Different sensitivity of ATP + Mg + Na (I) and Pi + Mg (II) dependent types of ouabain binding to phospholipase A2

Summary The effect of phospholipase A₂ and of related agents on ouabain binding and Na,K-ATPase activity were studied in intact and detergent-treated membrane preparations of rat brain cortex and pig kidney medulla. It was found that phospholipase A₂ (PLA₂) may distinguish or dissociate ouabain binding complexes I (ATP+Mg+Na) and II (P_i+Mg), stimulating the former and inhibiting the latter. Procedures which break the permeability barriers of vesicular membrane preparations, such as repeated freezing-thawing, sonication or hypoosmotic shock failed to mimic the effect of PLA₂, indicating that it was not acting primarily by opening the inside-out oriented vesicles. The detergent digitonin exhibited similar effects on ouabain binding in both ATP+Mg+Na and P_i+Mg media. Other detergents were ineffective.The ability of PLA₂ to distinguish between ouabain binding type I and II can be manifested even in SDS-treated, purified preparations of Na,K-ATPase. The number of ATP+Mg+Na-dependent sites is unchanged, while the P_i+Mg-dependent sites are decreased in number in a manner similar to that seen in original membranes. This inhibition is completely lost in the reconstituted Na,K-ATPase system, where the ATP- as well as P_i-oriented ouabain sites are inhibited by PLA₂.