An autoinhibitory peptide from the erythrocyte Ca-ATPase aggregates and inhibits both muscle Ca-ATPase isoforms

We have studied the effects of C28R2, a basic peptide derived from the auto inhibitory domain of the plasma membrane Ca-ATPase, on enzyme activity, oli gomeric state, and E1-E2 conformational equilibrium of the Ca-ATPase from s keletal and cardiac sarcoplasmic reticulum (SR). Time-resolved phosphoresce nce anisotropy (TPA) was used to determine changes in the distribution of C a-ATPase among its different oligomeric species in SR. C28R2, at a concentr ation of 1-10 mu M, inhibits the Ca-ATPase activity of both skeletal and ca rdiac SR (CSR), In skeletal SR, this inhibition by C28R2 is much greater at low (0.15 mu M) than at high (10 mu M) Ca2+, whereas in CSR the inhibition is the same at low and high Ca2+. the effects of the peptide on the rotati onal mobility of the Ca-ATPase correlated well with function, indicating th at C28R2-induced protein aggregation and Ca-ATPase inhibition are much more Ca-dependent in skeletal than in CSR. In CSR at low Ca2+ phospholamban (PL B) antibody (functionally equivalent to PLB phosphorylation) increased the inhibitory effect of C28R2 slightly. Fluorescence of fluorescein 5-isothioc yanate-labeled SR suggests that C28R2 stabilizes the El conformation of the Ca-ATPase in skeletal SR, whereas in CSR it stabilizes E2, After the addit ion of PLB antibody, C28R2 still stabilizes the E2 conformational state of CSR. Therefore, we conclude that C28R2 affects Ca-ATPase activity, conforma tion, and self-association differently in cardiac and skeletal SR and that PLB is probably not responsible for the differences.