Abstract
Synthetic peptides corresponding to the calmodulin-binding domain of the human erythrocyte Ca2+ pump were prepared representing residues 2-29 (C28W), 2-21 (C20W), 2-16 (C15W), and 16-29 (C14) of the sequence (James, P., Maeda, M., Fisher, R., Verma, A. K., Krebs, J., Penniston, J. T., and Carafoli, E. (1988) J. Biol. Chem. 263, 2905-2910). Peptides C28W, C20W, and C15W bound to calmodulin with an apparent 1:1 stoichiometry in the presence of Ca2+ and inhibited the activation of the Ca2+ pump by calmodulin, while C14 was ineffective. Substituting tyrosine (C28Y) or alanine (C28A) for the tryptophan residue lowered the affinity for calmodulin. The estimated Kd values for the calmodulin-peptide complexes were 0.1 nM for C28W, 5-15 nM for C20W, C28Y, and C28A, and 700-1700 nM for C15W. The Ca2+ pump in inside-out erythrocyte membrane vesicles was activated by proteolytic removal of the endogenous calmodulin-binding domain. Addition of C20W or C28W then inhibited calmodulin-independent Ca2+ transport, while a calmodulin-binding peptide from another enzyme had no effect. The inhibition of the pump by C20W was purely competitive with Ca2+, while C28W decreased the Vmax and increased the K1/2 for Ca2+, restoring the pump activity nearly to its low basal level. The results suggest that a calmodulin-binding peptide from any enzyme has two kinds of specificity: it shares with peptides from other enzymes the ability to bind to calmodulin, but only it has the specificity to interact with its own (proteolytically activated) enzyme.
Highlights
The Calmodulin Binding Domain of the PlasmaMembrane Ca2’ Pump Interacts Both with Calmodulin and withAnother Part of the Pump*
Synthetic peptides corresponding to the calmodulin-1985; Kemp et al, 1987), phosphofructokinase (Buschmeier binding domain of the human erythrocyte Ca2+pump et al, 1987),type I1 calmodulin-dependent protein kinase were prepared representing residu2e-s29 (C28W), 2- (Bennett and Kennedy, 1987), and phosphorylase b kinase
The differences between the sequences of these domains presumably occur because each one is specialized tointeract with calmodulin and with the active site within the enzyme in nM for C28W, 5-15 nM for c 2 0 w, C28Y, and C28A, question
Summary
The proteolysis medium contained 0.6 mg/ml IOV membrane protein, Chemi~als-[[y-~~P]ATwP as purchased from Du Pont-New Eng- 0.1 mM EGTA, 0.16 M KCl, and mM Tris-HC1, pH 7.2. N2-p-Tosyl-L-lysinechloromethyl ketone-treated a- Ca2+ Transport Measurements-Calcium influx into IOVs was chymotrypsin (EC 3.4.21.1), ~-tosylamido-2-phenylethylchloro-measured for 5 min at 37 'C by rapid filtration through Millipore methyl ketone-treatedtrypsin(EC 3.4.21.4), trypsin inhibitor, and membrane filters(0.45pm pore size, type HA)as described by Sarkadi aprotinin were obtained from Sigma. IOVs (either proteolyzed or control) at a concentration of 15-20 pg Purification of the Ca2+-ATPasferom Human Erythrocytes-Eryth- of membrane protein/ml were preincubated withthe required amount rocyte ghosts deficient in calmodulin were prepared as described by of calmodulin and/or peptides for 3 min at 37 "C before initiating. The Ca'+- calcium uptake by the addition of 0.5 mM ATP
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