BINDING OF CALCIUM BY CALMODULIN - INFLUENCE OF THE CALMODULIN BINDING DOMAIN OF THE PLASMA-MEMBRANE CALCIUM-PUMP

The interaction between calmodulin and synthetic peptides correspondin g to the calmodulin binding domain of the plasma membrane Ca2+ pump ha s been studied by measuring Ca2+ binding to calmodulin. The largest pe ptide (C28W) corresponding to the complete 28 amino acid calmodulin bi nding domain enhanced the Ca2+ affinity of calmodulin by more than 100 times, implying that the binding of Ca2+ increased the affinity of ca lmodulin for the peptide by more than 10(8) times. Deletion of the 8 C -terminal residues from peptide C28W did not decrease the affinity of Ca2+ for the high-affinity sites of calmodulin, but it decreased that for the low-affinity sites. A larger deletion (13 residues) decreased the affinity of Ca2+ for the high-affinity sites as well. The data sug gest that the middle portion of peptide C28W interacts with the C-term inal half of calmodulin. Addition of the peptides to a mixture of tryp tic fragments corresponding to the N- and C-terminal halves of calmodu lin produced a biphasic Ca2+ binding curve, and the effect of peptides was different from that on calmodulin. The result shows that one mole cule of peptide C28W binds both calmodulin fragments. Interaction of t he two domains of calmodulin through the central helix is necessary fo r the high-affinity binding of four Ca2+ molecules.