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Protein composition of erythrocytes and skeletal muscle cells in malignant hyperthermia Scrubb, Jennifer Ann

Abstract

The purposes of this study were to; 1. compare the protein composition of erythrocyte (RBC) ghosts from whole blood (fresh and frozen) and frozen ghosts extracted from Yorkshire (Y) and Pietrain (P) swine and 2. determine the amount of calpain-mediated degradation of beta-spectrin from Yorkshire and Pietrain ghosts. In addition protein composition of purified myofibrils from Yorkshire, Heterozygote (H) and Pietrain skeletal muscle was compared. Longissimus Dorsi (LD) muscle and whole venous blood was obtained from Yorkshire (N=10), Pietrain (N=10), and Heterozygote (N=8) fasted swine (50-70 kg) housed at the University of Guelph (Guelph, Ontario) experimental farm. RBCs were hemolysed in a hypotonic buffer (imidazole, EDTA, PMSF) and ghosts were obtained by ultracentrifugation and stored at -70°C. Myofibrils were prepared by differential centrifugation and stored at -70°C. The exogenous low calcium requiring isoform of calpain (uCANP) was prepared from rabbit skeletal muscle by anion exchange chromatography and purified on a phenylsepharose column. RBC protein yields were 1.4 7 + 0.3 5 (standard deviation) and 1.59 + 0.24 mg/g for frozen Yorkshire and Pietrain ghost samples (p>0.05). The protein composition (% of total protein) of frozen Yorkshire alpha-spectrin (6.62 + 0.58%), anion channel (25.31 + 6.06%) and actin (25.44 + 0.00) and frozen Pietrain alpha- spectrin (19.81 + 2.4%), anion channel (10.59 + 1.58%), and actin (32.79 + 2.63%) were different from fresh Yorkshire ghosts (alpha-spectrin = 25.88 + 1.82%, anion channel = 23.27 + 1.32%, actin = 3.71 + 0.43%) (p0.05). Calpain mediated degradation (1.5 U/ml) of beta-spectrin associated with the ghosts was not different after 60 minutes for Yorkshire and Pietrain samples (p>0.05); however, the percentage of beta-spectrin degraded between 10 and 60 minutes was greater for Pietrain (p0.05). Myofibril protein yields of Yorkshire, Heterozygote, and Pietrain samples were not significantly different (p>0.05). With respect to myofibril protein composition a 35.9 kDa protein was less in the Pietrain and Heterozygote as compared to the Yorkshire (p

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