the enzyme for the cardiac glycoside (on the example of calixarene С-97).
Key words: Na+,K+-ATPase, Mg2+-ATPase, plasma membrane, smooth muscle cells, myometrium, enzymatic hydrolysis of ATP, kinetic properties of ATPase, calixarenes, complexones, methylene-bis-phosphonic acid, aminophosphonic acid, hydroxyphosphonic acid.
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