Electron microscopic visualization of cathepsin D using mercury-labeled pepstatin as an enzyme inhibitor.

Abstract

Pepstatin, a specific inhibitor of pepsin, cathepsin D (E), renin, etc., was used in a new method to demonstrate the sites of enzymes. Pepstatin (Pst) was covalently attached to glutathione (GSH), using a dicyclohexylcarbodiimide, and CH3Hg+ was then put in the residue of SH for electron microscopic observation. Pst-GS-HgCH3 thus obtained was nonisotopical, had a very low molecular weight (about 1,200 daltons), and still almost completely retained its inhibitory activity. Sections of rat liver (less than 1 mm3 thick), prefixed by glutaraldehyde, were incubated in the prepared reaction medium. Cathepsin D, located in the lysosomes, was demonstrated by this compound, but when the sections were preincubated with pepstatin, this was not the case. These results demonstrate that mercury-labeled pepstatin is a useful reagent for the histochemical staining of acid proteases for electron microscope.