AJP - Renal Physiology, Vol 248, Issue 4 472-F481, Copyright © 1985 by American Physiological Society
High-affinity calcium binding sites in luminal and basolateral renal membranes
Z. Talor, G. Richison and J. A. Arruda
We evaluated Ca binding by highly purified luminal (L) and basolateral (BL)
tubular membranes prepared from beef kidney. Ca binding was measured by
using 45Ca and a rapid-filtration technique. After Ca uptake reached
equilibrium, the vesicles were lysed and the amount of 45Ca retained in the
membranes was considered the bound Ca. Ca binding in both membranes
accounted for approx. 80% of total Ca uptake. Analysis of binding data by
Scatchard plot revealed the presence of two distinct types of binding sites
in both L and BL membranes. The high-affinity binding sites showed a
similar affinity constant of 10(-5)M for both L and BL membranes, but the
maximum number of binding sites was 0.75 and 1.6 nmol/mg protein,
respectively. In contrast, the low-affinity binding sites were similar
regarding affinity constant and maximum number of binding sites in the two
membranes. In L and BL membranes, high-affinity binding sites were
selective for Ca, as high concentrations of divalent cations were required
to inhibit Ca binding. In both membranes Ca binding was inhibited by
ruthenium red, LaCl3, and detergents, and it was stimulated by calmodulin
inhibitors (trifluoperazine, calmidazolium), ionophore A-23187, and ATP.
These results demonstrate that L and BL membranes possess high-affinity
binding sites with different capacities but similar characteristics as
regards affinity constant and stimulation and inhibition of binding. The
data further demonstrate that most of Ca uptake by these membranes
represents binding.
