Cupric ion-dependent inhibition of lysosomal acid cholesteryl ester hydrolase in the presence of hydroxylamine.

Abstract

In the presence of hydroxylamine or ascorbic acid, the inhibitory effects of Cu2+ on lysosomal acid cholesteryl ester hydrolase (acid CEH) partially purified from rat liver were studied. Hydroxylamine stimulated the inhibition of acid CEH activity by Cu2+ but not that by Zn2+, Fe2+, Co2+, Mn2+, Ca2+, Mg2+ and Hg2+. This Cu2+-dependent inhibition of acid cholesterol ester hydrolase (CEH) activity was completely prevented by ethylenediamine tetraacetic acid (EDTA), EGTA and o-phenanthroline, a chelator with a stability constant for Cu2+, and also by sulfhydryl agents and cytoplasmic reducing agents such as cysteine, glutathione and mercaptoethanol. In addition, the stimulative effects of hydroxylamine on Cu2+-dependent inhibition were maintained even after preincubation of Cu2+ with hydroxylamine. On the other hand, ascorbic acid was found to replace the stimulation by hydroxylamine of the Cu2+-dependent inhibition of acid CEH activity but the effects of ascorbic acid progressively became smaller with prolongation of the preincubation time. Moreover, addition of chemical radical scavengers to the reaction mixture did not prevent the Cu2+-dependent inhibition of acid CEH activity in the presence of ascorbic acid. These results suggest that Cu2+ causes inhibition of lysosomal acid CEH activity through the formation of Cu1+ in a reductive medium.