SARCOPLASMIC-RETICULUM CALCIUM-ATPASE - LABELING OF A PUTATIVE MG2+ SITE BY REACTION WITH A CARBODIIMIDE AND A SPIN-LABEL

The sarcoplasmic reticulum Ca2+-ATPase loses hydrolytic activity and the ability to be phosphorylated by P-i following incubation with EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide]. 4 nmol of tempamine per mg SR protein can be coupled to either a glu or an asp side chain through the EDC reaction. Mg2+ protects against loss of activity and tempamine labeling with a mid-point of about 3 mM in the absence of Ca2+. This is similar to the K-d for a Mg2+ that serves as a cofactor in enzyme phosphorylation. The Mg2+ protection constant is lowered by an order of magnitude when Ca2+ is bound to the transport sites. It is suggested that control of the Mg2+ binding site affinity may be part of the mechanism of enzyme activation by Ca2+.