UBC Theses and Dissertations
Protein composition of erythrocytes and skeletal muscle cells in malignant hyperthermia Scrubb, Jennifer Ann
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%) (p<0.05). The proportion of beta-spectrin in fresh ghosts (16.23 + 1.84 %) was not different from that of frozen ghosts from Yorkshire (13.21 + 1.64%) and Pietrain (18.90 + 2.53%)(p>0.05). 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 (p<0.05). The RBC yield, composition and/or amount of degradation were not different in the presence of halothane (7 mM) (p>0.05). 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<0.05). In contrast, there was more of a 34.8 kDa protein in the Pietrain and Heterozygote breeds as compared to the Yorkshire breed (p< 0.05). The RBC results showed that the total protein composition for Yorkshire and Pietrain were qualitatively and quantitatively different when comparing ghosts from fresh and frozen samples. Because the proportion of beta-spectrin was the most stable in these samples and was susceptible to calpain degradation, its use in a diagnostic assay is suggested. The myofibril protein composition differences may be relevant as a diagnostic tool.
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