Photoaffinity labeling of brush-border membrane proteins which bind phosphonoformic acid

H A Al-Mahrouq,S A Kempson

doi:10.1016/s0021-9258(18)52311-5

Abstract

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.

Highlights

Identification and characterizationof the Na+/Pico- T h e lack of a specific covalent label has hindered attempts transporter in therenal brush-bordemr embrane to identify and characterize the Na+/Pi co-transporteirn the (BBM) has proved to be difficultin partbecause of the renal Brush-border membrane (BBM)
In contrast to masses of 97 and 70 kDa. Labelingof these bands was other specific inhibitors, such as phosphonoformicacid (9, markedly reduced in the presence of Na+ and phos- lo), NAD has the additional advantage of being a relatively phonoformicacid, a specific inhibitor of Na+/Pi co- large molecule so that a reactive groupcan be attached without transport. chromatography of solubilized BBM proteins indicated that the protein fraction whiicshphomarked changes in its inhibitory action
Where there was marked incorporation of '"P in controls ( C ) hut laheling was reduced in the presence of phosphonoformic acid and Attachment of the highly reactive azido group to NAD did not interfere with the inhibitory action of NAD on Na'/P, (Table 111)

Summary

Azidobenzoic acid

Binding of [14CJphosphonoformiaccid to solubilized BBM proteins in column fractions was determined asfollows. 120 p ~fo)r 20 s a t 20 "C before the addition of the uptake mixture. Solute transport was determined NaCl or 10 mM KCl. Fractions of 0.3 ml were collected, and "C was by the rapid filtration techniqudescribed and used previously After addition of the uptake mixture, the final composition of All experiments were repeated at least twice. Differences between the mixture was 100 mM NaC1, 100 mM mannitol, 5 mM Tris-Hepes control and experimental groups were analyzed with the Student t (pH 7.4), 0.5 mM EDTA, 1 mM nicotinamide,andeither 0.1 mM test for groupcomparisons.

RESULTS

PhotoaffinityLabeling of Renal RRM

DISCUSSION

PhotoaffinityLabeling of Renal BBM