UBC Theses and Dissertations
The influence of gamma radiation on catheptic activity and on ultrastructure of lysosomes and postmortem skeletal muscle of poultry (Gallus domesticus) Ali, Mumtaz
A three part study is presented dealing with radiation-induced release of cathepsins from isolated lysosomes, irradiation inactivation of cathepsins, and ultra-structural changes in irradiated lysosomes and skeletal muscle. Chicken liver lysosomes were isolated by differential centrifugation and sucrose density gradient technique. Isolated lysosomes were irradiated with doses in the range of 0.10 to 1.0 Mrad of gamma radiation. Irradiation resulted in a decrease in absorbance (540 nm) of lysosomal suspensions incubated at 37°C and a corresponding pronounced increase in free enzyme activity due to release of cathepsins. Rate of release of cathepsin D from lysosomes was considerably slower when incubated at 4°C compared with 37°C. Cathepsins A, B, C, and D showed a differential release under the influence of gamma radiation. Cathepsins C and D were more readily released as compared with cathepsins A and B. After 72 hours of incubation at 4°C, free activity of cathepsins A, B, C, and D in 1.0 Mrad irradiated lysosomal suspensions reached 80.2, 70.5, 85.5, and 81.6 percent of total enzyme content; free enzyme activity of control samples was 14.0, 16.7, 27.7, and 26.6 percent respectively. Total activity of cathepsins A, B, C, and D declined as a result of irradiation, due to apparent partial in-activation of the enzymes. Cathepsin A was most radiation resistant and cathepsin B was comparatively sensitive to radiation inactivation. Cathepsins exhibited higher radiation resistance when irradiated in lysosomal suspension compared with soluble enzyme form. Radiation sensitivity of cathepsins was higher at pH 4.0 and 5.5 compared with pH 7.0 and 8.5. Irradiation induced changes in hemoglobin substrate, rendering it resistant to catheptic digestion. Changes in electrophoretic pattern as well as visible spectra of irradiated hemoglobin were indicative of alterations in the substrate. Isolated chicken liver lysosomes and chicken pectoralis muscle were subjected to 1.0 Mrad of gamma radiation. Sections of embedded samples were studied by transmission electron microscopy and surface ultrastructure details were examined by scanning electron microscopy. Irradiation enhanced the release of inner dense material from lysosomes. After 4 8 and 72 hours of incubation most of the irradiated particles appeared as hollow rings of lysosomal membrane. In some irradiated particles, leakage of lysosomal contents from "weak points" in the lysosomal membrane was observed. There were indications that irradiation weakened the membrane structure which caused leakage of material from lysosomes and eventual disruption of particles on prolonged incubation. Scanning electron microscopy provided further evidence that lysosomal material leaked from "weak points" in the lysosomal membrane causing lysosomes to appear as "empty sacks" rather than totally disrupted particles. Transmission electron microscopy of cryofractured skeletal muscle revealed that irradiation caused an increase in interfibrillar spaces and some breaks in the myofibres especially at the I band region. Scanning electron micrographs of irradiated muscle showed fissures between the myofibrils. Control samples had a smooth surface at the transverse breaks, while irradiated tissue had spike-like structures at the surface of these breaks. These studies provide evidence that irradiation caused structural changes in lysosomes resulting in increased leakage of lysosomal contents and release of lysosomal enzymes. Structural changes at the fibre and fibril level of irradiated muscle are also indicated. The effects are likely fundamental to textural alterations of muscle subjected to post-mortem irradiation.