Autolysis and biochemical properties of a lobster muscle calpain-like proteinase
✍ Scribed by Beyette, Jill R.; Mykles, Donald L.
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 169 KB
- Volume
- 277
- Category
- Article
- ISSN
- 0022-104X
No coin nor oath required. For personal study only.
✦ Synopsis
Lobster skeletal muscle contains four Ca 2+ -dependent cysteine proteinases that completely degrade myofibrillar proteins to acid-soluble products and may be involved in moltinduced claw muscle atrophy. One of the four enzymes, designated Ca 2+ -dependent proteinase IIb (CDP IIb), degrades the myofibrillar proteins actin, troponin, tropomyosin, and myosin in situ and in vitro. Lobster CDP IIb (195 kDa) is a homodimeric protein (submit M r = 95 kDa) that autolyzes in the presence of 5 mM Ca 2+ . This study examined the autolytic mechanism and effects of autolysis on the Ca 2+ sensitivity of the enzyme. Autolysis produced fragments of 87, 83, 80, and 74 kDa in sequential order (fragments A, B, C, and D, respectively). Cleavage of 95 kDa polypeptide to fragment A and fragment A to fragment B was not inhibited by enzyme dilution or casein, showing that autolysis involves an intramolecular mechanism. Casein hydrolysis at 5 mM Ca 2+ preceded autolysis, which suggests that 95 kDa polypeptide is proteolytically active. However, once autolysis was initiated, casein accelerated intramolecular autolytic cleavages. Autolysis of fragments B, C, and D was inhibited by substrate, indicating that cleavages were intermolecular (i.e., active enzyme degrading inactive fragments). The half-maximal Ca 2+ requirement for autolysis was 1.9 mM and was unaffected by 80 µM phosphatidylinositol. Autolysis had little effect on Ca 2+ sensitivity. The half-maximal Ca 2+ requirement for proteolytic activity was 2.3 mM; autolysis decreased by 26% the required Ca 2+ concentration to 1.7 mM. The lack of effect of phosphatidylinositol and autolysis on Ca 2+ sensitivity, in addition to its ability to completely degrade endogenous proteins, suggests functional and/or structural differences between the CDP IIb and vertebrate calpains. The initial autolytic cleavage may be involved in altering intracellular location and/or substrate specificity rather than activating hydrolytic breakdown of substrate. The second cleavage may be involved in inactivating the enzyme to restrict hydrolysis to a specific intracellular location.