Purification and Properties of an Adenosine Triphosphatase from Sarcoplasmic Reticulum

Abstract An adenosine triphosphatase has been purified approximately 6-fold from sarcoplasmic reticulum through the use of deoxycholate and salt fractionation. Gel electrophoretic profiles indicate that the preparation consists largely of one protein component. Protein purification and the increase in ATPase activity appear to be commensurate. The enzyme, which is soluble by virtue of bound detergent, becomes insoluble when deoxycholate is removed. In both soluble and insoluble states the ATPase activity is stable under appropriate storage conditions. The enzyme contains phospholipid in an amount comparable to that found in intact reticulum. Since the ATPase activity is inhibited by digestion with phospholipase C but can be reactivated by further addition of phospholipid, the phospholipid is believed to be essential to activity. The properties of the purified enzyme appear to be the same as those of the enzyme in sarcoplasmic reticulum. In both cases the ATPase activity requires Mg++ and is stimulated by Ca++. The activity in the presence of 5 mm Mg++ is completely inhibited by the addition of 0.1 to 0.5 mm ethylene glycol bis(β-aminoethyl ether)-N,N'-tetraacetate, a chelator with great specificity for Ca++. The enzyme is inhibited by mersalyl acid at the same levels required for inhibition in sarcoplasmic reticulum. ATPase activity is insensitive to ouabain or oligomycin. The purified enzyme catalyzes an ATP-ADP exchange and is phosphorylated by ATP labeled in the terminal position with 32P. The rate of ATP-ADP exchange and the level of phosphorylation are both increased by a factor commensurate with the increase in ATPase activity. The purified enzyme, depleted of deoxycholate, binds calcium in the presence of ATP and oxalate, but the rate is only a few per cent of that catalyzed by the sarcoplasmic reticulum.