Nucleotide binding properties of a P-glycoprotein homologue from Plasmodium falciparum.
The Plasmodium falciparum P-glycoprotein homologue 1 (PGH1) is structurally similar to several members of the ATP-binding cassette (ABC) superfamily of membrane transporters. We have examined whether the nucleotide binding domains predicted from the deduced amino sequence are functional by photoaffinity labeling of purified parasite digestive vacuoles with the analogue 8-azido-alpha-[32P]ATP (8-N3-ATP). This reagent labels a 160-kDa protein in vacuoles from both a chloroquine sensitive and a chloroquine-resistant parasite isolate. The 160-kDa protein could be immunoprecipitated with affinity-purified antibodies against the P. falciparum P-glycoprotein homologue (PGH1). Inhibition of photoaffinity labeling of PGH1 could be achieved with ATP, ADP, GTP and GDP but not with AMP or GMP. In order to map the 8-N3-ATP binding sites on PGH1, photoaffinity-labeled PGH1 was digested with trypsin and immunoprecipitated with site-specific antibodies. Taken together, these results indicate that 8-N3-ATP specifically labels PGH1 and that one binding site resides within the amino terminal half of the molecule. This supports the contention that PGH1 is involved in a nucleotide-regulated transport function across the membrane of the digestive vacuole.
Karcz
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SR
Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia.
Galatis
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D
Cowman
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AF
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D009711:Nucleotides / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D009711:Nucleotides; D015800:Protozoan Proteins; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate