Photoaffinity labeling of brush-border membrane proteins which bind phosphonoformic acid.
Identification and characterization of the Na+/Pi co-transporter in the renal brush-border membrane (BBM) has proved to be difficult in part because of the lack of a specific covalent label. NAD is a competitive inhibitor of Na+/Pi co-transport, and we have explored its potential use as a specific label. We describe the synthesis and use of a highly reactive azido derivative of NAD. This derivative (AB-NAD), like the parent NAD molecule, acts as a competitive inhibitor of Na+/Pi co-transport by isolated BBM vesicles. After photoirradiation, the inhibition changes to noncompetitive, as would be expected if the label was bound covalently. This was confirmed by use of [3H]AB-NAD. Photoirradiation produced a 4-fold increase in acid-stable incorporation of 3H into BBM vesicles compared to controls which were not exposed to light. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that photoirradiation with [32P]AB-NAD produced labeling of several different protein bands, but almost one-half of the 32P was recovered in two bands corresponding to molecular masses of 97 and 70 kDa. Labeling of these bands was markedly reduced in the presence of Na+ and phosphonoformic acid, a specific inhibitor of Na+/Pi co-transport. Chromatography of solubilized BBM proteins indicated that the protein fraction which is photolabeled by AB-NAD is co-eluted with the protein fraction which exhibits Na(+)-dependent binding of phosphonoformic acid. The 97- and 70-kDa polypeptide bands may contain components of the intact Na+/Pi co-transport system.
al-Mahrouq
H A
HA
Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis 46202-5120.
Kempson
S A
SA
D000345:Affinity Labels / Q000737:chemistry; D000818:Animals; D001386:Azides; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D017245:Foscarnet; D007672:Kidney Cortex / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism*; D008970:Molecular Weight; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D010777:Photochemistry; D012964:Sodium / Q000378:metabolism*; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters
D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D008565:Membrane Proteins; D010710:Phosphates; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; D009243:NAD; D017245:Foscarnet; D012964:Sodium; D010746:Phosphonoacetic Acid