Differential degradation of myofibrillar proteins by four calcium‐dependent proteinase activities from lobster muscle

Abstract Four calcium‐dependent proteinases (CDPs I, IIa, IIb, and III) were isolated from lobster claw and abdominal muscles. In order to determine the substrate specificities of the CDPs, each enzyme was incubated with either native purified myosin or actomyosin from lobster deep abdominal muscles. After incubation in the presence or absence of 5 mM Ca2+, reaction mixtures were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and examined in 2 ways: (1) the fragment patterns from the degradation of the myosin heavy chain were identified with immunoblotting, and (2) gels were stained with Coomassie blue and densitometrically scanned to quantify the degradation of the major myofibrillar proteins. CDPs IIa and IIb were the most effective in degrading myosin heavy chain, actin, troponin‐T2, tropomyosin, troponin‐I1 and ‐I5, troponin‐C1, and myosin β‐light chain; the major difference between the 2 enzymes was that CDP IIa also degraded myosin α‐light chain. CDP III effectively hydrolyzed myosin heavy chain, actin, troponin‐T2, tropomyosin, troponin‐I5, troponin‐C1, and myosin β‐light chain. CDP I was the least efficient of the 4 CDPs in degrading myofibrillar proteins; only actin, troponin‐T2, and troponin‐I5 were readily hydrolyzed. All the CDPs recognized similar cleavage sites within the myosin heavy chain, since the proteolytic fragment pattern produced by each enzyme on the myosin heavy chain in reaction mixtures containing either actomyosin or myosin was essentially identical. These results show that the 4 CDPs share catalytic properties but differ in substrate specificities, particularly when presented with a complex assemblage of potential substrates. © 1993 Wiley‐Liss, Inc.