The activity and kinetic parameters of endoplasmic reticulum Ca2+,Mg2+-ATPase (SERCA) of blood lymphocytes in patients with ovarian cancer (OC) have been examined. It is known that cancer cells usually have the disturbance of calcium homeostasis. Although the changes of Ca2+-signaling can not be considered as a prerequisite for the initiation of cancer, the consequences of the changes of the structure and functioning of Ca2+-channels or other Ca2+-transporting systems in cancer cells can be significant in the tumor progression. SERCA is an intracellular Ca2+ -store and a multifunctional organelle that performs a variety of Ca2+-dependent functions involved in the homeostatic and signaling mechanisms. Plasma membrane Ca2+, Mg2+-ATPase and endoplasmic reticulum Ca2+,Mg2+-ATPase are two Ca2+-transporting structures which regulate Ca2+-concentration in cytosol. It is assumed that the endoplasmic reticulum Ca2+,Mg2+-ATPase in the lymphocytes plays a leading role in the process of reducing the Ca2+ level in cytosol. It has been found that SERCA activity of blood lymphocytes in the almost healthy individuals has been (2.25 ± 0.17) μmol Рі / min per 1 mg of protein. According to the stats, the SERCA activity of blood lymphocytes in the patients with OC has been significantly different from the control group. Its value has been decreased by 1.5 times (p < 0.001), compared to the almost healthy subjects. The decrease of the plasma membrane Ca2+,Mg2+-ATPase activity that has been demonstrated earlier and in SERCA activity of blood lymphocytes in the patients with OC, indicates the increase of [Ca2+]i in cytosol of lymphocytes.

Both Ca2+,Mg2+-ATPase and Na+,K+-ATPase use the energy of ATP hydrolysis to carry ions against their electrochemical gradient. Therefore, the changed concentration of ATP in the incubation medium may affect the rate of ATP-hydrolysis reaction. The dependence of Ca2+,Mg2+-ATPase activity on the substrate concentration (ATP) in the incubation medium has been determined by the value of the affinity constant of the substrate (KATP). It has been calculated by determining the Ca2+,Mg2+-ATPase activity in the incubation medium containing the substrate in the concentration ranging from 0.1 to 5.0 mM (with a constant concentration of Ca2+ - 0.05 mM and Mg2+- 5 mM).

It has been found that the increase of ATP concentration in the incubation medium in the concentration ranging from 0.1 to 4.0 mM has led to the gradual monotonic increase of SERCA activity of lymphocytes in the almost healthy subjects. The maximum ATP-hydrolase activities of SERCA of blood lymphocytes in the healthy donors and patients with OC have been noticed when ATP concentration in the incubation medium has been 4 mM. Researching of the concentration dependence of Ca2+, Mg2+-ATPase activity on ATP concentration shows that throughout the range of the substrate concentrations, the SERCA activity in the patients with OC has been decreased compared to the control group.

In order to determine the main kinetic parameters of ATP hydrolysis using SERCA and to elucidate a possible mechanism of the change of the enzyme activity of blood lymphocytes in the patients with OC, the curves of concentration dependencies have been depicted on the Lineweaver-Burk plot.

It has been found that the maximum rate of ATP-hydrolysis of SERCA in the healthy subjects has been (2.2 ± 0.2) μmol Рі/min per 1 mg of protein. The maximum rate of ATPase reaction catalyzed by SERCA in the patients with OC has decreased in 1.3 times (p < 0.05) compared to the control group. According the the stats, the affinity constants of SERCA for ATP in both groups have been also significantly different, what indicates their different affinity for the substrate. Specifically, SERCA affinity to ATP in the healthy donors has been (0.13 ± 0.04) mM and its value in the patients with OC has been in 2.15 times higher.

The obtained data indicate that inhibition of SERCA activity in lymphocytes occurs caused by both reduction of the maximum rate of ATP hydrolysis and reduction of SERCA affinity for the substrate (the affinity constant for ATP has been increased).

Article recieved: 08.05.2018

Ключові слова: ovarian cancer, lymphocytes, endoplasmic reticulum, Ca2+, Mg2+-ATPase

Повний текст: PDF (Ukr)

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