DEPARTMENT OF MUSCLE BIOCHEMISTRY
Head – Professor SerhiyO. Kosterin,
Dr. Sc. (Biol.)., Academician of the National Academy of Sciences of Ukraine
Tel.:+(044) 234 16 53, E-mail:
The Department of Muscle Biochemistry was created in 1944. Professor David L. Ferdman, Corr. Member of AS of the USSR and Member of AS of the Ukr.SSR, was a founder and permanent head of the Department up to January, 1970. Varvara A. Grygorieva, Cand. Sc. (Biol.), headed the Department from January,1970 to May, 1973, and from May, 1973 the Department was headed by professor Mikhaylo D.Kurskyi, Dr.Sc.(Biol.). In September, 1988, in accordance with the resolution of the Scientific Council, a new Department – the Department of Biochemical Kinetics was created after the staff reorganization of the Department of Muscle Biochemistry. The Department of Biochemical Kinetics was headed by Serhiy O. Kosterin. In July 1996,, after structural reorganization of the Institute, the Department of Muscle Biochemistry was broken up, and the Department of Biochemical Kinetics was renamed into the Department of Muscle Biochemistry.
At present, the staff of the Departments includes 7 Doctors of sciences, 8 Candidates of sciences and 3 Engineers.
Main results (2010-2015)
During 2010-2015 the Department researches were aimed at studying the biochemical mechanisms of excitation-contraction coupling in smooth muscles (mainly – in the smooth muscle of the uterus). In particular, the comparative study of energy-dependent transporting systems of Са and Na ions that were localized in subcellular membrane structures was conducted.
It has been proven that the Са ions induce depolarization of uterus smooth muscle mitochondria membrane. This process is inhibited by ruthenium red, or in case of Mg2+ and АТР presence in the incubation medium. The increase of organic polycation spermine concentration (1-10 mМ) also resulted in depolarization of myometrium mitochondria membrane. The antagonists of calmodulin (trifluoperazine, calmidazolium) inhibited the activity of calcium uniporter and caused mitochondria membrane depolarization.
In rat myometrium mitochondria the existence of K+ transporting system was shown. A suggestion has been made that this system is K+ATP channel which is blocked by glibenclamide and ATP and activated by diazoxide.
It has been shown that nitrogen oxides stimulate myometrium Na+,К+-АТРase activity through cGMP-dependent pathway. In case of the direct action they inhibited Na+,К+-АТРase with high affinity and in concentration-dependent manner according to noncompetitive or mixed mechanism.
A mathematical model that imitates the behavior of smooth muscle cell in conditions of its stimulation by agonist has been created. It is set that, depending on the values of model parameters, it is possible to look after both single splashes of Ca2+ concentration in cytosol and its periodic fluctuation.
In collaboration with employees of the Institute of Organic Chemistry of NAS of Ukraine (V.I.Kalchenko, Corresponding Member of NAS of Ukraine, and colleagues) new data were obtained in relation to the study of kinetic behavior and mechanism of calixarenes effects on АТР-hydrolyzing systems of smooth muscles and on their contractile activity.
Biochemical technologies, necessary for the search, study of properties and action mechanism for new bioactive reversible effectors (on the example of calixarenes, in particular, calix[4]arenes), were worked out. These compounds are potentially possible regulators of ions transmembrane exchange, foremost – ions of Са, in the uterus smooth muscles and of "contraction-relaxation" cycle of the latter.
It was proven that calixarene С-91 stimulated Mg2+,АТР-dependent Ca2+ accumulation in mitochondria, but did not practically influence the activity of calcium pumps of the sarcoplasmic reticulum and plasma membrane. The preliminary incubation of digitonin-permeabilized myometrium cells with calixarenes С-136 and С-137 increased polarization of the inner mitochondrial membrane. Calix[4]arenes С-136 and С-137 possess two chalcone amide moieties at the lower rim. In case of calix[4]arenes С-136 and С-137 take place, accordingly, the absence or presence of phenolic hydroxyl groups at the lower rim on the calix[4]arene skeleton. It was shown that calix[4]arenes С-136 and С-137 form micelles in a water medium and in the dimethylformamide (DМF). The irradiation of micelles with argon laser on flow cytometer results in the appearance of autofluorescence. In the water medium calix[4]arene micelles interact with positively charged potentialsensitive fluorescent probe TMRM, that can testify to the presence of negative charge in these structures. However calix[4]arene micelles in DMF solution do not interact with TMRM. Mitochondrial membrane potential was measured using fluorescent dyes MTG and TMRM with confocal microscopy and fluorescent dye TMRM with flow cytometry. Experiments were conducted on myometrium cells in culture and on suspension of digitoninpermeabilized uterus myocytes. A maximal effect was 173%.
It has been shown, that the laser confocal microscopy method can be used with the aim of visualization of distribution of potential-sensitive fluorescent dye DіО(С6)-3 in the smooth muscle mitochondria. This method was used to confirm the ability of calixarenes С-99 and С-107 to cause the increase of mitochondria membrane potential. These experimental data can be useful for further developments, when studying the role of mitochondria in the control of intracellular calcium homoeostasis in smooth muscles, biochemical aspects of excitation-relaxation coupling in them.
In plasma membranes of the uterus myocytes and suspension of spermatozoa, it was found, that calixarenes С-97 and С-107 considerably more effectively inhibits the activity of Na+,K+-АТРase, than ouabain. At the same time calixarene С-99, that inhibit Na+,K+-АТРase activity of the smooth muscle cells, practically does not influence it in case of spermatozoa. In general, these results indicate, that the mentioned calixarenes take no influence on the "basal" Mg2+-АТРase activity, are effective inhibitors of Nа+,К+-АТРase activity in the plasma membrane (at least, in case of contractive and movable cells).
It was found, that the presence of calixarenes С-99 and С-107, and also of nitrogen oxides, the characteristic sizes of the uterus myocytes, that was measured with the use of photon correlation spectroscopy method, grow. This effect correlates with the ability of the investigated compounds to cause relaxation of the myometrium.
Results obtained in comparative experiments from catalytic titration by calixarenes С-97 and С-99 of actomyosin АТРase and plasma membrane Nа+,К+-АТРase specify a considerably greater sensitiveness of Nа+-pump to these effectors as compared with АТР-ase of the contractive complex. The size of the apparent constant for calixarenes С-97 and С-99 inhibitory effects on actomyosin ATPase (88+9 and 86+8 mkМ accordingly) is by three orders more than in case of plasma membrane Nа+,К+-АТРase (33+4 and 98+8 nМ accordingly). Even in the 100 mkМ concentration calixarenes practically take no influence on activity of "basal" plasma membrane Mg2+-АТРase. The action of calix[4]arenes C-91, C-97, C-99, C-107 and C-160 on solvent-containing planar bilayer membranes made of cholesterol and egg phosphatidylcholine (egg PC) or synthetic 18-carbon-tail phospholipid DOPC has been investigated in a voltage-clamp mode (in cooperation with Dr. O.Shaturskiy). Within the range of calix[4]arenes tested, a steady-state voltage-dependent transmembrane current was achieved only after the addition of calix[4]arene C-99 (calix[4]arene-bis-hydroxymethylphosphonic acid) from the side of the membrane the positive potential was applied to. This current exhibited anion selectivity passing more chloride at negative potentials applied from the side of the membrane to which calix[4]arene C-99 was introduced. The kinetics and temperature dependence determined for calix[4]arene C-99-mediated ionic transport suggest a carrier mode of facilitated diffusion.
It has been proven that calixarene С-90 can be used as selective inhibitor of plasma membrane Са2+,Mg2+-АТРase: value of I0.5 20-30 µМ, the effect on other plasma membrane АТР-hydrolases is practically absent. The influence of calix[4]arene C-90 on the Ca2+,Mg2+-ATPase activity was caused by the cooperative action of four sulfonylamidine substituents on the upper rim of the calixarene bowl. We consider that the calcium pump inhibitor calix[4]arene C_90 will be useful for subsequent investigation of membrane mechanisms of calcium homeostasis in smooth muscle cells and also for investigation of the participation of the calcium pump in electromechanical coupling in myocytes.
The researchers also succeeded to show, that calixarenes take influence on ATPase activity of contractive proteins by cooperation with subfragment-1 of myosin. Using computer design methods (docking, molecular dynamics involving the Grid) the molecular model of complex formation between subfragment-1 of myosin and calixarenes С- 99 and С- 97 has been developed. Structural bases of intermolecular cooperation of calixarenes with subfragment-1 have been found out. It has been shown, that hydrophobic and electrostatic forces have a substantial value in stabilizing of complex "calixaren-subfragment-1", π-π cooperations, and also hydrogen bonds. Amino acid residues that participate in the binding of calixarenes С- 99 and С- 97 with the substrate-binding domain of subfragment-1 of myosin were identified.
And, finally, it was proven that calixarene С-107, functionalized by two tret-butyl and two amino-(2-pyridyl) methylphosphone groups that are in distal positions of overhead crown of calixarene molecule and are able to bind АТР in solutions and to hydrolyze this nucleoside triphosphate.
In a fundamental aspect the accumulated data are substantial for understanding the biochemical and biophysical mechanisms of ionic (foremost calcium) control of functioning of smooth muscles as a complex receptor electromechanical system. In a practical aspect the obtained data is promising for further development of new nontoxic (low-toxic) reversible effectors (on the basis of calix[4]arenes) – selective and high affinity regulators (inhibitors, activators) of ATP-hydrolases catalytic and transport activity. Potentially such effectors can serve as "supramolecular platforms" for creation of medications of new generation - modifiers of activity of АТР-hydrolyzing systems in case of their violation at pathologies (hypo- and hypertonus of the uterus, hypo- and hypertension, changes in the contraction of gastrointestinal, etc.).