Phospholipid requirement of the vanadate-sensitive ATPase from maize roots evaluated by two methods

The activation of the vanadate-sensitive ATPase from maize (Zea mays L.) root microsomes by phospholipids was assessed by two different methods. First, the vanadate-sensitive ATPase was partially purified and substantially delipidated by treating microsomes with 0.6% deoxycholate (DOC) at a protein concentration of 1 milligram per milliliter. Vanadate-sensitive ATP hydrolysis by the DOC-extracted microsomes was stimulated up to 100% by the addition of asolectin. Of the individual phospholipids tested, phosphatidylserine and phosphatidylglycerol stimulated activity as much as asolectin, whereas phosphatidylcholine did not. Second, phospholipid dependence of the ATPase was also assessed by reconstituting the enzyme into proteoliposomes of differing phospholipid composition. In these experiments, the rate of proton transport and ATP hydrolysis was only slightly affected by phospholipid composition. DOC-extracted microsomes reconstituted with dioleoylphosphatidylcholine had rates of proton transport similar to those found with microsomes reconstituted with asolectin. The difference between the two types of assays is discussed in terms of factors contributing to the interaction between proteins and lipids.