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Limited autolysis reduces the Ca2+ requirement of a smooth muscle Ca2+-activated protease
Abstract:
Chicken gizzard smooth muscle contains large amounts of Ca2+-activated protease activity. Approximately 15 mg of purified enzyme can be obtained from 1 kg of fresh muscle. The enzyme consists of two subunits (Mr = 80,000 and 30,000) present in a 1:1 molar ratio. In the presence of CaCl2, the 80,000/30,000-dalton heterodimer (form I) is rapidly converted by limited autolysis to a 76,000/18,000-dalton species (form II). Both the 80,000- and 30,000-dalton subunits are degraded simultaneously. Moreover, the Ca2+ dependence for autolysis (K0.5 = 300 microM) is identical for both subunits. Neither the time course nor the Ca2+ dependence of the autolytic conversion reaction is altered by 10- and 20-fold molar excesses of substrate. Limited autolysis markedly reduces the Ca2+ requirement for substrate degradation. Using N-[ethyl-2-3H]maleimide-labeled 27,000-dalton cardiac myosin light chains as substrate, the Ca2+ requirement of form I was found to be quite high (K0.5 = 150 microM). Under similar conditions, the Ca2+ requirement of form II was 30-fold lower (K0.5 = 5 microM). Limited autolysis did not alter the specific activity of the enzyme. Our results demonstrate that smooth muscle contains an abundant amount of Ca2+-activated protease. Moreover, autolysis of this enzyme may play an important regulatory role by converting the native form to a species that is fully active at physiological levels of intracellular calcium ion.
Insights
Chicken gizzard smooth muscle has abundant Ca2+-activated protease. Autolysis converts this enzyme, reducing its calcium requirement and potentially regulating its activity at physiological calcium ion levels.
Area of Science:
- Biochemistry
- Muscle Physiology
- Enzymology
Background:
- Smooth muscle possesses significant Ca2+-activated protease activity.
- This protease is abundant, with 15 mg purified enzyme per kg muscle.
- The enzyme exists as an 80,000/30,000-dalton heterodimer.
Purpose of the Study:
- To investigate the properties and regulation of Ca2+-activated protease in chicken gizzard smooth muscle.
- To determine the effect of autolysis on the enzyme's calcium sensitivity and activity.
Main Methods:
- Purification of Ca2+-activated protease from chicken gizzard smooth muscle.
- Analysis of enzyme subunits and autolysis using SDS-PAGE.
- Determination of Ca2+ dependence (K0.5) for autolysis and substrate degradation using labeled myosin light chains.
Main Results:
- Limited autolysis converts the native heterodimer (form I) to a smaller species (form II).
- Autolysis significantly reduces the Ca2+ requirement for substrate degradation (from 150 microM to 5 microM).
- Specific activity of the protease remains unchanged after autolysis.
Conclusions:
- Chicken smooth muscle contains a readily available Ca2+-activated protease.
- Autolysis is a key regulatory mechanism, enhancing protease activity at physiological intracellular calcium concentrations.
- This process may be crucial for modulating smooth muscle function.