Temperature-Sensitivity of Sodium-Potassium ATPase Activity in Crab Hearts
Luis Arreguin
Department of Chemistry & Biochemistry
Faculty Supervisor: Jonathon Stillman
The study goal is to determine the exact failure temperature of sodium potassium ATPase in hearts of the intertidal zone porcelain crab, Petrolisthes cinctipes. Frozen crab hearts were weighed and homogenized in 75-200 volumes of homogenization buffer, briefly centrifuged and the supernatant used in assays. Assays consisted of ATP, KCN to inhibit mitochondrial ATPases, and some tubes of assay cocktail contained ouabain, a selective inhibitor of sodium-potassium ATPase. To commence the reaction, a small volume of each homogenate was added to each assay tube. Assay tubes were incubated at 10 different temperatures at ~0.9 °C intervals between 25.3 and 34.4°C. After incubation, the assay tubes were frozen and stored at -80°C until use in a malachite green-based colorimetric assay to measure inorganic phosphate (Pi). Colorimetric assays were done using a series of Pi standards in a Tecan Infinite 200 microplate reader at 595nm. Gradually, with temperature ATPase activity increases till a peak at 32.5°C,however a drop at higher temperatures citing failure of ATPase activity. From the two assays with and without ouabain with different Pi concentrations verifies that decline was specific to sodium-potassium ATPase.