Transport studies with brush border membrane vesicles: choice of experimental parameters

1. We have studied different parameters, in their effects on a transport system chosen as a model: the Na+-phosphate symporter of the renal brush border membrane. 2. Ionic strength was found to be a critical factor in the retention capacity of the filter. 3. When high ionic strength solutions containing 150 mM NaCl or KCl were used, less than 8% of the membrane proteins were lost through filtration. 4. Lowering the ionic strength by replacing NaCl or KCl by 300 mM mannitol, however, caused a 52% loss of protein. 5. Addition of 15 mM NaCl to this low ionic strength solution was sufficient to restore full retention of the vesicles by the filter. 6. The presence of arsenate, a competitive inhibitor, in the stop solution did not improve the retention of phosphate by the vesicles in high ionic strength media, but caused a pronounced temperature dependent loss of the vesicle content, as a function of time of incubation in low ionic strength solutions. 7. Addition of 5 mM phosphate in the stop solution caused a 31 and 37% loss for KCl and NaCl stop solutions, respectively, while no effect was observed for the mannitol stop solution. 8. The presence of HgCl2 gave a 32% stimulation for the mannitol solution and a 35 or 22% inhibition for the KCl or NaCl solutions. 9. Addition of NaCl in the stop solution caused an overaccumulation of 75%, after 60 sec of incubation at 25 degrees C. 10. Phosphate transport by renal vesicles is thus highly affected by the composition of the stop solution.(ABSTRACT TRUNCATED AT 250 WORDS)