Abstract
The Na-Ca exchange inhibitory peptide (XIP), which corresponds to residues 251-270 of the Na-Ca exchange protein, specifically inhibits exchange activity (Li, Z., Nicoll, D. A, Collins, A., Hilgemann, D. W., Filoteo, A. G., Penniston, J. T., Weiss, J. N., Tomich, J. M., and Philipson, K. D. (1991) J. Biol. Chem. 266, 1014-1020). We have found that XIP decreased Na+i-dependent Ca2+ uptake to 46 and 20% of control in mixed and inside-out bovine sarcolemmal (SL) vesicles, respectively, and to 22% of control in ferret red cell vesicles. XIP inhibited uptake in bovine SL vesicles after proteolytic digestion. XIP also inhibited Na+o-dependent Ca2+ efflux in bovine SL vesicles but did not inhibit Ca2+ uptake in reconstituted proteoliposomes. Extracellular XIP did not inhibit Ca2+ uptake into intact ferret red cells. Inhibition of uptake in bovine SL vesicles was reduced as the ionic strength was increased. 125I-labeled XIP (1 microM) was cross-linked to proteins of bovine SL vesicles, ferret red cell vesicles, and intact ferret red cells. Labeling of bands at approximately 75, 120, and 220 kDa (in bovine SL vesicles) and bands at 55 and 85 kDa (in ferret red cell vesicles) was detected. No cross-linking was detected in intact ferret red cells. We conclude that XIP inhibition is insensitive to proteolytic digestion and is partially dependent on charge association and conformation of the exchanger. XIP binds to and interacts with the intracellular side of the Na-Ca exchanger.
Highlights
Labeling of bands at ap- In thisreport we further characterize the inhibition of Naproximately 75, 120, and 220 kDa (in bovine SL ves- Ca exchange by the exchange inhibitory peptide (XIP) in icles) and bands at 55 and 8 5 kDa was detected
A t 500 mM KC1 (Table I)we found Na+,dependent Ca2+uptake levels in bovine SL vesiclesto be 87.2% of control when 5 p~ XIP was included in the uptake solution
A peptide corresponding to residues 251-270 was found to be the most potent of the four specific peptides examined and was referred to as the exchange inhibitory peptide (XIP)
Summary
The tubes were centrifuged at 16,000rpm for 20 min This procedure was repeated until the membranes had been washed XIP by incubating 0.5 mg of XIP with 1.5 mCi of lZ5I(as NaI) for 15. A solution containing 10 mM Tris, 2 mM EDTA, pH 7.4, was added and mixed with the suspension, which was centrifuged 20 min an 16,000 rpm. The uptake reaction was stopped at the indlcated time by addition of 3 ml of ice-cold solution, which contained 200 mM KC], 20 mM MOPS/Tris, pH 7.4, 1mM EGTA. Miscellaneous Procedures-Reconstitution of detergent solubilized bovine SL proteinswas achieved by the detergent dilution method as after thawing the vesicles and incubation in 10 volumes of 150 mM NaC1,20 mM HEPES,pH 7.4, a t 37 "C for 15min. At appropriate times,aliquots were removed and diluted into a stop solution containing 2 mM EGTA, 150 mM LiCl, and 20 mM HEPES. This procedure was repeated twice with the finalpellet being resuspended in 160 mM
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