J P Bai1, L L Chang. 1. Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis 55455, USA.
Abstract
PURPOSE: The purpose of this study was to determine the existence of insulin-degrading enzyme (EC 3.4.22.11) (IDE) in rat intestinal enterocytes. METHODS: Subcellular fractionation, biochemical characterization, immunoprecipitation, and western blots were employed. RESULTS: Insulin-degrading activity was localized in the cytosol, constituting 92% of total insulin-degrading activity. Cytosolic insulin-degrading activity had a pH optimum of 7.5, was almost completely inhibited by IDE inhibitors (N-ethylmaleimide, 1,10-phenanthroline, EDTA, p-chloromercuribenzoate, bacitracin), but was not or only weakly inhibited by others (aprotinin, chymostatin, leupeptin, and diisopropyl phosphofluoridate.) Further, cytosolic insulin-degrading activity had a Km of 78 nM, sharing a similar Km value with insulin-degrading enzyme in non-purified forms. Approximately, 87 +/- 1.7% of cytosolic insulin-degrading activity was removed by the monoclonal antibody to IDE. On the SDS gel, the molecular weight of cytosolic IDE was 110 KD which is the same as that of human IDE. CONCLUSIONS: IDE is the major enzyme which degrades insulin in enterocytes.
PURPOSE: The purpose of this study was to determine the existence of insulin-degrading enzyme (EC 3.4.22.11) (IDE) in rat intestinal enterocytes. METHODS: Subcellular fractionation, biochemical characterization, immunoprecipitation, and western blots were employed. RESULTS:Insulin-degrading activity was localized in the cytosol, constituting 92% of total insulin-degrading activity. Cytosolic insulin-degrading activity had a pH optimum of 7.5, was almost completely inhibited by IDE inhibitors (N-ethylmaleimide, 1,10-phenanthroline, EDTA, p-chloromercuribenzoate, bacitracin), but was not or only weakly inhibited by others (aprotinin, chymostatin, leupeptin, and diisopropyl phosphofluoridate.) Further, cytosolic insulin-degrading activity had a Km of 78 nM, sharing a similar Km value with insulin-degrading enzyme in non-purified forms. Approximately, 87 +/- 1.7% of cytosolic insulin-degrading activity was removed by the monoclonal antibody to IDE. On the SDS gel, the molecular weight of cytosolic IDE was 110 KD which is the same as that of humanIDE. CONCLUSIONS:IDE is the major enzyme which degrades insulin in enterocytes.
Authors: Malcolm A Leissring; Carlos M González-Casimiro; Beatriz Merino; Caitlin N Suire; Germán Perdomo Journal: Int J Mol Sci Date: 2021-02-24 Impact factor: 5.923
Authors: Carlos M González-Casimiro; Beatriz Merino; Elena Casanueva-Álvarez; Tamara Postigo-Casado; Patricia Cámara-Torres; Cristina M Fernández-Díaz; Malcolm A Leissring; Irene Cózar-Castellano; Germán Perdomo Journal: Biomedicines Date: 2021-01-17