Properties of calcium binding by Myxicola axoplasmic protein

Abstract

The 45Ca 2+ binding properties of axoplasmic protein from the Myxicola giant axon have been investigated using a centrifugal/concentration-dialysis technique. Scatchard plot analysis of these binding data suggest that Ca 2+ is attached to a site with an equilibrium dissociation constant of 7.7 ± 0.5 μM and a capacity of 4.4 ± 0.2 μmol/g axoplasmic protein (n = 11). Addition of other cations — Cd 2+, Mn 2+, Al 3+, Cu 2+, Ba 2+, and Zn 2+ — at concentrations up to 10 μM did not displace 0.2 μM 45Ca 2+ from its binding site, probably because of buffering of these cations by amino acid residues within the protein solutions. The protein could be stored at 4°C for up to 16 days with no appreciable change in the number of calcium sites. Ca 2+ binding equilibrium took place in less than 30 min of incubation. Increasing the incubation temperature from 4°C to 37°C reduced the number of Ca 2+ sites. The binding capacity was reduced by one-half when the protein was dialyzed with 4 M urea or high ionic strength KCl (2 M). Calcium binding was examined as a function of pH. When the protein was dialyzed overnight at different pH values and all the binding was done at pH 7.0, the apparent number of Ca 2+ sites decreased as the pH of the dialysis medium was increased. When the protein was dialyzed overnight at pH 7.0 and the binding was done at different pH values, the apparent binding capacity increased as pH increased. When the protein was dialyzed overnight at the same pH at which the binding was done, the apparent binding capacity reached a maximum at pH 7.0. The histidine-specific reagent diethyl pyrocarbonate (DEP) reduced the apparent binding capacity.