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Published on: August 31, 2017
Microsomal T system: a stereological analysis of purified microsomes derived from normal and dystrophic skeletal
Abstract:
Heterogeneous populations of microsomes obtained from normal and dystrophic chicken pectoralis muscle were separated into two subfractions by an iterative loading technique. The buoyant density of the sarcoplasmic reticulum (SR) microsomes was increased after loading them with calcium oxalate. Several incubations in the transport medium were necessary to load all of the SR. The fraction that did not form a pellet contained microsomes which displayed freeze-fracture faces that had a low density of particles. A stereological analysis was used on membrane fracture faces of intact muscle to generate reference particle density distributions, which were compared with the distributions measured on the microsomal fracture faces. The concave microsomal fracture faces of purified microsomes which did not load calcium oxalate had particle distributions nearly identical to the distributions of intact P-face T tubules. The morphological data suggest that this subfraction is microsomal T system. Biochemical measurements show negligible amounts of specific Na+, K+-ATPase activity, suggesting that there was little contamination from the surface membrane in this subfraction. Furthermore, an active Ca2+-ATPase is demonstrated in both normal and dystrophic T-tubular membranes.
Insights
Researchers isolated T-tubule microsomes from chicken muscle, distinguishing them from sarcoplasmic reticulum. This purified fraction showed active Ca2+-ATPase, crucial for muscle function in both normal and dystrophic states.
Area of Science:
- Muscle physiology
- Cellular biology
- Biochemistry
Background:
- Microsomes from chicken pectoralis muscle present heterogeneous populations.
- Distinguishing sarcoplasmic reticulum (SR) from T-tubules is essential for studying muscle function.
Purpose of the Study:
- To separate and characterize microsomal subfractions from normal and dystrophic chicken muscle.
- To identify the T-tubule system within these microsomes and assess its biochemical properties.
Main Methods:
- Iterative loading technique using calcium oxalate to increase SR microsome buoyant density.
- Stereological analysis of freeze-fracture electron microscopy images of intact muscle and microsomes.
- Biochemical assays for Na+, K+-ATPase and Ca2+-ATPase activity.
Main Results:
- Two distinct microsomal subfractions were obtained.
- The non-pelleting fraction exhibited particle densities similar to intact T-tubules, identifying it as the T-system.
- This T-tubule subfraction showed negligible Na+, K+-ATPase but active Ca2+-ATPase in both normal and dystrophic muscle.
Conclusions:
- The study successfully isolated and identified microsomal T-tubules from chicken muscle.
- Active Ca2+-ATPase in T-tubular membranes is present in both normal and dystrophic conditions, suggesting its role in muscle function and potential disease mechanisms.
