Linked Life Data

9575193

Source:http://linkedlifedata.com/resource/pubmed/id/9575193

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
20
pubmed:dateCreated
1998-6-12
pubmed:abstractText
Experimental data indicate that the relatively hydrophilic carboxyl-terminal domains of Na+-H+ exchangers mediate the regulation of transporter activity through interactions with cytoskeletal effectors. It has therefore been assumed that this entire domain lies on the cytoplasmic surface of the plasma membrane. The purpose of the present study was to determine the membrane orientation of the COOH-terminal 131 amino acids of Na+-H+ exchanger isoform NHE3 by use of three monoclonal antibodies that recognize at least two distinct epitopes within this region. Enzyme-linked immunosorbent assay studies demonstrated binding of these monoclonal antibodies (mAbs) to intact right-side-out renal brush border membrane vesicles in the absence of detergent. Moreover, when coupled to an affinity matrix to isolate membrane vesicles, the anti-NHE3 mAbs bound structures that were morphologically identical to intact microvilli. To confirm the identity of the exoplasmic antigen bound by the antibodies, immunoprecipitation studies were performed. Intact right-side-out brush border membrane vesicles were incubated with the mAbs in the absence of detergent. The membranes were pelleted, supernatant with unbound antibody was removed, the pellet was solubilized, and then immunoprecipitation with secondary antibody was performed. Immunoblot analysis indicated that NHE3 was precipitated after binding of the mAbs to intact membranes. Finally, the localization of the mAb epitopes was determined using high resolution immunocytochemistry. Ultrathin cryosections of rat kidney were labeled with the mAbs and bound antibody detected with the colloidal gold technique. Labeling was restricted to the exoplasmic surface of microvilli of the proximal tubule. Taken together, these findings indicate that epitopes within the carboxyl terminus of the Na+-H+ exchanger isoform NHE3 are exposed to the outside of the plasma membrane.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
273
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
12391-6
pubmed:dateRevised
2011-11-17
pubmed:meshHeading
pubmed:year
1998
pubmed:articleTitle
Membrane topology of NHE3. Epitopes within the carboxyl-terminal hydrophilic domain are exoplasmic.
pubmed:affiliation
Departments of Internal Medicine and of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8029, USA. daniel.biemesderfer@yale.edu
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.