Structure of UDP-glucuronosyltransferases in membranes.

This chapter presents the most recent experimental approaches to the investigation of UDP-glucuronosyltransferase (UGTs) in membranes. The first topic described is the subcellular localization of UGTs with special emphasis on the association of these proteins with the endoplasmic reticulum (ER). Experimental methods include subfractionation of tissue for microsome preparation, evaluation of the purity of the membrane fraction obtained, and measurement of UGT activity in the presence of detergents. Next, the recently demonstrated formation of UGT homo- and heterodimer formation and its functional relevance is discussed and the appropriate methods used to characterize such interactions are given (radiation inactivation, size exclusion chromatography, immunopurification, cross-linking, two-hybrid system). The structural determinants of UGTs in relation to membrane association, residency, and enzymatic activity are the next topic, supplemented by a description of the appropriate methods, including the design and expression of chimeric proteins, membrane insertion, and subcellular localization by immunofluorescence. Also presented is new information on the structure and function of UGTs obtained by molecular modeling, bioinformatics (sequence alignment), and comparison with selected crystallized glycosyltransferases. Finally, we discuss the important, and still not fully developed, issue of UGT active site architecture and organization within the ER. This is addressed from two perspectives: (1) chemical modification of UGT active sites by amino acid-specific probes and (2) photoaffinity labeling of UGTs. The detailed synthesis of a photoaffinity probe for an aglycon-binding site is provided and the use of this probe and direct photoaffinity labeling with retinoids is discussed. The application of proteomics techniques, including proteolytic digestion and protein sequencing by liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption ionization/time of flight, to the identification of crucial amino acids of the active sites, and subsequent site-directed mutagenesis of identified amino acids, is discussed in detail.