Date of Award
Doctor of Philosophy
F. C. Parrish
The protease [mu]-calpain degrades the myofibrillar proteins titin, nebulin, filamin, desmin and troponin-T. Titin and nebulin have been reported to be affected by sample preparation. This study showed that there was little difference in the rate of degradation of the intact forms due to sample preparation (whole muscle preparations vs. purified myofibrils). The postmortem degradation of titin, nebulin, filamin, desmin, and troponin-T was monitored using SDS-PAGE and Western blotting techniques at 0, 1, 3, 7, 14, 28, and 56 days postmortem. Postmortem aged samples with lower shear force values exhibited faster degradation of the five myofibrillar proteins. Similar degradation products were detected in Western blots of [mu]-calpain digested myofibrils incubated at 4° C, pH 5.6, 165 mM NaCl, and 100 [mu] M Ca[superscript]2+. When [mu]-calpain was incubated with purified myofibrils at 4° C and 100 [mu]M CaCl[subscript]2 and (1) pH 7.0, 165 mM NaCl, (2) pH 7.0, 330 mM NaCl, (3) pH 5.6, 165 mM NaCl, (4) pH 5.6, 330 mM NaCl, [mu]-calpain degraded titin, nebulin and desmin faster than it degraded filamin and troponin-T under all four conditions. The degradation of myofibrillar proteins in general by [mu]-calpain was greatest at pH 7.0, 165 mM NaCl, and was lowest at pH 7.0, 330 mM NaCl. The proteins troponin-T and filamin were not noticeably degraded under the conditions of pH 7.0, 330 mM NaCl. When the same four pH and ionic strength conditions were examined at 37° and 100 [mu]M CaCl[subscript]2, there was no detectable degradation at pH 7.0, 165 mM NaCl or at pH 7.0, 330 mM NaCl. When the CaCl[subscript]2 concentrations were raised to 200 [mu]M, rapid degradation of the majority of the five proteins was noted. This study illustrates that under conditions of low temperature and pH, [mu]-calpain can produce many of the same degradation products that are seen under normal postmortem aging conditions. Physiological temperatures and pH in the presence of 100 mM CaCl[subscript]2 appear to inhibit the action of [mu]-calpain against these five myofibrillar proteins, illustrating the need to carefully evaluate incubation conditions on the action of [mu]-calpain.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Elisabeth Jane Huff Lonergan
Lonergan, Elisabeth Jane Huff, "The relationship between the calpain enzyme system and the postmortem degradation of selected myofibrillar proteins " (1995). Retrospective Theses and Dissertations. 10928.