UBC Theses and Dissertations
The relationship between Mg+Ca-AtPase and active calcium transport in researled human erythrocyte ghosts Quist, E. E. (Eugene Edwin)
Human red blood cell ghosts were prepared by a modification of the procedure of stepwise hemolysis (57). EDTA (1.0 mM) was included in the washing procedure to remove endogenous ATP and divalent cations. Ghosts resealed with appropriate amounts of ATP, calcium and magnesium were found to have Mg+Ca-ATPase activity and linearity was maintained up to thirty minutes. Active calcium transport could be studied in these ghosts by measuring the change ln the cellular concentration of calcium over time by atomic absorption spectrophotometry. Variation in the concentration of calcium in the loading medium resulted in an activation of Mg+Ca-ATPase and two peaks were evident on the activation curve. The high and low affinity Mg+Ca-ATPase were maximally stimulated at 0.25 and 5.0 mM calcium in the loading medium,respectively. The velocity of calcium transport was also found to be dependent on the concentration of calcium in the loading medium and was activated over the concentration range of 0.1 to 5.0 mM calcium. A change in the concentration of cellular calcium was not evident in the absence of added ATP. In contrast to the activation of Mg+Ca-ATPase two peaks were not obtained, and the activation curve had a sigmoidal appearance. Comparison of the calcium activation curves of Mg+Ca-ATPase and calcium transport revealed, a similarity in the shape and position of the low affinity part of the Kg+Ca-ATPase and calcium transport activation curves. A stoichiometry of two (Ca :ATP) was obtained, in the low affinity activity range. Ruthenium red (0.05 to 0.4 mM) selectively inhibited the low affinity Mg+Ca-ATPase and inhibited calcium transport over the same concentration range to a similar degree. Both low affinity Mg+Ca-ATPase and calcium transport were inhibited by external ruthenium red with an I₅₀ of 0.2 mM. Propranolol, qulnidine and quinine (10⁻⁵ to 10⁻³M) were found to be Ineffective in stimulating or Inhibiting Mg+Ca-ATPase when added to the Internal and external aspects of the ghosts. Manganese, added to the loading medium over a wide concentration range, was unable to substitute for calcium in activating Mg+Ca-ATPase. External divalent cations calcium and magnesium further increased Mg+Ca-ATPase activities when added to the external medium. Maximal stimulation occurred at a concentration of approximately 3.0 mM and calcium was almost twice as effective as magnesium.
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