Ca²⁺ Binding and Transport Studied with Ca²⁺/EGTA Buffers and ⁴⁵Ca²⁺

Abstract

The chapter describes procedures useful for determination of Ca²⁺ binding by membranous Ca²⁺-ATPase based on the correction for the removal of Ca²⁺ present in a non-bound state in the suspension medium. This is done by a filtration procedure that retains the membranous material on the Millipore filters. With suitable sucking devices it is possible to gently remove without dehydration nearly all medium from the Ca²⁺ containing membranes, except that required for wetting of the filters on which they are deposited. Correction for this effect can be done with a double-filter where the radioactive content of the lower (protein-free) filter is subtracted from that present in the upper filter for calculation of Ca²⁺ binding. This methodology can be used to study the effect of inhibitors on Ca²⁺ binding and –transport, and with Ca²⁺/EGTA buffers to explore the Ca²⁺ binding affinities and cooperative aspects of the two transport sites.