Purification and partial characterization of candidate antidiuretic hormone water channel proteins of M(r) 55,000 and 53,000 from toad urinary bladder.

H W Harris,C Hosselet,L Guay-Woodford,M.L Zeidel

doi:10.1016/s0021-9258(18)41643-2

H W Harris, C Hosselet + Show 2 more

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https://doi.org/10.1016/s0021-9258(18)41643-2

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Abstract

Antidiuretic hormone (ADH) increases toad bladder granular cell apical membrane osmotic water permeability (Pf) by insertion of cytoplasmic vesicles containing water channels into the apical membrane. Termination of ADH stimulation results in endocytosis of water channel-containing membrane. In previous work, we have purified water channel-containing vesicles and demonstrated that they contain 12 major protein bands when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). On the basis of vectorial labeling studies of granular cells and purified vesicles, we have proposed previously that vesicle proteins of 55, 53, and 17 kDa are ADH water channel components. In this report, we have purified and analyzed these three proteins using a combination of SDS-PAGE, peptide mapping, amino acid composition, and amino-terminal analyses. The 55- and 53-kDa proteins are distinct protein species possessing a high degree of structural similarity. Both possess a large content of cysteine. The 17-kDa protein appears to be a proteolytic fragment of the 53-kDa protein. None of these three proteins is phosphorylated or contains large amounts of covalently linked carbohydrate. ADH-elicited Pf is inhibited by the organic mercurial reagent fluorescein mercuric acetate (FMA). Exposure of water channel-containing vesicles to FMA labels selectively four vesicle proteins of 92, 55, 53, and 29 kDa while reducing vesicle Pf by 82%. The combination of FMA and 2-mercaptoethanol or exposure to another mercurial reagent, n-ethylmaleimide, does not inhibit vesicle Pf. Together, these data provide additional evidence for the role of the 55- and 53-kDa proteins as components of the ADH water channel. These candidate ADH water channel proteins are distinct from a 28-kDa candidate water channel protein (CHIP 28) isolated recently from human erythrocyte membranes and kidney proximal tubule by Agre and co-workers (Preston, G. M., Carroll, T. P., Guggino, W. B., and Agre, P. (1992) Science 256, 385-387).

Highlights

Antidiuretic hormone (ADH) increases toad bladder ADH’ stimulation of epithelial cells in themammalian granular cell apical membraneosmotic water permea- collecting tubule and anuran urinary bladder cause an inbility ( P f )by insertion of cytoplasmic vesicles contain- creaseintheir apical membrane permeabilities to sodium, ing water channels into the apicaml embrane
On the basis of these data, we have proposed that these proteins are ADH water channel components [1,27]
Purification of 55,53, and 17-kDa Proteins-Previous data producedfrom LPO-mediatedapicalmembraneand intraendosomal ""I-labeling of ADH-stimulated granularcells [20] as well as quantitation of proteins from purified water channel-containing vesicles (WCV) [27] revealed that candidate ADH water channel proteins of 55 and 53 kDa appear as distinct sharp bands,while the 17kDa band is quite broad, stains poorly with Coomassie Blue, and extends from 20 to 15.5 kDa

Summary

Introduction

Antidiuretic hormone (ADH) increases toad bladder ADH’ stimulation of epithelial cells in themammalian granular cell apical membraneosmotic water permea- collecting tubule and anuran urinary bladder cause an inbility ( P f )by insertion of cytoplasmic vesicles contain- creaseintheir apical membrane permeabilities to sodium, ing water channels into the apicaml embrane. The 17-kDa protein microscopy have related water channel insertion to the apappears to be a proteolytic fragment of the 53-kDa pearance of structures called particle aggregates in the apical protein. None of these three proteinis phosphorylated membrane of ADH-responsive cells [14, 15].

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