Autolysis and biochemical properties of a lobster muscle calpain-like proteinase

Abstract

Lobster skeletal muscle contains four Ca 2+ -dependent cysteine proteinases that completely degrade myofibrillar proteins to acid-soluble products and may be involved in molt- induced 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 Mr = 95 kDa) that autolyzes in the presence of 5 mM Ca 2+ . This study examined the autolytic mechanism and ef- fects 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. Au- tolysis of fragments B, C, and D was inhibited by substrate, indicating that cleavages were inter- molecular (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 intracellu- lar 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 spe- cific intracellular location. J. Exp. Zool. 277:106-119, 1997. © 1997 Wiley-Liss, Inc. In association with the molting process, the claw closer muscles of decapod crustaceans un- dergo a cyclical atrophy and restoration (for re- view see Mykles and Skinner, '90b). During this molt-induced muscle atrophy, there is a reduction of at least 40% in protein content associated with a fourfold decrease in myofibrillar cross-sectional area (Skinner, '66; Mykles and Skinner, '81). Experiments with crab claw muscles cultured in vitro demon- strate that myofibrillar protein turnover is accom- plished by Ca 2+ -dependent cysteine proteinases (CDPs), although other nonlysosomal proteolytic systems, such as the ATP/ubiquitin-dependent pro- teolytic pathway, may also contribute (Mykles, '90; Shean and Mykles, '95). CDP activity, as well as muscle protein ubiquitination and polyubiquitin expression, increases during claw muscle atrophy (Mykles and Skinner, '82; Shean and Mykles, '95). The CDP activity responsible for myofibrillar protein turnover is composed of four different cys-