Figure 6. Assessment of endosomal acidic insulinase activity after a gel-filtration HPLC protocol and immunodepletion procedures. Endosomal acidic insulinase activity was partially purified using a TSK-gel G3000 HPLC column (see Fig. 5, A and B) and incubated with[ ¹²⁵I]Tyr^A14-HI (75 fmol) at 37 C and pH 4 with the indicated concentrations of unlabeled HI or H2-analog peptides (A). The amount of degraded radiolabeled HI was determined by precipitation with TCA, and results are expressed as in Fig. 4A. In Panel B, partially purified EAI was incubated at different pH values for 10 min with [¹²⁵I]Tyr^A14-HI or -H2-analog, after which the amount of degraded radiolabeled ligand was determined by precipitation with TCA. C, ENs was immunodepleted of various proteases using independently rabbit antirat procathepsin B (CB), -rat cathepsin L (CL), -rat tripeptidylpeptidase II (TPP II), -rat Thimet Oligopeptidase (TOP), and mouse antihuman insulin-degrading enzyme (IDE) at a dilution of 1/500. After immunoprecipitation using protein G-Sepharose and centrifugation, the resultant supernatants were tested for the ability to degrade[ ¹²⁵I]Tyr^A14-HI or[ ¹²⁵I]-glucagon at pH 4 using the TCA-precipitation assay. Identification of Insulin Domains Important for Binding to and Degradation by Endosomal Acidic Insulinase**This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale and the Fondation pour la Recherche Médicale (Grant 10000320-01, to F.A.). Endocrinology. 2001;142(1):276-289. doi:10.1210/endo.142.1.7916 Endocrinology | Copyright © 2004 by the Endocrine Society 1