title: Plasma membrane Ca(2+)-ATPase sulfhydryl modifications: implication for oxidized red cell.
abstract: A common perturbation found in cells under oxidative stress is alteration in cellular Ca2+ homeostasis. In order to understand the effects of such oxidative damage, human red cell plasma membrane Ca(2+)-ATPase (PMCA) was studied by measuring PMCA activity, both in the presence and absence of calmodulin (CaM), following treatment with sulfhydryl agents, N-ethylmaleimide, iodoacetate and diamide. PMCA activity of washed red cell membrane was measured by coupling with pyruvate kinase, using phosphoenolpyruvate as substrate, and lactate dehydrogenase to convert pyruvate to lactate employing beta-NADH as co-factor. All treatments inhibited basal and CaM-stimulated activity in a dose-dependent manner (0.01-1 mM), but at low concentrations, basal Ca(2+)-ATPase activity was inhibited whereas CaM-stimulated activity was unaffected. Inhibition by diamide, a disulfide-forming agent, was reversed with dithiotreitol (DTT). Treatment with calpain, a calcium-dependent protease, elevated basal PMCA activity to CaM-stimulated level, but abolished response to CaM. Further treatment with diamide inhibited PMCA activity, which could be restored by DTT, but only to basal and not CaM-stimulated level. These studies indicated that it is necessary to protect against both sulfhydryl and proteolytic damages to red cell PMCA if perturbation to Ca2+ homeostasis is to be minimized. This has implications for membranes under oxidative stress, such as in the hereditary anemia, thalassemia, where membrane-bound unmatched hemoglobin chains cause oxidative damage to red blood cells.
authors: Department of Biochemistry, Faculty of Science, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
authors: Namphaung
authors: N
authors: Pengpanichpakdee
authors: Tanapon
authors: T
authors: Thadtapong
authors: Saranya
authors: S
authors: Auparakkitanon
authors: Prapon
authors: P
authors: Wilairat
mesh_terms: D002147:Calmodulin / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism
chemical_list: D002147:Calmodulin; D013438:Sulfhydryl Compounds; D053499:Plasma Membrane Calcium-Transporting ATPases