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Some properties of Ca-binding microsomal subfractions isolated from rabbit colon muscle
Abstract:
From a homogenate of rabbit colon smooth muscle a microsomal fraction was isolated, which was divided into subfractions by centrifugation on a discontinuous sucrose gradient. The Ca-binding properties of the subfractions were investigated under different conditions. In the presence of 0.35 mM ATP the Ca binding of the fractions amounted to 4--8 nmol/mg protein. The 35% fraction bound more Ca per mg protein than the 35--45% fraction. The Ca accumulation was comparatively higher both in the presence of 5 mM ATP and in the presence of 5 mM oxalate. The two fractions showed about the same sensitivity for oxalate. This substance stimulated the Ca uptake at 5 mM but not at lower concentrations. The amount and the rate of Ca binding were more dependent on variations in the exogenous ATP concentration in the 35% fraction than was the case for the 35--45% fraction. The Ca binding was completely inhibited by salyrgan when the microsomal fractions were pretreated with this agent. Sodium azide did not influence the Ca-binding capacity of the fractions. It is suggested that the microsomal subfractions of the rabbit colon muscle represent physiologically important parts of the Ca sequestering system of the muscle, since Ca binding takes place at Ca- and ATP-concentrations which are believed to be present in the myoplasm.
Insights
Rabbit colon smooth muscle microsomes bind calcium (Ca) and adenosine triphosphate (ATP). Specific subfractions show varying Ca-binding capacities, suggesting a role in the muscle's Ca sequestering system.
Area of Science:
- Muscle Physiology
- Cellular Biology
- Biochemistry
Background:
- Smooth muscle contraction is regulated by intracellular calcium (Ca) levels.
- Microsomal fractions are key in cellular Ca sequestration and release.
- Understanding Ca-binding properties in rabbit colon smooth muscle is crucial for physiological insights.
Purpose of the Study:
- To isolate and characterize Ca-binding properties of microsomal subfractions from rabbit colon smooth muscle.
- To investigate the influence of adenosine triphosphate (ATP) and oxalate on Ca binding.
- To determine the physiological relevance of these subfractions in the muscle's Ca sequestering system.
Main Methods:
- Isolation of a microsomal fraction from rabbit colon smooth muscle homogenate.
- Subfractionation using discontinuous sucrose gradient centrifugation.
- Measurement of Ca-binding capacities under varying conditions (ATP, oxalate).
Main Results:
- Microsomal subfractions exhibited significant Ca-binding capacities (4-8 nmol/mg protein) in the presence of 0.35 mM ATP.
- The 35% sucrose fraction demonstrated higher Ca binding than the 35-45% fraction.
- Ca accumulation was enhanced by 5 mM ATP and 5 mM oxalate, with similar oxalate sensitivity in both fractions.
- Ca binding was ATP-dependent and inhibited by salyrgan, but not affected by sodium azide.
Conclusions:
- Rabbit colon smooth muscle microsomes contain physiologically important Ca-sequestering components.
- Ca binding occurs at physiologically relevant Ca and ATP concentrations found in the myoplasm.
- These findings contribute to understanding Ca regulation in smooth muscle tissue.