Primary structure of a novel 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS)-binding membrane protein highly expressed in Torpedo californica electroplax

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Item Type:	Article
Title:	Primary structure of a novel 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS)-binding membrane protein highly expressed in Torpedo californica electroplax
Creators Name:	Jentsch, T.J., Garcia, A.M. and Lodish, H.F.
Abstract:	Polyclonal rabbit antibodies were raised against 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS), an inhibitor of a variety of anion transport proteins. These antibodies specifically recognize SITS-reacted erythrocyte band 3 in immunoprecipitations and Western blots. In Western blots of SITS-reacted membrane proteins derived from vesicles of the electric organ of Torpedo californica (known to express a SITS-sensitive Cl- channel) the antibodies recognized two major species of approximately 93 kDa and approximately 105 kDa. The approximately 93 kDa protein was identified as the alpha-subunit of the Na,K-ATPase. The approximately 105 kDa protein (designated sp105) is a glycoprotein which binds to wheat-germ agglutinin and concanavalin A and is present as a disulphide-linked homodimer under non-reducing conditions. A partial amino acid sequence and a polyclonal antibody were used to clone the corresponding cDNA. sp105 is encoded in electroplax by two abundant mRNAs of approximately 6 and approximately 6.8 kb. A hybridizing mRNA of approximately 5 kb was over 200-fold and over 500-fold less abundant in brain and heart respectively. Sequence analysis of the cDNA predicted a novel protein of 697 amino acids containing eight potential N-linked glycosylation sites. Analysis of hydrophobicity indicated the presence of at least one, and possibly three, putative membrane-spanning domains. When expressed from the Sp6 message in Xenopus laevis oocytes, the protein was inserted into membranes, glycosylated and processed to form a dimer. However, no increase in 36Cl uptake or in membrane conductance could be detected. We found no effect of hybrid depleting the specific message on expression of the Torpedo electroplax Cl- channel in oocytes. Thus we conclude that this novel electroplax membrane protein is probably not a functional part of the chloride channel.
Keywords:	Amino Acid Sequence, Base Sequence, Carrier Proteins, Electric Organ, Membrane Proteins, Molecular Sequence Data, Torpedo, Animals
Source:	Biochemical Journal
ISSN:	0264-6021
Publisher:	Portland Press
Volume:	261
Number:	1
Page Range:	155-166
Date:	1 July 1989
Official Publication:	http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1138795/
PubMed:	View item in PubMed

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