[35] Preparation of reconstitutively active Rutamycin (cas 1404-59-7)-sensitive ATPase from bovine heart mitochondria

Add time:09/06/2019    Source:sciencedirect.com

Publisher SummaryThe assay used for the purification of reconstitutively active rutamycin-sensitive ATPase (RSA) preparation is the 32Pi–ATP exchange. The most convenient procedure for the reconstitution of vesicles catalyzing the exchange is the cholate dilution method. Soybean phospholipids (asolectin) are partially purified and stored in chloroform–methanol (4:1) at −20°C. Aliquots are dried under N2 and sonicated at 10% concentration in 3% cholate–20 mM Tris-sulfate–0.4 mM ethylenediaminetetraacetic acid (EDTA) (pH 8) until clarified (5–10 min). After incubation for 10 minutes at 30°C, the reaction is stopped with 0.1 ml of 50% trichloroacetic acid and, after centrifugation, 0.5 ml of the supernatant is assayed for radioactive ATP.

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