[44] Preparation of microsomal β-glucuronidase and its membrane anchor protein, egasyn

Publisher Summary Acid hydrolases, such as β-glucuronidase are typically located in lysosomes, but in certain tissues of mammals, substantial amounts of the enzyme are also present in the membranes of the endoplasmic reticulum. This chapter provides an overview of the preparation of microsomal β-glucuronidase and its membrane anchor protein, Egasyn. Egasyn serves to stabilize the binding of β-glucuronidase to membrane, and a mouse strain that lacks egasyn is unable to maintain fl-glucuronidase in endoplasmic reticulum membrane. Thus, egasyn appears to function as a membrane anchor for the highly polar glucuronidase tetramer. The β-glucuronidase purification assay involves electrophoresis, lysosomal (L form) β-glucuronidase, raising antisera, antibody affinity chromatography, purification of microsomal glucuronidase from mouse liver or kidney, and rat microsomal β-glucuronidase. Isolation and assay of egasyn involves isolation from mouse tissues, glucuronidase binding protein from rat tissues, antiserum against egasyn, labeling with 125 I, titration of labeled egasyn with anti-egasyn antibody, and radioimmunoassay of egasyn.