KINETICS OF INHIBITORY EFFECT OF HYDROGEN PEROXIDE ON ACTIVITY OF PLASMA MEMBRANE TRANSPORTING Cа2+, Mg2+-ATPase OF SPERM CELLS

Decreased potential of spermatozoa fertility is closely associated with the development of oxidative stress and dysfunction of ion-transporting ATPases. Oxidative stress may have negative impact on the activity of membrane-bound enzymes, such as Са2+,Мg2+-АТPase that is involved in maintaining calcium homeostasis in sperm cells. The aim of present work was to evaluate the exogenous H2O2 effect on the main kinetic parameters of ATP hydrolysis by plasma membrane Са2+,Мg2+-АТPase of spermatozoa of fertile (normozoospermia) and infertility (asthenozoospermia) men. Since Са2+,Mg2+-АТPase is one of the targets for the reactive oxygen species and is directly involved in oxidative stress, spermatozoa obtained from normo- and asthenozoospermic samples were subjected to oxidative stress in the form of exogenous H2O2. Then ATP hydrolysis by thapsigargin-resistant Ca2+,Mg2+-ATPase in media with different Ca2+ concentrations was measured. An effective inhibitory effect of H2O2 on the activity of the thapsigargin-resistant component of Са2+,Мg2+-АТPase of sperm cells was demonstrated. In order to elucidate possible mechanisms of change in Ca2+,Mg2+-ATPase activity under H2O2-induced oxidative stress, the concentration curves were linearized using Hanes–Woolf plot {[S]/V; [S]}. The apparent activation constant for Ca2+ (KCa2+) in sperm cell obtained from men with proven fertility was not changed, whereas in the asthenozoospermic samples, it was decreased almost twice under H2O2-induced oxidative stress. These results indicate that in normozoospermic samples H2O2 implements its inhibitory action through the mechanism of uncompetitive inhibition of plasma membrane Ca2+,Mg2+-ATPase activity. According to formal features of kinetics in the asthenozoospermic samples a mixed type of enzyme inhibition occurs under the oxidative stress induced by H2O2. Strategies to protect against a loss in Cа2+,Mg2+-ATPase activity may be useful to prevent the harmful biochemical cascades leading to Ca2+ overload and dysfunction of spermatozoa as a result of the oxidative stress.