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

The sarcoplasmic reticulum Ca2+-ATPase loses hydrolytic activity and t he ability to be phosphorylated by P-i following incubation with EDC [ 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide]. 4 nmol of tempamine pe r mg SR protein can be coupled to either a glu or an asp side chain th rough the EDC reaction. Mg2+ protects against loss of activity and tem pamine 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 en zyme phosphorylation. The Mg2+ protection constant is lowered by an or der of magnitude when Ca2+ is bound to the transport sites. It is sugg ested that control of the Mg2+ binding site affinity may be part of th e mechanism of enzyme activation by Ca2+.