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 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 microM, 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 microM) than at high (10 microM) 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.
Reddy
L G
LG
Department of Biochemistry, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
Shi
Y
Y
Kutchai
H
H
Filoteo
A G
AG
Penniston
J T
JT
Thomas
D D
DD
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000097:blood / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D004910:Erythrocyte Membrane / Q000201:enzymology*; D016650:Fluorescein-5-isothiocyanate; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D018482:Muscle, Skeletal / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology
C097865:C28R2 peptide; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D010455:Peptides; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate