Sequential use of detergents for solubilization and reconstitution of a membrane ion transporter.
Solubilization and reconstitution of the cardiac sarcolemmal Na+/Ca2+ exchanger by use of the anionic detergent cholate and its application for reconstitution of the exchanger following solubilization with zwitterionic or nonionic detergents is described. Solubilization and reconstitution with cholate provided a 32.6-fold enrichment of Na+/Ca2+ exchange activity over sarcolemmal vesicles (5.2 to 170 nmol/mg/s) with 202% recovery of total activity. In combination with asolectin, the cholate dilution technique (H. Miyamoto and E. Racker, J. Biol. Chem. 255, 2656, 1980) offers a rapid and simple means for reconstitution and provides good recovery of total and specific Na+/Ca2+ exchange activity. However, the use of anionic detergents for solubilization precludes the use of certain chromatographic procedures for protein purification. Conversely, nonionic and zwitterionic detergents permit effective use of available chromatographic techniques, but can be troublesome during reconstitution. We have combined the advantages of solubilization with nonionic and zwitterionic detergents with the advantages of reconstitution by cholate dilution. Reconstitution of the exchanger, after solubilization with 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (Chaps) or n-octyl-beta-D-glucoside, was accomplished by the addition of a cholate/asolectin medium followed by dilution. Na+/Ca2+ exchange activity was enriched 30.7-fold with 196% recovery with Chaps and 34.1-fold with 204% recovery with n-octyl-beta-D-glucoside. The presence of Chaps was found to shift the optimal asolectin concentration for reconstitution from 15 mg/ml (cholate alone) to 25 mg/ml. In addition, pelleting of proteoliposomes subsequent to reconstitution resulted in greatest recovery of total activity when volumes were kept below 1.0 ml.(ABSTRACT TRUNCATED AT 250 WORDS)
Ambesi
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Department of Pharmacology, Medical University of South Carolina, Charleston 29425.
VanAlstyne
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Bagwell
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Lindenmayer
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GE
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002793:Cholic Acids; D003902:Detergents; D004285:Dogs; D005960:Glucosides; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification; D000644:Quaternary Ammonium Compounds; D012508:Sarcolemma / Q000737:chemistry*; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger; D012995:Solubility
D002352:Carrier Proteins; D002793:Cholic Acids; D003902:Detergents; D005960:Glucosides; D008565:Membrane Proteins; D000644:Quaternary Ammonium Compounds; D019831:Sodium-Calcium Exchanger; C034730:zwittergent 3-12; C018619:octyl-beta-D-glucoside; D012964:Sodium; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D002118:Calcium