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The presence and binding characteristics of calmodulin in microsomal preparations enriched in sarcoplasmic reticulum
Abstract:
ATP-dependent oxalate facilitated calcium transport in sarcoplasmic reticulum (SR) preparations obtained from rabbit vastus lateralis muscle (fast skeletal muscle; Fsr) and soleus (slow skeletal muscle; Ssr) was determined. Addition of exogenous calmodulin did not stimulate calcium transport in either Fsr or Ssr preparations. Fsr and Ssr previously washed in 1 mM EGTA demonstrated a reduced capacity to transport Ca2+; the exogenous addition of calmodulin (0.24 microM) under these conditions, did not restore uptake activity but significantly decreased the steady-state level of Ca2+ uptake. Extracts of skeletal SR prepared by treatment with 0.2 mM EDTA and boiling produced significantly more stimulation of red cell Ca2+ATPase activity than extracts prepared by boiling alone. This stimulation of red cell Ca2+-ATPase was inhibited to a significant extent by 48/80, a known anti-calmodulin agent. Radioimmunoassay revealed that extracts prepared by boiling or EDTA-treatment followed by boiling contained considerable amounts of calmodulin. Washing with 1 mM EGTA, though, did not release any calmodulin from SR. These studies reveal that calmodulin is present in both Fsr and Ssr and can only be removed by harsh treatments. The role of calmodulin in skeletal muscle Ca2+-transport remains to be determined.
Insights
Sarcoplasmic reticulum (SR) preparations from fast and slow skeletal muscles contain calmodulin, but it doesn't directly stimulate calcium transport. Calmodulin removal requires harsh conditions, and its precise role in muscle calcium transport needs further investigation.
Area of Science:
- Muscle Physiology
- Cellular Biology
- Biochemistry
Background:
- Sarcoplasmic reticulum (SR) is crucial for calcium (Ca2+) handling in muscle cells.
- Calmodulin is a calcium-binding protein involved in various cellular processes.
- The role of calmodulin in skeletal muscle SR Ca2+ transport is not fully understood.
Purpose of the Study:
- To investigate ATP-dependent oxalate-facilitated Ca2+ transport in fast (Fsr) and slow (Ssr) skeletal muscle SR.
- To determine the effect of exogenous calmodulin on Ca2+ transport in Fsr and Ssr.
- To assess calmodulin presence and extractability from skeletal muscle SR.
Main Methods:
- Preparation of SR from rabbit vastus lateralis (fast) and soleus (slow) muscles.
- Measurement of ATP-dependent oxalate-facilitated Ca2+ uptake.
- Treatment of SR with EGTA, EDTA, and boiling.
- Assay of red cell Ca2+-ATPase activity.
- Radioimmunoassay for calmodulin detection.
Main Results:
- Exogenous calmodulin did not stimulate Ca2+ transport in Fsr or Ssr.
- EGTA washing reduced Ca2+ transport capacity and exogenous calmodulin did not restore it.
- EDTA and boiling treatments released calmodulin and stimulated red cell Ca2+-ATPase activity.
- Calmodulin was present in both Fsr and Ssr but resistant to EGTA washing.
Conclusions:
- Calmodulin is present in both fast and slow skeletal muscle SR.
- Calmodulin is not readily removed by standard washing procedures (EGTA).
- The direct role of calmodulin in skeletal muscle SR Ca2+ transport remains to be elucidated.