Degradation of canine cardiac myosin and actin by cathepsin D isolated from homologous tissue

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Abstract
The ability of canine cardiac cathepsin D to catalyze hydrolysis of myosin and actin isolated from homologous tissue was monitored using both SDS [sodium dodecyl sulfate] polyacrylamide gel electrophoresis and the release of trichloroacetic acid soluble radioactivity from the radioactively (14C) labeled proteins. Cathepsin D readily catalyzed the hydrolysis of myosin and actin. The pH-activity profile for myosin hydrolysis exhibited 2 optima with a major peak at pH 2.6 together with a smaller peak at pH 3.4. This was distinct from the pH-activity profile for actin hydrolysis where a single optimum at pH 4.2 was observed. Incubation of cathepsin D with myosin (pH 2.6, 37.degree. C) resulted in complete degradation of myosin light chains (MW = 28,000 .+-. 700 and 18,600 .+-. 350, n = 4) within 10 min while heavy chains (MW = 190,400 .+-. 2500, n = 4) were degraded into 3 proteins of MW 32,000 .+-. 750; 37,000 .+-. 1000 and 49,000 .+-. 750 (n = 4) within 4 h. Further incubation (24 h) of cathepsin D with myosin resulted in 2 proteins of MW 29,000 .+-. 2000 and 48,800 .+-. 3100 (n = 4). In the case of actin (MW 44,800 .+-. 1600, n = 6) polypeptides produced as a result of hydrolytic cleavage by cathepsin D (pH 4.2, 37.degree. C) were not observed on SDS gels, although the overall size of the actin band was markedly decreased at 24 h. Cathepsin D catalyzed the hydrolysis of myosin at near neutral pH (6.5) although the overall rate of degradation was considerably reduced compared to that observed at pH 2.6. Pepstatin A was a potent inhibitor of the hydrolysis of myosin and actin at all pH values investigated indicating that cathepsin D and not a contaminant was responsible for the observed degradation.

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