ATP-binding proteins on the external surface of synaptic plasma membranes: identification by photoaffinity labeling.
8-Azidoadenosine triphosphate labeled in the alpha or gamma position with 32P was used as a photoaffinity reagent for identifying ATP binding sites on the external surface of intact rat brain synaptosomes. As revealed by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns. UV irradiation of intact synaptosomes in the presence of the above radioactive compounds at 5-10 microM resulted in the formation of several major radioactive conjugates with approximate molecular masses of 29, 45/46, 58, and 93 kDa. Minor bands of 20, 39, 52/54, 82/84, 120, and 140 kDa were also consistently labeled in these experiments. The possibility that labeling of these proteins was due to the presence of contaminating subcellular particles or intrasynaptosomal proteins was excluded. The major 8-azidoadenosine [alpha-32P]triphosphate-labeled protein complex of approximately 45/46 kDa was resolved into several subbands that are labeled differently depending on the type of divalent cations added to the photoaffinity reaction. In the presence of magnesium only, the major labeled band appeared at 45 kDa. With calcium, two additional subbands (43 and 46 kDa) could be distinguished. In the presence of 1 mM EDTA, a band at 44 kDa was labeled within this ATP-binding complex. The labeling pattern of the subbands of this 45/46-kDa complex is consistent with these bands being extracellular ATP-binding proteins on the surface of the synaptosome.
Nagy
A K
AK
Department of Neurology, UCLA School of Medicine, USA.
Shuster
T A
TA
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001345:Autoradiography; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013570:Synaptic Membranes / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate