Proteolytic activity within lysosomes and turnover of pinocytic vesicles. A kinetic analysis

Degradation of exogenous [125I] ribonuclease by renal lysosomes follows first-order kinetics in ribonuclease concentration. To demonstrate this, it was necessary to apply corrections for the presence of labeled but digestively inactive particles, either pinocytic vesicles or lysosomes damaged during preparation. Such kinetics were not observed under conditions favoring lysosomal breakdown, i.e., in isotonic KCl, or in the absence of EDTA. The kinetic analysis allows determination of half-times for lysosomal protein digestion. This facilitates comparison of different lysosome preparations, or of in vitro degradation rates with results of in vivo metabolism studies. Degradation of [125I] ribonuclease showed a half-time of about 11 1/2 minutes in isotonic sucrose or saline media. This is less than the half-time for decrease of kidney radioactivity in vivo after uptake of [125I] ri-onuclease. The proportion of exogenous, labeled protein contained within secondary lysosomes was determined as a function of time after injection of ribonuclease, to monitor transfer of the protein from pinocytic vesicles to lysosomes. Ribonuclease molecules remained in pinocytic vesicles for approximately three minutes after uptake, before passage into the lysosomes.