Reconstitution and partial purification of cardiac sarcolemmal Na+/H+ (K+/H+) antiporter.
Most of the plasma membrane Na+/H+ antiporters that have been well characterized do not transport K+. Our previous studies showed that cardiac plasma membrane contains a nonselective alkali cation/H+ antiporter that transports either Na+ or K+. The purpose of this work was to develop a convenient assay for the cardiac antiporter to be used in the course of its purification. Sarcolemmal membranes prepared from bovine heart were solubilized with Triton X-100 in the presence of a mixture of phospholipids, and the solubilized proteins were passed through Bio-Beads to form proteoliposomes. Exchange activities were assayed either by measuring H+ gradient-dependent uptakes of 22Na+ and 86Rb+ by proteoliposomes or by recording H+ release from proteoliposomes as reported by the fluorescence of a pH probe that was trapped in the proteoliposomes during reconstitution. Properties of the reconstituted nonselective antiporter were found to be similar to those of the native sarcolemmal antiporter. Using a DEAE-cellulose column, 20-fold purification of the antiporter was achieved, demonstrating the suitability of the reconstitution assay procedures for further purification of the functional antiporter.
Periyasamy
S M
SM
Department of Pharmacology, Medical College of Ohio, Toledo 43699.
Paucek
P
P
Garlid
K D
KD
Askari
A
A
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009206:Myocardium / Q000737:chemistry*; D011510:Proteolipids; D012508:Sarcolemma / Q000737:chemistry; D017923:Sodium-Hydrogen Exchangers / Q000302:isolation & purification* / Q000378:metabolism
D011510:Proteolipids; D017923:Sodium-Hydrogen Exchangers; C020485:proteoliposomes; D000584:Amiloride