Susceptibilities of cardiac myofibrillar proteins to cathepsin D-catalyzed degradation.
The binding of various myofibrillar proteins to cathepsin D and consequent susceptibility to degradation was assessed by determination of apparent Michaelis constant (Km) values for the purified enzyme using myosin, alpha-actinin, actin, and tropomyosin as substrates. Cathepsin D, purified 1,000-fold to homogeneity from canine cardiac tissue, was incubated (0-100 min at 37 degrees C) with myofibrillar proteins isolated from homologous tissue and labeled radioactively with 14C by reductive alkylation. The reaction was terminated by addition of trichloroacetic acid (10% wt/vol), and radioactivity in the supernatant fraction was determined after centrifugation (5 min) at 100,000 g. Double reciprocal graphs (1/reaction velocity vs. 1/substrate concn) were constructed for each substrate from the linear portion of graphs of disintegrations. min-1 X ml-1 100,000-g supernatant vs. time (min). Apparent Km values (+/- SD) calculated for myosin, alpha-actinin, actin, and tropomyosin were found to be 2.7 +/- 0.3 X 10(-6) (n = 6), 10.0 +/- 2.3 X 10(-6) (n = 13), 13.0 +/- 1.3 X 10(-6) (n = 7), and 45.5 +/- 9.0 X 10(-6) (n = 6) mol/l, respectively. The results show that myofibrillar proteins differ in their binding and thus susceptibility to hydrolysis by cathepsin D in order of molecular weight (i.e., myosin greater than alpha-actinin greater than actin greater than tropomyosin). Because the relative turnover rates of myofibrillar proteins are known to be independent of molecular weight, our results suggest that the degradation of these proteins by cathepsin D is not rate limiting and that other factors including lysosomal or nonlysosomal enzymes must determine the rate-limiting steps of myofibrillar protein degradation in cardiac tissue.
D000185:Actinin / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D002402:Cathepsin D; D002403:Cathepsins / Q000378:metabolism*; D003285:Contractile Proteins / Q000378:metabolism*; D004285:Dogs; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D013379:Substrate Specificity; D014335:Tropomyosin / Q000378:metabolism
D000199:Actins; D003285:Contractile Proteins; D014335:Tropomyosin; D000185:Actinin; D002403:Cathepsins; D002402:Cathepsin D; D009218:Myosins