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

Abstract

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⁺ (K_Ca2+) 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.