Stability of rainbow trout (Salmo gairdneri) muscle lysosomes Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/qf85nf666

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  • Studies were conducted to determine the post-mortem stability of lysosomes in the white muscle of rainbow trout (Salmo gairdneri) and to determine whether lysosomal stability was related to rigor mortis and its subsequent dissolution. In addition, isolated lysosomes were subjected to different environments in an attempt to detect some of the factors that might influence their stability. After slaughter, fish were aged at either 4 or 15. Muscle samples were excised from each fish 1 hr post-mortem, during rigor mortis, soon after the dissolution of rigor mortis, and after a period of aging. Lysosonnes were extracted by blending the muscle in 0. 25 M sucrose-0.175 M KC1-1 mM EDTA in a solution:muscle ratio of 4:1. After blending, the mixture was centrifuged at 1000 x G to sediment the nuclear-debris pellet, followed by centrifugation of the supernatant at 27,000 x G to obtain a lysosomal pellet and a supernatant containing the soluble enzymes. The distribution of the lysosomal enzymes, cathepsin and α-glucosidase, was measured in each of the above fractions at each sampling interval. The stability of lysosomes was thus followed by monitoring the release of these lysosomal enzymes with time. The lysosomal pellet extracted at 1 hr post-naortem contained 23% of the catheptic activity of the muscle. This percentage decreased to approximately 15% by the end of rigor mortis with small changes noted thereafter. The nuclear-debris pellet contained a relatively constant amount of catheptic activity, generally 55-65%, while the soluble activity increased with decreasing lysosomal activity. Approximately one-third of the extractable lysosomes appeared to rupture and release catheptic enzymes before or during rigor mortis. Freezing and thawing the muscle four times reduced the catheptic activity in the lysosomal pellet to 8% of the total activity and increased the soluble activity to 51%. Isolated lysosomes rapidly released their enzymes in response to environmental conditions, but once adjusted they were relatively stable. Seventy-five percent of the cathepsins from lysosomes suspended in 0.25 M sucrose-0.005 M phosphate buffer (pH 6.5) at 4° remained sedimentable for 105 hr. Increasing the sucrose concentration toward 0.5 M and decreasing the incubation temperature increased the stability. The minimum release of the enzymes occurred between pH 6 and 7. Addition of salts (NaCl, KCl, CaCl₂) decreased the initial solubilization of the enzymes, but appeared to increase solubilization over a 70 hr incubation. Ca⁺² was more effective in producing this pattern than Na⁺. Fish saline had little protective effect relative to distilled water. The detergent Triton X-100 caused nearly complete solubilization of the enzyme. The solubilization of α-glucosidase in response to incubation conditions paralleled that of cathepsin but was invariably greater. This suggested that the enzymes have different binding affinities with the membrane surface. However, when the changing distribution of the two enzynnes was determined in aging muscle, an identical pattern resulted.
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