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Sarcoplasmic reticulum vesicles and glycogen-protein particles in microsomal fraction of skeletal muscle
Abstract:
1. Heavy microsomal fraction (HM) of rabbit skeletal muscle obtained by differential centrifugation between 8 000-30 000 g and consisting of sarcoplasmic reticulum (SR) vesicles contains variable amounts of glycogen and reveals some activity of phosphorylase b. The monomer of this enzyme of mol. wt. about 100 000 co-migrates in SDS-polyacrylamide gel electrophoresis with the main SR protein--Ca2+,Mg2+--dependent ATPase. 2. The highest specific activity of phosphorylase and the highest content of glycogen is present in the light microsomal (LM) fraction (30 000-100 000 g). 3. Contrary to the ATPase, phosphorylase b is released from the microsomal fraction by treatment with EDTA and is resistant to trypsin. 4. Both HM and LM fractions can be further fractionated on continuous sucrose density gradient at high speed. Main fraction of HM consists of highly purified SR vesicles. The second, small fraction of HM is identical with the main fraction of LM and consists of two populations: vesicles of structure and properties different from those of SR vesicles, and the particles of a complex of glycogen with some glycolytic enzymes.
Insights
Rabbit skeletal muscle microsomes contain glycogen and phosphorylase b enzyme. The enzyme co-migrates with Ca2+,Mg2+-dependent ATPase, and is released by EDTA treatment, indicating distinct properties from SR vesicles.
Area of Science:
- Muscle physiology
- Biochemistry
- Cellular biology
Background:
- Skeletal muscle sarcoplasmic reticulum (SR) is crucial for calcium regulation.
- Microsomal fractions contain various cellular components, including enzymes and glycogen.
Purpose of the Study:
- To investigate the association and properties of phosphorylase b within rabbit skeletal muscle microsomal fractions.
- To characterize the composition of heavy (HM) and light (LM) microsomal fractions.
Main Methods:
- Differential centrifugation to isolate HM and LM fractions.
- SDS-polyacrylamide gel electrophoresis to analyze protein components.
- Sucrose density gradient centrifugation for further fractionation.
- Enzyme activity assays and sensitivity to EDTA and trypsin.
Main Results:
- Phosphorylase b activity and glycogen content varied between HM and LM fractions.
- The monomer of phosphorylase b co-migrated with Ca2+,Mg2+-dependent ATPase in SDS-PAGE.
- Phosphorylase b was released from microsomes by EDTA treatment and was trypsin-resistant, unlike ATPase.
- Further fractionation revealed purified SR vesicles in HM and a distinct fraction in LM containing glycogen-enzyme complexes.
Conclusions:
- Rabbit skeletal muscle sarcoplasmic reticulum (SR) vesicles are associated with glycogen and phosphorylase b.
- Phosphorylase b exhibits distinct properties from the SR Ca2+,Mg2+-dependent ATPase.
- A complex of glycogen with glycolytic enzymes exists in the light microsomal fraction.