Purification of the cardiac Na+-Ca2+ exchange protein.
We have used fractionation procedures to enrich solubilized cardiac sarcolemma in the Na+-Ca2+ exchange protein. Sarcolemma is extracted with an alkaline medium to remove peripheral proteins and is then solubilized with decylmaltoside. Next, the exchanger is applied to DEAE-Sepharose and eluted with high salt. The DEAE fraction is applied to WGA-agarose, and a small fraction of protein, enriched in the exchanger, can be eluted by changing the detergent to Triton X-100. This fraction is reconstituted into asolectin proteoliposomes for measurement of Na+-Ca2+ exchange activity and gel electrophoresis. The purified fraction has a Na+-Ca2+ exchange activity of 600 nmol Ca2+/mg of protein per s at 10 microM Ca2+ and a purification factor of about 30 as compared with control reconstituted sarcolemmal vesicles. Ca2+-Ca2+ exchange and Na+-Ca2+ exchange activities were both present in the same final reconstituted vesicles indicating that the same protein is responsible for both transport activities. SDS-PAGE reveals two prominent protein bands at 70 and 120 kDa. After mild chymotrypsin treatment (1 microgram/ml), there is no loss of exchange activity, but the 120 kDa band disappears and the 70 kDa band becomes more dense. This suggests that the 70 kDa band is due to an active proteolytic fragment of the 120 kDa protein. Under non-reducing gel conditions, only a single protein band is seen with an apparent molecular weight of 160 kDa. Antibodies to the purified exchanger preparation are able to immunoprecipitate exchange activity and confirm that the 70 kDa protein derives from the 120 kDa protein. We propose that both the 70 and 120 kDa proteins are associated with the Na+-Ca2+ exchanger.
Philipson
K D
KD
Department of Medicine, University of California, Los Angeles.
Longoni
S
S
Ward
R
R
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002918:Chymotrypsin / Q000378:metabolism; D004285:Dogs; D008970:Molecular Weight; D009206:Myocardium / Q000032:analysis* / Q000378:metabolism; D011233:Precipitin Tests; D012508:Sarcolemma / Q000032:analysis; D019831:Sodium-Calcium Exchanger
D002352:Carrier Proteins; D019831:Sodium-Calcium Exchanger; D002918:Chymotrypsin; D002118:Calcium