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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Title: Identification and characterization of membrane-associated polypeptides in Torpedo nicotinic acetylcholine receptor-rich membranes by hydrophobic photolabeling
Author: BLANTON, MP; LALA, AK; COHEN, JB
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 .
URI: http://dx.doi.org/10.1016/S0005-2736(01)00321-2
http://dspace.library.iitb.ac.in/xmlui/handle/10054/6662
http://hdl.handle.net/10054/6662
Date: 2001


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