MEMBRANEOUS ULTRASTRUCTURAL DEMONSTRATION OF AMINOPEPTIDASE AND γ-GLUTAMYL TRANSPEPTIDASE ACTIVITIES WITH A NEW DIAZONIUM SALT THAT YIELDS A LIPOPHOBIC, OSMIOPHILIC AZO DYE

When 4-aminophthalhydrazide is freshly diazotized and used as the coupling agent in demonstrating aminopeptidase activity or γ-glutamyl transpeptidase activity, with substrates that yield 4-methoxy-2-naphthylamine or even 2-naphthylamine, azo dyes are produced which are readily osmiophilic. Although the distribution of the reddish azo dyes is similar to that noted with earlier methods in light microscopy, the distribution of the deposits in electron microscopic preparations is distinctly membraneous in location in contrast to the droplet distribution of previous methods. We attribute membraneous localization of enzymatic activity with the new methods presented here to the lipophobic properties of the azo dyes provided by their 4-aminophthalhydrazide (4-APH) moiety. The new agent 4-aminophthalhydrazide was designed with this in mind.