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Sarcoplasmic reticulum. IX. The permeability of sarcoplasmic reticulum membranes
Abstract:
Fragmented sarcoplasmic reticulum (FSR) membranes isolated from rabbit skeletal muscle are impermeable to inulin-(14)C (mol wt 5,000), and dextran-(14)C (mol wt 15,000-90,000) at pH 7.0-9.0, yielding an excluded space of 4-5 microl/mg microsomal protein. In the same pH range urea and sucrose readily penetrate the FSR membrane. EDTA or EGTA (1 mM) increased the permeability of microsomes to inulin-(14)C or dextran-(14)C at pH 8-9, parallel with the lowering of the FSR-bound Ca(++) content from initial levels of 20 nmoles/mg protein to 1-3 nmoles/mg protein. EGTA was as effective as EDTA, although causing little change in the Mg(++) content of FSR. The permeability increase caused by chelating agents results from the combined effects of high pH and cation depletion. As inulin began to penetrate the membrane there was an abrupt fall in the rate of Ca(++) uptake and a simultaneous rise in ATPase activity. At 40 degrees C inulin penetration occurred at pH 7.0 with 1 mM EDTA and at pH 9.0 without EDTA, suggesting increased permeability of FSR membranes. This accords with the higher rate of Ca(++) release from FSR at temperatures over 30 degrees C. The penetration of microsomal membranes by anions is markedly influenced by charge effects. At low ionic strength and alkaline pH acetate and Cl are partially excluded from microsomes when applied in concentrations not exceeding 1 mM, presumably due to the Donnan effect. Penetration of microsomal water space by acetate and Cl occurs at ionic strengths sufficiently high to minimize charge repulsions.
Insights
Fragmented sarcoplasmic reticulum (FSR) membranes show increased permeability to large molecules like inulin and dextran when treated with chelating agents at alkaline pH. This change correlates with reduced calcium content and altered ATPase activity in skeletal muscle microsomes.
Area of Science:
- Biochemistry
- Cell Biology
- Muscle Physiology
Background:
- Fragmented sarcoplasmic reticulum (FSR) is crucial for calcium storage and release in skeletal muscle.
- Understanding FSR membrane permeability is key to elucidating calcium handling mechanisms.
Purpose of the Study:
- To investigate the permeability of FSR membranes to various molecules.
- To determine the effects of pH, chelating agents, and temperature on FSR membrane permeability.
- To correlate changes in permeability with calcium content and ATPase activity.
Main Methods:
- Isolation of FSR membranes from rabbit skeletal muscle.
- Measurement of membrane permeability using radiolabeled molecules (inulin, dextran, urea, sucrose).
- Assessment of FSR-bound calcium and magnesium content.
- Monitoring of calcium uptake and ATPase activity.
- Experiments conducted at varying pH, temperature, and in the presence of chelating agents (EDTA, EGTA).
Main Results:
- FSR membranes are impermeable to inulin and dextran at neutral to alkaline pH, with limited excluded space.
- Urea and sucrose readily penetrate the FSR membrane across a similar pH range.
- EDTA and EGTA significantly increase FSR permeability to inulin and dextran at alkaline pH, correlating with decreased bound calcium.
- Increased temperature (above 30°C) also enhances FSR membrane permeability.
- Anion penetration is influenced by charge effects and ionic strength, with exclusion at low ionic strength and alkaline pH.
Conclusions:
- FSR membrane permeability is modulated by pH, cation levels, and temperature.
- Chelating agents, by reducing bound calcium, increase FSR permeability, impacting calcium uptake and ATPase activity.
- These findings provide insights into the dynamic nature of the sarcoplasmic reticulum membrane and its role in muscle function.