Tag Archives: Na(+)-K(+)-ATPase
Kinetic properties of Na(+),K(+)-АТРase of spermatozoa from fertile and infertile men under effect of calix[4]arene C-107
R. V. Fafula, O. I. Meskalo, A. S. Besedina,
Io. A. Nakonechnyi, D. Z. Vorobets, Z. D. Vorobets
Danylo Halytsky Lviv National Medical University, Ukraine;
e-mail: kaf_medicalbiology@meduniv.lviv.ua; roman_fafula@ukr.net
Received: 12 November 2018; Accepted: 14 March 2019
The calix[4]arene C-107 (5,17-diamino(2-pyridyl)methylphosphono-11,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxy-calix[4]arene) effects on the kinetic properties of Na+,K+-ATPase in spermatozoa of fertile (normozoospermia) and infertility men (oligozoospermia, and asthenozoospermia) were studied. It was shown that in spermatozoa of healthy men calix[4]arene С-107 inhibited Na+,K+-ATPase activity and decreased the maximum reaction rate of ATP hydrolase reaction without affecting the coefficient of (half-) activation by ATP and Hill coefficient nH. In оligo- and asthenozoospermic samples of spermatozoa almost a 2-fold decrease of cooperativity coefficient nH of ATPase inhibition with calyx[4]aren C-107 was observed. In normozoospermic samples of spermatozoa the KMgCl2 for Na+,K+-ATPase was decreased at calix[4]arene C-107 high concentrations (≥50 nM) in the incubation medium in contrast to oligozoospermic samples of spermatozoa where KMgCl2 was increased only at high calix[4]arene C-107 concentration (100 nM). The increase of the KMgCl2 in the entire range of investigated calix[4]arene concentrations and the decrease of cooperativity coefficient nH of MgCl2 activating effect were detected in asthenozoospermic samples of Na+,K+-ATPase.
The calixarene C-107 increases the affinity of the Na(+),K(+)-АТРase activity in plasmatic membrane of smooth muscle cells to the ouabain
T. O. Veklich1, A. A. Shkrabak1, R. V. Rodik2,
V. I. Kalchenko2, S. O. Kosterin1
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: kinet@biochem.kiev.ua;
2Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: vik@ioch.kiev.ua
In the experiments carried out with the suspension of the myometrium cell plasmatic membranes treated with 0.1% digitonin solution we investigated the influence of сalixarene С-107 (5,17-diamino(2-pyridyl)methylphosphono-11,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxycalix[4]arene) on the Nа+,K+-АТРase activity. It was shown that this calixarene increased the affinity of the enzyme for the sodium pump conventional inhibitor – ouabain: the magnitudes of the seeming constant of inhibition I0.5 changed from 26.9 ± 1.3 mM to 10.9 ± 0.6 mM. However the ouabain itself did not influence on the affinity of the Nа+,K+-АТРase for сalixarene С-107.
Comparative investigation of the effect of calix[4]arene C-99 and its analogs on Nа(+),K(+)-ATPase activity of uterus myocite plasma membrane
T. O. Veklich1, A. A. Shkrabak1, S. O. Cherenok2,
V. I. Kalchenko2, S. O. Kosterin1
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: kinet@biochem.kiev.ua; vik@ioch.kiev.ua
The aim of our investigation was to determine structural features of calix[4]arene C-99 which are important for its inhibition properties relative to Nа+,K+-ATPase of uterus myocite plasma membrane. Therefore we studied the effect of calix[4]-arenes С-296, С-297, С-424, С-425, С-426, С-427, which are structurally similar to this inhibitor, on the mentioned enzyme activity. We have shown that calixarenes С-296 and С-297 which have two additional propoxy groups on the lower rim of macrocycle are less effective inhibitors of Nа+,K+-ATPase relative to calixarene C-99. Calixarenes С-425 and С-427 which have on the upper rim of macrocycle three and four phosponic residues, respectively, also inhibit Nа+,K+-ATPase activity less effectively as compared to calixarene C-99. Both calixarenes: С-424, which has only two carbonate residues on the upper rim, and С-426, which has on the upper rim ketomethilphosphonate residues instead of hydroxymethilphosphonate residues of calixarene C-99, do not affect Nа+,K+-ATPase activity. We have made respective conclusions concerning the role of certain chemical groups of calixarene C-99 during its interaction with Nа+,K+-ATPase.
Kinetic properties of Na(+), K(+)-activated, Mg(2+)-dependent ATP-hydrolysis of blood lymphocytes in patients with rheumatoid arthritis and ankylosing spondyloarthritis
R. V. Fafula, U. P. Efremova, N. E. Lychkovska, Z. D. Vorobets
Danylo Halytski Lviv National Medical University, Ukraine;
e-mail: roman_fafula@ukr.net; vorobets@meduniv.lviv.ua
The comparative analysis of the kinetic properties of ouabain-sensitive Na+, K+-ATPase activity of saponin-perforated blood lymphocytes of donors and patients with rheumatoid arthritis (RA) and ankylosing spondyloarthritis (AS) was carried out. When analyzing the alterations in hydrolase activity of the examined enzyme it was shown that in the blood lymphocytes of patients with RA and AS the primary active transport of Na+ and K+ ions is less intensive in comparison with practically healthy donors, but it is characterized by almost the same capacity as in donors. The affinity constant of Na+, K+-ATPase for ATP in the blood lymphocytes in patients with RA and AS is greater 3.1 and 2.5 times, respectively, in comparison with healthy donor. It was found that in conditions of rheumatic pathology in immunocompetent cells the inhibition of Na+, K+-ATPase activity is not related to the reduction of maximum reaction rate, but is related to the decrease of Na+, K+-ATPase affinity to ATP. However, Mg2+-binding center of Na+, K+-ATPase in patients with RA and AS remains native. It was identified that the affinity constant of Na+, K+-ATPase to Na+ ions in the blood lymphocytes of patients with RA and AS is 2.75 times lower than its value in healthy donors. Na+, K+-ATPase of the blood lymphocytes of patients with RA and AS retains its native receptor properties and sensitivity to ouabain does not change.
Na(+), K(+)-ATPase, endogenous cardiotonic steroids and their transducing role
O. V. Tsymbalyuk1, S. O. Kosterin2
1Taras Shevchenko Kyiv National University, Ukraine;
2Palladin Institute of Biochemistry, National Academy of Science of Ukraine, Kyiv;
e-mail: otsimbal@univ.kiev.ua
Na+, K+-ATPase – a protein complex of plasmatic membrane, which performs the dual function: firstly, it supports the Na+ and K+ homeostasis, and also transmembrane potential gradient, secondly, it serves as the transducer of signals and as the regulator of the expression of many key genes. Endogenous cardiotonic steroids, which are synthesized in the adrenal glands and hypothalamus, serve as the signal molecules. New concepts about the mechanisms of the realization of the Na+, K+-ATPase signal function and their connection with cellular functions, apoptosis, and with pathologies of cardiovascular system and water-salt homeostasis are described in the survey.
The сalix[4]arene C-107 is highly effective supramolecular inhibitor of the Na+,K+-АТРase of plasmatic membrane
O. V. Bevza1, T. O. Veklich1, O. A. Shkrabak1, R. V. Rodik2, V. I. Kalchenko2, S. O. Kosterin1
1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: kinet@biochem.kiev.ua;
2Institute of Organic Chemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: vik@bpci.kiev.ua
The inhibition of the Na+,K+-АТРase activity of the myometrium cell plasma membranes with calixarene С-107 (5,17-diamino(2-pyridyl)methylphosphono-11,23-di-tret-butyl-26,28-dihydroxy-25,27-dipropoxycalix[4]arene) was investigated. It has been shown that calixarene С-107 reduced the Na+,K+-АТРase activity more efficiently than ouabain did, while it did not practically influence the “basal” Mg2+-АТРase activity of the same membrane. The magnitude of the cofficient of inhibition I0.5 was 33 ± 4 nМ, Hill coefficient was 0.38 ± 0.06. The model calixarene C-150 – the calixarene “scaffold” (26,28-dihydroxy-25,27-dipropoxycalix[4]arene), and the model compound М-3 (4-hydroxyaniline(2-pyridine)methylphosphonic acid) – a fragment of the calixarene С-107, had practically no influence on the enzymatic activity of Na+,K+-АТРase and Mg2+-АТРаse. We carried out the computer simulation of interaction of calixarenes C-107 and the mentioned model compound with ligand binding sites of the Na+,K+-АТРase of plasma membrane and structure foundation of their intermolecular interaction was found out. The participation of hydrogen, hydrophobic, electrostatic and π-π (stacking) interaction between calixarene and enzyme aminoacid residues, some of which are located near the active center of Na+,K+-АТРase, was discussed.
The influence of iron ions on ATP-hydrolases activity of cell membranes of rat colon smooth muscle and kidney
A. A. Kaplia
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: kaplya@biochem.kiev.ua
To elucidate the specific features of the АТР- hydrolases structural resistance in the membrane under the action of the prooxidants: Fe2+ and hydrogen peroxide, and N-ethylmaleimide (NEM) the colonic smooth muscle (CSM) Na+,K+-AТРase activity was compared with activities of the corresponding Mg2+-АТР-hydrolase and ATP-ases from kidney medullar layer of rats. The inhibition study of the CSM Na+,K+-AТРase by divalent iron shows the decrease of the activity by 30% at 0.1 µM FeSO4 and in the range of 0.1-10 µM – to 45% of residual activity. When comparing with kidney enzyme (represents exclusively α1-isozyme) the CSM Na+,K+-AТРase sensitivity to Fe2+ is reliably higher at its submicromolar concentration. CSM Mg2+-АТРase is much more resistant to iron ions effect, than kidney one. However for two tissues Mg2+-АТРase activity is always more resistant as compared with corresponding Na+,K+-AТРase activity. Against 1 mM EGTA Na+,K+-AТРase and Mg2+-АТРase activities of GMOK and kidneys are equally insensitive to effect of hydrogen peroxide in concentration up to 1 mM. But in the presence of 20 µM FeSO4 in the concentration range of 1 nМ – 1 mM of Н2О2 the Na+,K+-AТРase is inhibited to greater extent, than Mg2+-АТРase activity. NEM sensitivity of the two АТР-hydrolase systems corresponds to prooxidant sensitivity that indicates the distinct importance of SH-groups for their functioning. It is concluded that Na+,K+-AТРase can serve as a marker of membrane sensitivity to oxidation, Mg2+-АТРase is resistant to oxidation and can be considered as criterion of the oxidation resistance when comparing membrane enzyme complexes, especially in GMOK.