Abstract
Glucose-6-phosphate dehydrogenase (G6PDH) gates flux through the pentose phosphate pathway and is key to cellular antioxidant defense due to its role in producing NADPH. Good antioxidant defenses are crucial for anoxia-tolerant organisms that experience wide variations in oxygen availability. The marine mollusc, Littorina littorea, is an intertidal snail that experiences daily bouts of anoxia/hypoxia with the tide cycle and shows multiple metabolic and enzymatic adaptations that support anaerobiosis. This study investigated the kinetic, physical and regulatory properties of G6PDH from hepatopancreas of L. littorea to determine if the enzyme is differentially regulated in response to anoxia, thereby providing altered pentose phosphate pathway functionality under oxygen stress conditions. Several kinetic properties of G6PDH differed significantly between aerobic and 24 h anoxic conditions; compared with the aerobic state, anoxic G6PDH (assayed at pH 8) showed a 38% decrease in Km G6P and enhanced inhibition by urea, whereas in pH 6 assays Km NADP and maximal activity changed significantly between the two states. The mechanism underlying anoxia-responsive changes in enzyme properties proved to be a change in the phosphorylation state of G6PDH. This was documented with immunoblotting using an anti-phosphoserine antibody, in vitro incubations that stimulated endogenous protein kinases versus protein phosphatases and significantly changed Km G6P, and phosphorylation of the enzyme with 32P-ATP. All these data indicated that the aerobic and anoxic forms of G6PDH were the high and low phosphate forms, respectively, and that phosphorylation state was modulated in response to selected endogenous protein kinases (PKA or PKG) and protein phosphatases (PP1 or PP2C). Anoxia-induced changes in the phosphorylation state of G6PDH may facilitate sustained or increased production of NADPH to enhance antioxidant defense during long term anaerobiosis and/or during the transition back to aerobic conditions when the reintroduction of oxygen causes a rapid increase in oxidative stress.
Highlights
Glucose-6-phosphate dehydrogenase (G6PDH; E.C. 1.1.1.49) catalyzes the rate determining step of the pentose phosphate pathway (PPP) and is the first of two oxidative steps within that pathway: D-glucose-6-phosphate + NADP+ → D-6-phosphoglucono-δ-lactone + NADPH + H+.The pentose phosphate pathway has several important functions in cells including providing sugars for the synthesis of aromatic amino acids and nucleotides and producing NADPH reducing equivalents that are used in biosynthesis and antioxidant defense
Anoxia-induced enhancement of antioxidant defenses appears to result from the need to cope with a rapid, large increase in reactive oxygen species (ROS) formation associated with arousal from hypometabolism and the return to oxygen-based metabolism (Hermes-Lima, Storey & Storey, 1998) but a second reason may be involved
More recent work suggests that well-developed antioxidant defenses are important for long term protection of macromolecules during anaerobsis because the potential for degradation and resynthesis of oxidatively-damaged proteins is much reduced in the hypometabolic state (Storey & Storey, 2012)
Summary
The pentose phosphate pathway has several important functions in cells including providing sugars for the synthesis of aromatic amino acids and nucleotides and producing NADPH reducing equivalents that are used in biosynthesis and antioxidant defense. With respect to antioxidant defense the NADPH generated by the pentose phosphate pathway is the main source of cellular reducing equivalents that are used to regenerate the reduced forms of glutathione and thioredoxin after they have been oxidized in antioxidant reactions (Riganti et al, 2012). Protection against oxidative damage is important in organisms that experience wide fluctuations in environmental oxygen content, to deal with transitions from low to high oxygen because a rapid increase in oxygen availability and/or consumption is paralleled by a comparable increase in reactive oxygen species (ROS) generation (Hermes-Lima, Storey & Storey, 1998). When oxygen becomes available again, oxgen availability, oxygen consumption and ROS production all rise rapidly
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