Purification of cathepsin D from rabbit skeletal muscle and its action towards myofibrils

Abstract

Cathepsin D (EC 3.4.23.5) was purified from rabbit skeletal muscle using acetone-dried muscle powder as starting material. After the acetone-dried powder was extracted with 0.2 mM ATP, the extract was fractionated with acetone and subjected to DEAE-Sephadex A-50 and Sephadex G-100 column chromatography. Rechromatography on a Sephadex G-100 column resulted in a purified preparation. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed one major band of 42 000 daltons and some bands of contaminants. Since gel filtration also indicated a value of 42 000 daltons for the enzyme, it was concluded that muscle cathepsin D has no subunit structure. The enzyme acted optimally towards myofibrils around pH 3, resulting in the degradation of the myosin heavy chain and production of a 30 000-dalton component.