Title

Localization of type I iodothyronine 5'-deiodinase to the basolateral plasma membrane in renal cortical epithelial cells

UMMS Affiliation

Molecular Endocrinology Laboratory; Department of Medicine, Division of Endocrinology amp; Metabolism

Publication Date

6-15-1991

Document Type

Article

Subjects

Affinity Labels; Animals; Cell Line; Cell Membrane; Detergents; Epithelium; Iodide Peroxidase; Isoenzymes; Kidney Cortex; Kidney Glomerulus; Kinetics; Molecular Weight; Rats; Subcellular Fractions; Thyroxine

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

Type I iodothyronine 5'-deiodinase is an integral membrane protein catalyzing the phenolic ring deiodination of thyroxine. We recently showed that the substrate binding subunit of this approximately 50-kDa protein is selectively labeled with N-bromoacetyl-L-thyroxine, allowing ready identification of the type I enzyme without the need to maintain catalytic activity. In this study, we used both affinity labeling and catalytic activity to determine the regional distribution of this enzyme in rat kidney and to localize the enzyme to specific plasma membrane domain(s) of renal epithelial cells. The type I enzyme was present exclusively in tubular epithelial cells of the outer renal cortex and co-purified with basolateral plasma membranes; the renal medulla lacked activity. LLC-PK1 cells, derived from the proximal convoluted tubule, have abundant type I 5'-deiodinating activity. We used this homogenous cell line to verify that the type I enzyme was localized to the cytosolic surface of the basolateral membrane. Digitonin permeabilization increased affinity labeling of the enzyme 4-fold, and approximately 75% of the affinity label was incorporated into the 27-kDa substrate binding subunit. Affinity labeling of the type I enzyme in LLC-PK1 cells mimicked the affinity labeling of the substrate binding subunit of type I 5'-deiodinase in rat kidney (Kohrle, J., Rasmussen, U. B., Ekenbarger, D. M., Alex, S., Rokos, H., Hesch, R. D., and Leonard, J. L. (1990) J. Biol. Chem. 265, 6155-6163). Subcellular fractionation of LLC-PK1 cell homogenates showed that both affinity labeled and catalytically active type I enzyme were present on the cytosolic surface of the basolateral region of the renal cell membrane.

Rights and Permissions

Citation: J Biol Chem. 1991 Jun 15;266(17):11262-9.

Related Resources

Link to Article in PubMed

Journal/Book/Conference Title

The Journal of biological chemistry

PubMed ID

2040632