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. / Graduation date: 1974
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/26949 |
| Date | 30 November 1973 |
| Creators | Whiting, Richard Charles |
| Contributors | Anglemier, Allen F. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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