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

Abstract

The ability of canine cardiac cathepsin D to catalyse hydrolysis of myosin and actin isolated from homologous tissue was monitored using both SDS polyacrylamide gel electrophoresis and the release of trichloroacetic acid soluble radioactivity from the radioactively (14C) labelled proteins. Cathepsin D readily catalysed the hydrolysis of myosin and actin. The pH-activity profile for myosin hydrolysis exhibited two 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°C) resulted in complete degradation of myosin light chains (MW =28000±700 and 18600±350, n=4) within 10 min while heavy chains (MW= 190400±2500, n=4) were degraded into three proteins of molecular weight 32000 ±750; 37000 ±1000 and 49000 ±750 (n=4) within 4 h. Further incubation (24 h) of cathepsin D with myosin resulted in two proteins of MW 29000±2000 and 48800±3100 (n=4). In the case of actin (MW 44800± 1600, n=6) polypeptides produced as a result of hydrolytic cleavage by cathepsin D (pH 4.2, 37°C) were not observed on SDS gels, although the overall size of the actin band was markedly decreased at 24 h. Cathepsin D catalysed 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.