Limited autolysis reduces the Ca2+ requirement of a smooth muscle Ca2+-activated protease

• Published in: The Journal of biological chemistry 1982-08, Vol.257 (15), p.9072-9077
• Main Authors: Hathaway, D R, Werth, D K, Haeberle, J R
• Format: Article
• Language: English
• Subjects: Animals; Calcium - metabolism; Calpain; Chickens; Endopeptidases - metabolism; Enzyme Activation; Gizzard, Avian - enzymology; Kinetics; Macromolecular Substances; Muscle, Smooth - enzymology; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)34244-3

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.