Ca2+-dependent proteolytic activity in crab claw muscle. Effects of inhibitors and specificity for myofibrillar proteins.

The claw closer muscle of the Bermuda land crab, Gecarcinus lateralis, undergoes a sequential atrophy and restoration during each molting cycle. We describe here the role of Ca2+-dependent proteinases in the turnover of myofibrillar protein in normal anecdysial (intermolt) claw muscle. Crab Ca2+-dependent proteinase degrades the myofibrillar proteins actin, myosin heavy and light chains, paramyosin, tropomyosin, and troponin-T and -I. Ca2+-dependent proteinase activity in whole homogenates and 90,000 X g supernatant fractions from muscle homogenates has been characterized with respect to Ca2+ requirement, substrate specificity, and effects of proteinase inhibitors. The enzyme is inhibited by antipain, leupeptin, E-64, and iodoacetamide; it is insensitive to pepstatin A. The Ca2+-dependent proteinase is a sarcoplasmic cysteine proteinase that shows maximal activation at 1 mM Ca2+ at neutral pH. Since approximately 28% of the activity remains at 1.5 microM Ca2+, the enzyme is partially active at physiological Ca2+ concentrations. The specificity of crab Ca2+-dependent proteinase was examined with native myosin with normal ATPase activity as well as with radioiodinated myosin and radioiodinated hemolymph proteins. Hydrolysis of 125I-myosin occurs in two phases, both Ca2+-dependent: 1) heavy chain (Mr = 200,000) is cleaved into four large fragments (Mr = 160,000, 110,000, 73,000, 60,000) and numerous smaller fragments; light chain (Mr = 18,000) is cleaved to a 15,000-Da fragment; 2) the fragments produced in the first phase are hydrolyzed to acid-soluble material. Although radioiodinated native hemolymph proteins are not susceptible to the Ca2+-dependent proteinase, those denatured by carboxymethylation are degraded. These data suggest that crab Ca2+-dependent proteinase is involved in turnover of myofibrillar protein in normal muscle and muscle undergoing proecdysial atrophy.