Photoaffinity labelling of P-glycoprotein catalytic sites.
Photoaffinity labelling of hamster P-glycoprotein was carried out after trapping of radioactive Mg-8-azido-ADP in the catalytic sites by vanadate or beryllium fluoride. With either trapping agent the same labelled peptide was obtained in homogeneous form, with the sequence -FNEVVFNxPTRPDI-, corresponding to residues 1034-1037 in the C-terminal nucleotide binding site. The missing residue 'x' corresponds to Tyr-1041, which is therefore a primary reaction target of 8-azido-ADP. This tyrosine is conserved in all hamster, mouse and human P-glycoproteins. A second major labelled peptide fraction was also identified. The major sequence in this fraction was -NIHFSxPSR-, corresponding to residues 393-401 of hamster P-glycoprotein, where 'x' corresponds to Tyr-398 in the N-terminal nucleotide binding site. Therefore Tyr-398, which is also conserved in other P-glycoproteins, is also a reaction target for 8-azido-ADP. In sequence alignment of the two nucleotide binding sites, Tyr-398 exactly corresponds to Tyr-1041. The data indicate that these two tyrosines lie close to the adenine ring of bound substrate MgATP in the respective catalytic sites of P-glycoprotein.
Sankaran
B
B
Department of Biochemistry and Biophysics, University of Rochester Medical Center, NY 14642, USA.
Bhagat
S
S
Senior
A E
AE
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001386:Azides / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D006224:Cricetinae; D019920:Photoaffinity Labels; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides; D019920:Photoaffinity Labels; D014443:Tyrosine; D000244:Adenosine Diphosphate; D014357:Trypsin; C021622:8-azidoadenosine diphosphate