Abstract
Chicken myoblasts were cultured from the pectoralis muscles of dystrophic and normal 11-day-old embryos. Cells were allowed to grow to fusion (differentiation) and exposed to [35S]methionine for a short period. Subsequently, the decay of labeled proteins in the presence of cycloheximide was measured for various cellular fractions as well as individual proteins isolated from the sarcoplasmic reticulum and separated by gel electrophoresis. Some dystrophic material showed an increased decay when compared to normal material. The most significant (p less than 0.005) difference was found in a Mr = 65,000 component of the sarcoplasmic reticulum. This same component accumulates label at an accelerated rate in the presence of the protease inhibitor leupeptin. Increased turnover of this protein, possibly calsequestrin, may be a manifestation of the genetic disease.
Highlights
Cells were allowed to grow to fusion the possibility that abnormally functioning proteins contained and exposedto [“Slmethionine for a short period
Sequently, the decay of labeled protieninths e presence We proposed to investigate the process of development which of cycloheximide was measuredforvariouscellular involves myoblast fusion in cell cultures for evidence of abfractions as well as individual proteins isolated from the sarcoplasmic reticulum and separatedby gel electrophoresis
The unusualkinetics of synthesisand decay of a specific protein component of the sarcoplasmic reticulum which may result from agenetic alteration
Summary
Chemi~ak-[”~S]methionine (500-780 Ci/mmol) was obtained from New England Nuclear. Soybean trypsin inhibitor and marker proteins for electrophoresis standard were obtained from Sigma. Samples were taken of the cell homogenate for counting and for SDS-polyacrylamide gel electrophoresis. One-pl samples of the cell homogenates and isolated membrane fractions were spotted onto glass fiber fdters, and proteins were precipitated by boiling in 10%. They were subsequently layered onto a 7.5 to 15% gradient slab gel and subjected to electrophoresis according to the method of Laemmli [16]. The slab gels were subse- lane has incorporated label a t a greater rate than the normal tissue quently fixed, dried,and used to expose to KodakXR-5 film at over the 6-h interval.
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