Calcium- and magnesium-binding properties of a high affinity calcium-binding protein from crayfish sarcoplasm.

Equilibrium binding studies show that 6 atoms of calcium are bound by the dimeric molecule of the sarcoplasmic calcium-binding protein (SCP) from crayfish. SCP exhibits positive cooperativity in calcium-binding for four sites, as indicated by increasing intrinsic binding constants (1 to 5 x 10” M-l), and negative cooperativity for the other two sites (decreasing constants from 5 to 0.8 x lo7 M-‘). In the presence of 1 mM Mg’+, the binding constants are reduced by about 1 order of magnitude, but cooperativity is conserved. Mg2+ competes with Ca2+ for four out of six sites on SCP. In the absence of Ca’+, magnesium binds to SCP with positive cooperativity for two sites and with negative cooperativity for two others, the four intrinsic binding constants for Mg2+ ranging from 0.4 to 1.0 X 10” M-‘. MgSCP is essentially as stable as Ca-SCP, in contrast to the metal-free protein, which denatures readily. A purified preparation of fragmented sarcoplasmic reticulum removes bound calcium from SCP easily and, in the presence of physiological levels of Mg2+, the latter cation replaces Ca”+ on the Ca-Mg sites, suggesting that exchange of Ca2+ and Mg2’ takes place during muscular activity. With its two calcium-specific sites, two Ca-Mg sites that interact with positive cooperativity and two Ca-Mg sites that interact with negative cooperativity, crayfish SCP displays more complex metal-binding properties than does parvalbumin, the sarcoplasmic calcium-binding protein found in vertebrates which has only two noninteracting Ca-Mg sites. Furthermore, a comparison between the metal-binding properties of SCP’s and troponin C’s from invertebrate muscle reveals that the former are much more complex than the latter. This contrasts sharply with the situation prevailing in vertebrate skeletal muscle, where complexity is found in myofibrillar troponin C and not in sarcoplasmic parvalbumin.