AJP - Renal Physiology, Vol 249, Issue 6 806-F812, Copyright © 1985 by American Physiological Society
Radiation inactivation studies of renal brush border water and urea transport
A. S. Verkman, J. A. Dix, J. L. Seifter, K. L. Skorecki, C. Y. Jung and D. A. Ausiello
Radiation inactivation was used to determine the nature and molecular
weight of water and urea transport pathways in brush border membrane
vesicles (BBMV) isolated from rabbit renal cortex. BBMV were frozen to -50
degrees C, irradiated with 1.5 MeV electrons, thawed, and assayed for
transport or enzyme activity. The freezing process had no effect on enzyme
or transport kinetics. BBMV alkaline phosphatase activity gave linear
ln(activity) vs. radiation dose plots with a target size of 68 +/- 3 kDa,
similar to previously reported values. Water and solute transport were
measured using the stopped-flow light-scattering technique. The rates of
acetamide and osmotic water transport did not depend on radiation dose (0-7
Mrad), suggesting that transport of these substances does not require a
protein carrier. In contrast, urea and thiourea transport gave linear
ln(activity) vs. dose curves with a target size of 125-150 kDa; 400 mM urea
inhibited thiourea flux by -50% at 0 and 4.7 Mrad, showing that radiation
does not affect inhibitor binding to surviving transporters. These studies
suggest that BBMV urea transport requires a membrane protein, whereas
osmotic water transport does not.
