Reconstitution of the Na+/H+ antiporter: a new method for the determination of H+ efflux from Na+/H+ antiporter-reconstituted vesicles.
A Na+/H+ antiporter was solubilized with octyl-beta-D-glucopyranoside from rat renal brush border membrane and reconstituted in egg phosphatidylcholine liposomes. The activity of the antiporter was determined by a new method. Reconstitution was performed in a solution containing a pH-sensitive fluorescence dye, fluorescein isothiocyanate-dextran (FITC-dextran), at pH 5.5. The reconstituted vesicles, encapsulating FITC-dextran, were incubated at pH 8.0, and the intravesicular pH (pHi) was determined by monitoring fluorescence change of the dye. An increase in the fluorescence intensity which corresponded to an increase in pHi was observed. The rate of the increase was dependent on the concentration of the extravesicular Na+, and the efflux of H+ in the presence of 150 mM sodium gluconate was suppressed by the addition of amiloride. These results show that the Na+/H+ antiporter was functionally reconstituted and that its activity can be determined simply by the use of FITC-dextran.
Shibamoto
S
S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Osaka, Japan.
Yoshida
S
S
Oku
N
N
Hayakawa
M
M
Hori
T
T
Ito
F
F
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003911:Dextrans; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins; D066298:In Vitro Techniques; D007474:Ion Exchange; D007668:Kidney / Q000378:metabolism; D008081:Liposomes; D008871:Microvilli / Q000378:metabolism; D011522:Protons; D051381:Rats; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D003911:Dextrans; D005452:Fluoresceins; D008081:Liposomes; D011522:Protons; D017923:Sodium-Hydrogen Exchangers; C015219:fluorescein isothiocyanate dextran; D012964:Sodium; D016650:Fluorescein-5-isothiocyanate