Restoration of calcium transport in the trypsin-treated (Ca+ + Mg2+)-dependent adenosine triphosphatase of sarcoplasmic reticulum exposed th sodium dodecyl sulfate

Abstract

When sarcoplasmic reticulum vesicles are exposed to trypsin for 1 min the adenosine triphosphatase (Mr = 102,000) is cleaved to fragments of Mr = 45,000 and 55,000. The purified ATPase, containing both fragments, transports Ca2+ when incorporated into vesicles containing excess phospholipid. The two fragments can only be dissociated in solutions containing 1% sodium dodecyl sulfate (SDS). Ca2+ transport activity is restored in SDS-dissociated preparations in a series of steps involving dilution with 5 volumes of 5% phospholipids in 0.75% sodium cholate, incubation in ice for 30 min, and passage through an anion exchange column. Vesicles formed in this procedure regain high Ca2+ transport activity if they are incubated in SDS solution at 24 degrees for less than 20 min. However, the extent of renaturation diminishes if the vesicles are incubated for longer periods and little acitivity is recovered in vesicles incubated longer than 60 min at 24 degrees.