Identification and characterization of membrane-associated polypeptides in Torpedo nicotinic acetylcholine receptor-rich membranes by hydrophobic photolabeling

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Identification and characterization of membrane-associated polypeptides in Torpedo nicotinic acetylcholine receptor-rich membranes by hydrophobic photolabeling

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dc.contributor.author BLANTON, MP en_US
dc.contributor.author LALA, AK en_US
dc.contributor.author COHEN, JB en_US
dc.date.accessioned 2011-07-25T06:30:47Z en_US
dc.date.accessioned 2011-12-26T12:50:04Z en_US
dc.date.accessioned 2011-12-27T05:36:02Z
dc.date.available 2011-07-25T06:30:47Z en_US
dc.date.available 2011-12-26T12:50:04Z en_US
dc.date.available 2011-12-27T05:36:02Z
dc.date.issued 2001 en_US
dc.identifier.citation BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES, 1512(2), 215-224 en_US
dc.identifier.issn 0005-2736 en_US
dc.identifier.uri http://dx.doi.org/10.1016/S0005-2736(01)00321-2 en_US
dc.identifier.uri http://dspace.library.iitb.ac.in/xmlui/handle/10054/6662 en_US
dc.identifier.uri http://hdl.handle.net/10054/6662
dc.description.abstract To identify membrane-associated polypeptides present in Torpedo nicotinic acetylcholine receptor (AChR)-rich membranes, we used hydrophobic photolabeling with [H-3]diazofluorene ([H-3]DAF) and 1-azidopyrene (1-AP) to tag the membrane proteins which were then identified by amino-terminal sequence analysis of labeled fragments isolated from proteolytic digests by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reverse-phase high-performance liquid chromatography. In addition to AChR subunits, identified polypeptides include the 95 kDa alpha -subunit of the (Na++K+)-ATPase, the 89 kDa voltage-gated chloride channel (CLC-0), the 105 kDa SITS-binding protein, and 32 and 34 kDa polypeptides identified as Torpedo homologues of the mitochondrial membrane ATP/ADP carrier protein and the voltage-dependent anion channel (VDAC), respectively. Further, individual amino acids that reacted with [H-3]DAF and therefore likely to be in contact with lipid were identified in the transmembrane segment M3 of the alpha -subunit of the (Na++K+)-ATPase and in a putative transmembrane beta -strand in VDAC. Collectively these results demonstrate that [H-3]DAF/1-AP photolabeling provides an effective method for tagging the membrane-associated segments of polypeptides in a way that makes it easy to isolate the labeled polypeptide or polypeptide fragments by fluorescence and then to identify amino acids at the lipid-protein interface by H-3 release. (C) 2001 . en_US
dc.language.iso en en_US
dc.publisher ELSEVIER SCIENCE BV en_US
dc.subject gated chloride channel en_US
dc.subject amino-acid-sequence en_US
dc.subject mitochondrial porin en_US
dc.subject electric organ en_US
dc.subject postsynaptic membranes en_US
dc.subject skeletal-muscle en_US
dc.subject agrin receptor en_US
dc.subject ion-channel en_US
dc.subject protein en_US
dc.subject purification en_US
dc.subject.other [h-3]diazofluorene en_US
dc.subject.other nicotinic acetylcholine receptor en_US
dc.subject.other (na++k+)-atpase en_US
dc.subject.other voltage-dependent anion channel en_US
dc.subject.other photoaffinity en_US
dc.title Identification and characterization of membrane-associated polypeptides in Torpedo nicotinic acetylcholine receptor-rich membranes by hydrophobic photolabeling en_US
dc.type Article en_US


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