E. Bayer, M. Wilchek
1977
Citations
0
Influential Citations
8
Citations
Journal
Methods in Enzymology
Abstract
Publisher Summary In this chapter the biotin transport system has been examined. The chapter describes affinity labeling studies on intact cells using well characterized active transport system. The biotin transport system in yeasts has been characterized as a high-affinity, carrier-mediated, energy-requiring process. Since the carrier recognizes the ureido ring of the biotin molecule, a broad range of modifications of the valeric acid side chain are possible without affecting the inherent affinity. The chapter explains identification of biotinyl-p-nitrophenyl ester (pBNP) as the potent inhibitor of biotin transport. It also provides and expansion to the above approach, and describes the methodology in which a homologous series of affinity labeling reagents was synthesized in which the chemically reactive p-nitrophenyl ester group is located at increasing distances from the biologically active ureido moiety. The affinity labels were selected in order to encompass chain lengths corresponding to those of biotin and biocytin, both of which occurs in nature. The chapter details Preparation of Biotinyl-p-nitrophenyl Ester (BNP), Preparation of Model Affinity Labels, and Biotin Uptake Assay.