Kinetics of hydrolysis of endocytosed substrates by mammalian cultured cells: Early introduction of lysosomal enzymes into the endocytic pathway

Abstract

The kinetics of exposure of endocytosed material to two lysosomal enzymes were determined for a number of cultured cell lines using fluorogenic substrates. Hydrolysis of endocytosed substrates for cathepsin B and acid phosphatase was observed to begin within 3–10 min of substrate addition and to proceed linearly for up to 60 min thereafter. Hydrolysis of the cathepsin B substrate was not affected by inhibition of protein synthesis with cycloheximide, indicating that the enzymes present in early endosomes are not exclusively newly synthesized. As had been observed previously for a cathepsin B substrate (Roederer, M., Bowser, R., and Murphy, R.F., J. Cell. Physiol., 131:200–209, 1987), hydrolysis of the acid phosphatase substrate was not blocked at temperatures below 20°C. The results suggest that the endosome is the primary site of initial exposure of endocytosed material to hydrolytic enzymes.