Separation of Adenosine Triphosphatase of HK and LK Sheep Red Cell Membranes by Density Gradient Centrifugation

Abstract

Membrane fragments from high potassium (HK) and low potassium (LK) sheep red cells were separated by density gradient centrifugation. Three preparations were studied: HK membranes sonicated for 20 minutes, HK membranes sonicated for 3 minutes, and LK membranes sonicated for 3 minutes. The adenosine triphosphatase (ATPase) activity in the maximally disrupted preparation was not sensitive to Na + K and was recovered in relatively small but heavy (specific gravity 1.19) fragments which made up no more than 8% of the total membrane. Both Na + K-sensitive (S) and Na + K-insensitive (I) ATPase activity were found in the more gently broken up preparations, but the ratio of S- to I-ATPase was much greater in HK than in LK membrane fragments. S-ATPase activity in preparation was about 50% that observed in KH membranes prior to sonication. S-ATPase activity was recovered from the density gradient in relatively large but light (specific gravity 1.10) fragments. As was the case with the maximally disrupted preparation, I-ATPase activity in both preparations was recovered in small but heavy (specific gravity> 1.20) fragments. The possibility that sensitivity of sheep red cell membrane ATPase to Na + K depends on the association between units containing the enzyme (s) and large, light, phospholipid-containing components is discussed.