A sensitive assay is developed to assess the existence of an iodide channel in a fraction of solubilized membrane proteins. This step is critical when considering various procedures for purification of this channel. Sodium cholate is used as a detergent as it does not denature the iodide channel. A simple and rapid method involving gel-filtration chromatography is used simultaneously to remove the detergent and to adjust the buffer composition, before protein insertion into liposomes. The presence of an iodide channel is investigated by measuring the iodide conductance of these proteoliposomes at 4 degrees C. An outward iodide gradient is set up across the proteoliposomal membrane by anion-exchange chromatography, allowing uptake of radiolabelled iodide. This uptake is conductive as it is abolished by valinomycin in the presence of potassium. It is specifically mediated by a thyroid plasma-membrane protein inserted into liposomes, as its denaturation before insertion totally abolished uptake. It was observed only within a well-defined fraction of thyroid membrane proteins collected by size-exclusion chromatography (molecular mass between 100 and 200 kDa). Furthermore, it was not observed with other membrane proteins such as ileal brush-border-membrane proteins or bacteriorhodopsin. Like many anion channels, this conductance was also inhibited by N-phenylanthranilic acid. Optimization of the assay is described, validating the measurement of conductive iodide uptake at 30 s by proteoliposomes reconstituted in a ratio of 10 micrograms of protein to 90 micrograms of lipid, with an outward iodide gradient (KI 15 mM inside and 1 microM outside). This assay provides a test of the biological activity of the iodide channel at each step of the purification; it can be applied to any anionic channel.
Skip Nav Destination
Follow us on Twitter @Biochem_Journal
Article navigation
December 1995
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkAdvertising
Research Article|
December 01 1995
Methodology for assaying iodide conductance in proteoliposomes: specific induction by thyroid membrane protein Available to Purchase
P E Golstein;
P E Golstein
*Laboratory of Physiology and Pathophysiology, B 1070 Brussels, Belgium
Search for other works by this author on:
A Sener;
A Sener
†Laboratory of Experimental Medicine, School of Medicine, Université Libre de Bruxelles, B 1070 Brussels, Belgium
Search for other works by this author on:
R Beauwens
R Beauwens
*Laboratory of Physiology and Pathophysiology, B 1070 Brussels, Belgium
Search for other works by this author on:
Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1995 The Biochemical Society, London
1995
Biochem J (1995) 312 (2): 543–548.
Citation
P E Golstein, A Sener, R Beauwens; Methodology for assaying iodide conductance in proteoliposomes: specific induction by thyroid membrane protein. Biochem J 1 December 1995; 312 (2): 543–548. doi: https://doi.org/10.1042/bj3120543
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Could not validate captcha. Please try again.
Biochemical Society Member Sign in
Sign InSign in via your Institution
Sign in via your InstitutionGet Access To This Article
Cited By
Follow us on Twitter @Biochem_Journal
Open Access for all
We offer compliant routes for all authors from 2025. With library support, there will be no author nor reader charges in 5 journals. Check here |
![]() View past webinars > |