The temperature dependence of ATPase activities and stearic acid spin label motion in red blood cells of normal and MH-susceptible pigs have been examined. Arrhenius plots of red blood cell ghost Ca-ATPase and calmodulin-stimulable Ca-ATPase activities were identical for both normal and MH erythrocyte ghosts. Arrhenius plots of Mg-ATPase activity exhibited a break (defined as a change in slope) at 24°C in both MH and normal erythrocyte ghosts. However, below 24°C the apparent activation energy for this activity was less in MH than normal ghosts. To determine whether breaks in ATPase Arrhenius plots could be correlated with changes in the physical state of the red blood cell membrane, the spin label 16-doxyl-stearate was introduced into the bilayer of both erythrocyte ghosts and red blood cells. With both ghosts and intact cells, at each temperature examined, the mobility of the probe in the lipid bilayer, as measured by electron paramagnetic resonance, was greater in normal than in MH membranes. While there were no breaks in Arrhenius plots for probe motion in the erythrocyte ghosts, the apparent activation energy for probe motion was significantly greater in normal than in MH ghost membranes. While there was no break in the Arrhenius plot of probe motion in normal intact red blood cell membranes, there were breaks in the Arrhenius plot of probe motion at both 24 and 33°C in intact MH red blood cell membranes. Based on the altered temperature dependence of Mg-ATPase activity and spin probe motion in membranes derived from MH red blood cells, we conclude that there may be a generalized membrane defect in MH pigs which is reflected in the red blood cell as an altered membrane composition or organization.
Bibliographical noteFunding Information:
This work was supported in part by the Muscular Dystrophy Association, the American Heart Association, and the NIH (GM 31382, GM 27906, and AM 32961). We acknowledge the invaluable help of Dr. William Rempel, who provided us with the swine used in this study, the excellent technical assistance of Peggy Hogan and Nitin Phadke, and the secretarial help of the Veterinary Biology Office staff.