Localization and characterization of N-ethylmaleimide sensitive inhibitor(s) of thiol cathepsin activity from cultured nil and polyoma virus-transformed Nil hamster cells

Abstract

Exposure of cultured Nil (a stable line of fibroblast cells from Syrian hamsters) or polyoma virus-transformed (PyNil) hamster fibroblasts to 0.5 mM N-ethylmaleimide for 5 minutes resulted in striking increases in thiol cathepsin activity in unfractionated cell-free lysates. The paradoxical increase in activity of the normally N-ethylmaleimide-sensitive cathepsins apparently occurred as the result of the protective compartmentalization of the cathepsins in the lysosomes (20,000 × g sedimented fraction) and the unprotected localization of an inhibitor(s) in the soluble cytoplasm (175,000 × g supernatant fraction). Under continuous exposure of the cells to N-ethylmaleimide, a rapid increase in cathepsin activity (seen in the first 5 minutes) was followed by a steady decrease in activity (half inactivation time, 90 minutes). The relative difference in rates of N-ethylmaleimide inactivation of thiol cathepsins and thiol cathepsin inhibitors provides a means for estimating lysosomal cathepsin activity in whole cell extracts without the need for more time-consuming fractionation procedures. In reciprocal inhibition tests, it was found that, regardless of the source of cathepsins, the Nil and PyNil cathepsin inhibitor(s) inactivated the cathepsins to approximately the same extent. The inhibitors were heat stable (90–100°C for 15 minutes) at pH 4, but were totally inactivated when boiled at pH 8.5. On a calibrated Sephadex G-100 column, the relative molecular weight (M_r) of the inhibitor(s) was 13,000 daltons. On the same column, the M_r of the cathepsins was 24,000 daltons. Compared with the cathepsin activity from Nil cells, there was about five times less cathepsin activity recoverable from the PyNil cells.