We have studied the effects of C28R2, a basic peptide derived from the autoinhibitory domain of the plasma membrane Ca-ATPase, on enzyme activity, oligomeric state, and E1-E2 conformational equilibrium of the Ca-ATPase from skeletal and cardiac sarcoplasmic reticulum (SR). Time-resolved phosphorescence anisotropy (TPA) was used to determine changes in the distribution of Ca-ATPase among its different oligomeric species in SR. C28R2, at a concentration of 1-10 μM, inhibits the Ca-ATPase activity of both skeletal and cardiac SR (CSR). In skeletal SR, this inhibition by C28R2 is much greater at low (0.15 μM) than at high (10 μM) Ca2+, whereas in CSR the inhibition is the same at low and high Ca2+. The effects of the peptide on the rotational mobility of the Ca-ATPase correlated well with function, indicating that C28R2-induced protein aggregation and Ca-ATPase inhibition are much more Ca-dependent in skeletal than in CSR. In CSR at low Ca2+, phospholamban (PLB) antibody (functionally equivalent to PLB phosphorylation) increased the inhibitory effect of C28R2 slightly. Fluorescence of fluorescein 5-isothiocyanate-labeled SR suggests that C28R2 stabilizes the E1 conformation of the Ca-ATPase in skeletal SR, whereas in CSR it stabilizes E2. After the addition of PLB antibody, C28R2 still stabilizes the E2 conformational state of CSR. Therefore, we conclude that C28R2 affects Ca-ATPase activity, conformation, and self-association differently in cardiac and skeletal SR and that PLB is probably not responsible for the differences.
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The effects of C28R2 on the rotational dynamics of the Ca-ATPase show a strong correlation between activity and oligomeric state in skeletal and CSR, indicating that this peptide affects Ca-ATPase activity by causing self-association of the enzyme. C28R2 inhibits and aggregates the Ca-ATPase of both skeletal and CSR. Inhibition and aggregation are Ca-dependent in skeletal SR, but not in CSR. The effects of C28R2 on CSR Ca-ATPase are not significantly altered by PLB Ab. C28R2 stabilizes the E1 conformational state of the Ca-ATPase in skeletal SR, whereas in CSR it stabilizes E2. After the addition of PLB Ab, C28R2 still stabilizes the E2 conformational state in CSR. This suggests that PLB is probably not responsible for the differences observed between skeletal and CSR in response to C28R2. Despite the significant sequence homology between SERCA1-type (skeletal) and SERCA2-type (cardiac) Ca pumps, there are differences in their response to various inhibitory agents, such as general anesthetics and C28R2. On the other hand, it is remarkable that an autoinhibitory peptide from the PMCA enzyme, which has substantial differences in sequence from the SERCA enzymes, has such potent functional and physical effects on the SERCA pumps. Future studies on the structural bases of these inhibitory effects should shed light on the relationship between structure and function in these Ca-ATPase isoforms. We thank Dr. Joseph J. Feher, Medical College of Virginia, for the generous gift of CSR. We are also pleased to thank Dr. Larry Jones, Indiana University School of Medicine, for the anti-PLB Ab 2D12. We also thank Brad Karon, Razvan Cornea, Nicoleta Cornea, John Matta, and Robert L. H. Bennett for technical support. DDT was supported by National Institutes of Health grant GM27906. JTP was supported by National Institutes of Health grant GM28835, and HK by GM50764. LGR was supported by a grant from the American Heart Association.